Pregnancy Protein Research

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PREGNANCY PROTEIN RESEARCH

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

PREGNANCY PROTEIN RESEARCH

MARIE O’LEARY AND

JOHN ARNETT EDITORS

Nova Science Publishers, Inc. New York

Copyright © 2009 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Pregnancy protein research / [edited by] Marie O'Leary and John Arnett. p. ; cm. Includes bibliographical references and index. ISBN 978-1-61470-609-0 (eBook) 1. Pregnancy proteins. I. O'Leary, Marie. II. Arnett, John, 1959[DNLM: 1. Pregnancy Proteins. 2. Embryo Implantation--physiology. 3. Gestational Trophoblastic Neoplasms. 4. Pre-Eclampsia--diagnosis. WK 920 P9235 2009] QP552.P65P727 2009 612.6'4--dc22 2009002396

Published by Nova Science Publishers, Inc.  New York

Contents Preface

vii

Research and Reviews: Chapter I

Pregnancy Proteins as Markers for Preeclampsia Lenka Fialová and Ivan Matouš-Malbohan

Chapter II

Pregnancy in Buffalo Cows G. Campanile, G. Neglia, D. Vecchio, M. Russo and L. Zicarelli

Chapter III

The Dialogue between Blastocyst hCG and Endometrial hCG/LH Receptor: Impact in Implantation and Placentation S. Perrier d’Hauterive, M. Tsampalas, S. Berndt, C. Munaut, J. M.Foidart and V. Geenen

Short Communications:

1 3 31

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109

Short Communication A Gamma Interferon Production Correlates Negatively with Plasma Levels of Pregnancy-Associated Glycoprotein-1 (PAG-1) During Gestation in Dairy Cows Naturally Infected with Neospora CANINUM F. López-Gatius, S. Almería, J. L. Yániz, P. Santolaria, C. Nogareda, M. Mezo, M. Gonzalez-Warleta, J. A. Castro-Hermida, N. M. de Sousa and J. F. Beckers

111

Short Communication B Concentrations of Pregnancy-Associated Glycoproteins in Water Buffaloes Females (Bubalus Bubalis) During Pregnancy and Postpartum Periods O. Barbato, N. M. Sousa, A. Malfatti, A. Debenedetti, L. Todini, V. L. Barile, and J. F. Beckers

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Contents

vi Short Communication C

The Role of Human Chorionic Gonadotropin (HCG) in Gestational Trophoblastic Disease Caroline Wilson and B. W Hancock

135

Short Communication D Fusogenic Syncytin-1 and Transcription Factor Glial Cells MissingA: Presumed Regulators in Human Placental Physiology and Pathophysiology Christina Wich, Said Hashemolhosseini and Ina Knerr Index

149 163

Preface Chapter I - Pre-eclampsia is a serious disease affecting about 5 % of pregnant women. It is associated with significant perinatal and maternal morbidity and mortality. Despite a great effort devoted to the investigation of preeclampsia the specific tests for its early prediction with the high specificity and sensitivity remain to be determined. Besides uterine artery ultrasonography a variety of biochemical markers has been intensively explored in the first as well as the second trimester of pregnancy. The determination of pregnancy and placental proteins was analysed in the aspect if their maternal serum levels are altered in women who subsequently suffered with preeclampsia. Some of these proteins are used as maternal serum markers in the first or second trimester prenatal aneuploidy screening. The authors have concerned to the placenta-derived proteins which have been already used in the prenatal screening for Down syndrome – human chorionic gonadotrophin (hCG) and pregnancy-associated plasma protein A. Besides these proteins the authors have dealt with other placental proteins – placental protein 13 (PP13) and pregnancy-specific β1glycoprotein (PSβG/SP1). Their biochemistry, physiology and clinical usefulness especially from the point of view of their ability to discriminate women at risk of preeclampsia were reviewed. Chapter II - Immediately after parturition, buffaloes show several physiological modifications which are fundamental to sustain the new pregnancy. The first step is the resumption of ovarian cycle which is blocked during pregnancy by the effect of progesterone that exerts its action in avoiding other ovulations and maintaining hypotonic the uterus. In buffalo species, the resumption of ovarian activity is affected by the calving season and climate variation. Embryo development is faster in buffalo than in bovine. Therefore, the maintenance of pregnancy is due to either the embryo capacity of signalling its presence or the mother capacity of recognizing these signals and maintaining an adequate uterine environment. Embryo implantation commences around Day 30 in cattle and is presumed to be similar in buffalo. The advent of ultrasonography has contributed in the field of buffaloes reproduction, leading to new information on follicular development, pregnancy diagnosis, fetal sex determination, folliculocenteses, diagnosis of abnormalities of the reproductive organs, monitoring of treatment of ovarian cysts, monitoring of postpartum genital resumption, ultrasound-guided centesis and male genital ultrasonography. Recent applications include the use of doppler ultrasonography for ovarian, uterine and mammarian

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blood flow ultrasonography. In particular, the evaluation of early pregnancy, allowed it to establish an incidence of embryonic mortality of 20-40% between 28-60 days of gestation in buffaloes that conceive during increasing daylight length. A reduced capacity to secrete progesterone seems to explain in part this embryonic mortality, but other unidentified factors contribute between 40-50% to the embryonic losses. Treatments with hCG, GnRH agonist or progesterone on Days 5 after AI not always reduce embryonic mortality in buffalo species. Embryonic mortality in buffaloes appears to occur later (Day 25-40) than in cattle and P4 treatments should perhaps be applied later. Furthermore, few studies have demonstrated that new methods can be used for evaluating the pregnancy and, in particular, the presence of a live conceptus. In fact, also in buffalo species some pregnancy-associated glycoproteins (PAG) have been isolated and purified. PAG concentrations in pregnant buffaloes were almost 2 to 3 times higher than those observed in dairy cows from Day 30 till 60 after breeding. This rapid increase, associated with high maternal concentrations at early pregnancy period, are characteristic of caprine and ovine species and different from cattle, in which concentrations increase slowly and remain at low levels during early pregnancy gestation. Currently, strong efforts are made to develop accurate ELISA kits for PAG determination. In this review, several aspects regarding buffalo reproduction will be analysed. Chapter III - The success of implantation depends on a receptive endometrium, a functionally normal blastocyst, and a synchronized cross-talk between embryonic and maternal tissues. In addition to the hormonal control, a cascade of cytokines intervenes in the dialogue at the maternal-embryonic interface, which is a crucial step at the crossroad between immunology and endocrinology. This chapter relates to the very first phases of human embryo implantation, starting from the entry of the blastocyst in the uterine cavity, its arrival in the vicinity of the maternal endometrium, and the dialogue it engages with the latter according to adhesion and paracrine modes. Among the abundant and redundant mediators of the maternal-fetal cross-talk, the authors will focus on the specific and very early embryonic signal: human chorionic gonadotropin hormone (hCG). Data from literature show that through this signal, the embryo profoundly intervenes in its own implantation and favors immunological tolerance and active angiogenesis which are crucial prerequisites to successful implantation and placentation. From the authors’ previous studies, it appears that in vitro, dose-dependent hCG enhances the endometrial production of pro-implantatory leukemia inhibitory factor (LIF), proangiogenic VEGF, and reduces pro-inflammatory interleukine (IL) -6 secretion. This positive action is mediated by hCG binding to its cognate receptor (LH/hCG-R), which the authors have evidenced on endometrial epithelial cells. From our preliminary results, this epithelial expression on the endometrium varies along the menstrual cycle, with a higher expression at the time of implantation. It remains to be determined whether the embryo itself modifies the endometrial LH/hCG-R expression. Successful embryo development also requires an extensive endometrial angiogenesis in the proximity of the implantation site. In their work, the authors have also demonstrated an angiogenic effect of hCG in several in vivo (chick chorio-allantoïc membrane, matrigel plug assay, aortic ring assay) and in vitro experimental models. LH/hCG-R was detected in

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endothelial cells by RT-PCR and by Western blotting. In mice aortic ring assay, angiostimulation by hCG was abrogated by deletion of LH/hCG-R (LuRKO mice). Use of recombinant hCG and anti-hCG antibody further confirmed the specificity of this angiogenic activity. By using dibutyryl cAMP, adenylate cyclase or protein kinase A inhibitors, the authors have demonstrated that hCG-mediated angiogenesis involves adenylyl-cyclase– protein kinase A activation. Altogether, these data demonstrate that peritrophoblastic angiostimulation may result from a paracrine dialogue between trophoblast, epithelial and endothelial cells through hCG and VEGF. This chapter supports that, through a specific dialogue between hCG and LH/hCG-R, human blastocyst actively intervenes in implantation and placentation processes. The evaluation of the uterine receptivity, the impact of the embryo at the time of implantation and the interconnections between mother and embryo through this hCG/LH-R signaling remain further challenges for clinical practice. Short Communication A - Gamma interferon (IFN-γ) production has been ascribed a role in protecting cows infected with Neospora caninum against abortion. The present study analyzes the interaction between IFN-γ production and levels of plasma pregnancy-associated glycoprotein-1 (PAG-1), as a marker of placental/fetal well-being, throughout gestation in naturally Neospora-infected dairy cows. Data were obtained from 88 pregnant HolsteinFriesian cows in two herds: 62 seropositive and 26 seronegative for the parasite. Blood samples were collected on Days 40, 90, 120, 150, 180 and 210 of gestation. Plasma was tested for antibodies against N. caninum, PAG-1 and IFN-γ. Twenty five (28.4%) pregnancies were recorded after AI using Holstein-Friesian semen (19 in seronegative and 6 in seropositive animals), and 63 (71.6%) after AI using Limousin semen (7 in seronegative and 56 in seropositive animals). Gamma interferon was detected in the plasma of 14 (22.6%) of the 62 Neospora-seropositive cows and could not be detected in any of the 26 seronegative animals. All 14 cows producing IFN-γ became pregnant using Limousin semen. Our GLM repeated measures analysis revealed no effects of herd, lactation number, milk production at the time of pregnancy diagnosis and Neospora-seropositivity on plasma PAG-1 concentrations. Significant positive effects were observed of both the day of gestation (P<0.0001) and the interaction between day of gestation and breed of sire (P = 0.001) on PAG-1 values. Cows carrying twins had higher (P = 0.002) PAG-1 concentrations throughout gestation than cows carrying singletons. Interactions between breed of sire and Neosporaseropositivity (P<0.0001), and between IFN-γ production and Neospora-seropositivity (P = 0.04) were also detected. Thus, Neospora-seronegative cows inseminated with Limousin and Neospora-seropositive cows showing no IFN-γ production, exhibited higher PAG-1 concentrations during gestation than seropositive cows inseminated with Limousin semen and producing IFN-γ, respectively. Our data indicate that the production of IFN-γ correlates negatively and the production of antibodies against N. caninum is uncorrelated with plasma PAG-1 concentrations during gestation in Neospora-infected dairy cows. Our results also suggest that augmented Th1 cell-mediated immunity is related to a lower risk of abortion and therefore higher resistance to the parasite in cows inseminated with Limousin versus Holstein-Friesian semen. Short Communication B - The concentrations of pregnancy-associated glycoproteins (PAG) were determined in buffalo cows (Bubalus bubalis) using three different

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radioimmunoassay (RIA) systems (RIA-497, RIA-706, and RIA-708). Samples (10 mL) were collected by jugular venipuncture from Week 0 until Week 28 of pregnancy (9 females), and from parturition until Week 10 postpartum (13 females). During pregnancy, PAG molecules were detectable at Week 6 using the three aforementioned RIA systems (3.9 ± 1.3 ng/mL, 9.7 ± 1.3 ng/mL, and 9.9 ± 0.7 ng/mL for RIA-497, RIA-706, and RIA-708, respectively). These concentrations increased gradually until Week 28, reaching values of 39.6 ± 4.0 ng/mL (RIA497), 50.5 ± 11.9 ng/mL (RIA-706), and 68.2 ± 20.8 ng/mL (RIA-708). PAG concentrations determined by RIA-497, RIA-706, and RIA-708 were strongly correlated throughout the entire gestation period, RIA-708 giving the higher concentrations. At parturition, the mean concentrations ranged from 34.9 ± 4.0 (RIA-497) to 84.7 ± 10.6 ng/mL (RIA-708). Thereafter, the concentrations decreased steadily, reaching very low levels (< 1.0 ng/mL) at Week 8 postpartum. In conclusion, PAG concentrations measured by the above three RIA systems showed a profile similar to those previously described in bovine species, with higher concentrations being detected by RIA-706 and RIA-708. However, the patterns of PAG concentrations, studied using the three aforementioned PAG-RIA systems, differed around parturition, with very low levels being observed in the female buffaloes. Short Communication C - Gestational trophoblastic disease (GTD) encompasses a number of rare related tumours, including complete hydatidform mole (CHM), partial hydatidiform mole (PHM), invasive mole, choriocarcinoma and placental site trophoblastic tumour, all of which vary in their propensity for spontaneous resolution, local invasion and metastasis. The worldwide incidence of GTD varies, with an increased incidence in women from Asia, however UK incidence is around 1.5 cases/1000 live births and the incidence of choriocarcinoma is 1/50,000 live births. Persistent GTD (termed gestational trophoblastic neoplasia, GTN) commonly follows molar pregnancy, but can occur after any type of gestation. Several risk factors are recognised i.e. maternal age, previous mole and socioeconomic factors. Clinical presentation of GTN has changed over time due to earlier diagnosis and use of ultrasound, and includes abnormal bleeding during pregnancy, anaemia, hyperemesis, pre eclampsia, pain, intraperitoneal haemorrhage, symptomatic metastatic disease and rarely thyrotoxicosis due to cross reaction of a subunit of hCG and TSH. The mainstay of diagnosis involves the measurement of hCG and its variants, produced by trophoblast cells. The amount of hCG indicates the volume of tumour and therefore influences prognosis. Other diagnostic investigations include history/examination; blood screens; imaging including chest x-ray, computed tomography scan of the thorax, ultra sound scan of the pelvis, and histological review. All patients are staged/scored according to WHO and FIGO criteria. Those who are high risk on scoring, have βHCG > 50,000iu/L, or have multiple pulmonary metastasis, undergo a central nervous system evaluation. Treatment of GTD requires a team of specialists. Initial management requires blood loss replacement and usually suction curettage of molar tissue. GTD is managed with either second evacuation, hysterectomy if family complete or chemotherapy chosen according to risk. In the UK, less than 10% patients require chemotherapy with a cure rate of virtually 100% in low risk disease and 86% in high risk disease. Measurements of hCG are essential to monitor response to treatment; an adequate treatment response is defined by a 50% reduction in hCG on weekly serum analysis. Once hCG becomes normal treatment continues to ensure elimination of residual tumour following biochemical remission. The choice of chemotherapy depends upon

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risk; low risk receiving intra muscular methotrexate and if salvage is required a combination of chemotherapy is used. High risk receive a combination chemotherapy from the outset. Follow up is essential and involves serial measurements of HCG at specified intervals. The bespoke management by specialist teams ensures that GTD remains one of the most curable of all human cancers. Short Communication D - The differentiation process and cell-cell fusion of human trophoblasts in the human placenta are controlled by a variety of regulatory genes and key molecules. In this article, the authors focus on the fusogenic glycoprotein syncytin-1, which originally derived from the human endogenous retrovirus HERV-W. In addition, they look at its transcription factor GCMa (glial cells missing-a or Gcm1). The authors propose that GCMa-driven syncytin-1 expression is a key mechanism for syncytiotrophoblast formation in the human placenta. Besides the physiological significance of syncytin-1 and GCMa, the authors discuss their pathophysiological role in pre-eclampsia and intrauterine growth restriction (IUGR). In addition, the authors focus on the effects of hypoxia on the expression of syncytin-1 and GCMa in trophoblastic cells because changing oxygen availability contributes to abnormal placental development. Basically, any alteration of the cAMP signaling cascade involving protein kinase A (PKA), GCMa and syncytin-1 can be considered a major risk factor for diminished trophoblast differentiation and impaired syncytiotrophoblast formation, followed by placental dysfunction, for example, in the course of hypoxia. Furthermore, hypoxia-related down-regulation of syncytin-1 can, to a great extent, be compensated by stimulating the cAMP-driven PKA pathway. Considering that preeclampsia is unique to humans and that syncytin-1 is derived from the HERV-W family exclusively found in humans and in higher primates, syncytin-1 is an interesting candidate for research into human placental physiology and altered placental function. Similarly, the authors describe a putative mode of action at the cellular level. Using a cell culture model of syncytin-1 overexpressing cells it has been shown that syncytin-1 is capable of anti-apoptotic functions. A lower apoptotic response, such as a lower level of caspase 3 along with higher amounts of anti-apoptotic Bcl-2 have been found in syncytin-1 transfected cells compared with controls. In conclusion, the authors propose that fusogenic syncytin-1 may function as an antiapoptotic glycoprotein during cell-cell fusion processes. Conversely, alterations in the syncytin-1/GCMa system may, in certain circumstances, be followed by placental disturbances and disorders of pregnancy such as pre-eclampsia and IUGR.

Research and Reviews

In: Pregnancy Protein Research Editor: Marie O’Leary and John Arnett

ISBN 978-1-60692-396-2 © 2009 Nova Science Publishers, Inc.

Chapter I

Pregnancy Proteins as Markers for Preeclampsia

1

Lenka Fialová1 and Ivan Matouš-Malbohan1,2

Institute of Medical Biochemistry, First Faculty of Medicine, Charles University, Prague, Czech Republic 2 Institute of Clinical Chemistry and Laboratory Medicine, General Teaching Hospital, First Faculty of Medicine, Charles University, Prague, Czech Republic

Abstract Pre-eclampsia is a serious disease affecting about 5 % of pregnant women. It is associated with significant perinatal and maternal morbidity and mortality. Despite a great effort devoted to the investigation of preeclampsia the specific tests for its early prediction with the high specificity and sensitivity remain to be determined. Besides uterine artery ultrasonography a variety of biochemical markers has been intensively explored in the first as well as the second trimester of pregnancy. The determination of pregnancy and placental proteins was analysed in the aspect if their maternal serum levels are altered in women who subsequently suffered with preeclampsia. Some of these proteins are used as maternal serum markers in the first or second trimester prenatal aneuploidy screening. We have concerned to the placenta-derived proteins which have been already used in the prenatal screening for Down syndrome – human chorionic gonadotrophin (hCG) and pregnancy-associated plasma protein A. Besides these proteins we have dealt with other placental proteins – placental protein 13 (PP13) and pregnancy-specific β1-glycoprotein (PSβG/SP1). Their biochemistry, physiology and clinical usefulness especially from the point of view of their ability to discriminate women at risk of preeclampsia were reviewed.

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Introduction Preeclampsia which affects about 5 % pregnancies is a multisystem disorder unique to human pregnancy. It is associated with significant maternal and neonatal morbidity and mortality. The diagnosis of preeclampsia is based on the new onset hypertension, proteinuria and oedema after 20 weeks of gravidity in previously normotensive and non-proteinuric women (Davison et al., 2004). Preeclampsia is a heterogeneous disorder. It had been differentiated into mild, moderate and severe forms. A more recently subclassification distinguished early and late onsets of preeclampsia. The early preeclampsia leading to onset before 34 weeks´ gestation comprises the most severe cases and it is usually associated with intrauterine growth restriction of foetus (Huppertz, 2008). Although the research of aetiopathogenesis of preeclampsia has been subject of many studies, its causes are not fully understood. The presence of placenta with or without foetus is unnecessary condition for the development of preeclampsia. It is believed that a failure of trophoblastic invasion of the maternal spiral arteries followed by alteration of villous trophoblasts plays a central role in the pathophysiology of preeclampsia. The cascade of other events such as a decrease of uteroplacental perfusion, placental ischemia, oxidative stress and vascular endothelial dysfunction may contribute to the development of preeclampsia (Huppertz 2008, Shah 2007). The trophoblast abnormalities associated with preeclampsia precede the appearance of clinical symptoms. The consequent defect of placental function may manifest by the changes in the circulating levels of a variety of products synthesised by placenta. Many proteins and hormones as well as other mediators have been tested in the effort to find potential biomarkers able to predict preeclampsia (Conde-Agudelo et al., 2004, Papageorghiou and Campbell 2006, Sibai et al., 2005). They included markers reflecting placental dysfunction, endothelial and coagulation activation, systemic inflammation or angiogenesis. A special attention has been paid to the markers used for the first and second trimester screening of chromosomal aneuploidy. It has been shown that their abnormal levels could be associated with adverse pregnancy outcomes including preeclampsia. So far published studies have brought new information about maternal serum levels of pregnancy-related proteins. In this review we have summarized the current knowledge about some placental proteins and hormones with respect to the preeclampsia. We have concerned to the placenta-derived proteins which had been already used in the prenatal screening for Down syndrome – human chorionic gonadotrophin (hCG) measured as a free β-hCG subunit in the first trimester or as hCG in the second trimester and pregnancy-associated plasma protein A (PAPP-A) assayed in the first trimester. Besides these proteins we have dealt with other placental proteins – placental protein 13 (PP13) which seems to be a perspective marker for screening preeclampsia, and pregnancy-specific β1-glycoprotein (PSβG/SP1) explored predominantly in the late pregnancy.

Pregnancy Proteins as Markers for Preeclampsia

5

1. Human Chorionic Gonadotrophin (HCG) Human chorionic gonadotrophin (hCG) has been known since 1927 when the first assay based on its biological activity had been described (Ascheim and Zondek, 1927). Today hCG testing is widely used especially as a pregnancy test, for monitoring of pregnancy and as a marker for gestational trophoblastic diseases or some other tumours. HCG as well as free βhCG are the important biomarkers for the first and second trimester Down´s syndrome screening (Stenman et al., 2006, Wald et al., 2005).

1.1. Physical and Chemical Characteristics Human chorionic gonadotrophin, a glycoprotein hormone of molecular weight of 37 900, is a heterodimer of an α- and β-subunit, joined noncovalently. The α-subunit similar to that of LH (luteinising hormone), FSH (follicle stimulating hormone) and TSH (thyroid stimulating hormone) is composed of 92 amino acid while β-subunit containing 145 amino acids distinguishes hCG from the other glycoprotein hormones. Molecule of hCG is heavily glycosylated. One third of molecular weight of hCG is formed by carbohydrates (Stenman, et al., 2006).

1.2. Function The major function of hCG is the stimulation of progesterone production in the corpus luteum during early pregnancy (Stenman et al., 2006). More recently findings suggest that hCG may have an impact on placental vasculogenesis and angiogenesis. The action of hCG on the process of angiogenesis may be mediated either directly through hCG/LH receptors or indirectly by increasing the synthesis of angiogenic factors or other growth factors (Herr et al., 2007, Zygmunt et al., 2002). HCG induced expression of VEGF (vascular endothelial growth factor) a primary angiogenic factor in a variety of tissues (Reisinger et al., 2007). In vitro experiments showed that hCG had angiogenic and strongly vasodilatatory effects on uterine endothelial cells where hCG/LH receptors are expressed in high levels. It could participate in the adaptation of uterine vasculature to the rising needs of the foetus via VEGF secretion. Furthermore, hCG may cause the elevation of functional IGF (insulin-like growth factor)-II by a local downregulation of IGFBP (insulin-like growth factor binding protein)-1 (Licht et al., 2007). These effects suggest a possible role of hCG as a modulator of vascular development in the fetoplacental unit which is important particularly in the human implantation and placentation (Licht et al., 2007).

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1.3. Physiological Pregnancy HCG is produced and secreted by the placental syncytiotrophoblast. Also cytotrophoblast participates in the synthesis of hCG in pregnancy. The serum concentration of hCG starts to increase 4 – 7 days after implantation. In the first trimester hCG rises exponentially doubling on average every 1.5 – 2 days and the maximum of concentration is reached at 7 – 10 weeks of pregnancy. After this the hCG levels decrease to the 13 – 15 weeks of gestation and then remain around this concentration until term (Cole 1997, Stenman et al., 2006). Free β-hCG concentrations peaks at around 10th week of pregnancy (Cole 1997).

1.4. HCG in Preeclampsia Measurement of hCG together with α1-fetoprotein in maternal serum in the mid-second trimester is widely used as a test for screening for Down syndrome. The exploitation of second-trimester screening for the further purposes such as a prediction of adverse pregnancy outcomes might be very favourable. Studies evaluating the contribution of the first-trimester hCG measurement as free β-hCG for the prediction of preeclampsia started to be published particularly after the screening for Down´s syndrome in the first trimester had been introduced. 1.4.1. First Trimester Maternal serum levels of free β-hCG were significantly decreased in pregnancies that subsequently developed proteinuric pregnancy-induced hypertension but not in nonproteinuric outcomes (Ong, et al., 2000). Similar results have been reported in a recent study of Canini et al. (Canini et al., 2008). This study has also found an association between low levels of maternal serum free β-hCG and a subsequent development of preeclampsia, however the magnitude of this difference was quite small. The results of multivariable analysis showed that free β-hCG was an independent predictor of gestational hypertension and preeclampsia. Further studies failed to confirm the findings of low first-trimester free β-hCG in patients with pregnancy-induced hypertension. They consented that this biomarker is a poor early predictor of preeclampsia. Yaron et al. (2002b) and Smith et al. (2002) demonstrated that levels of free β-hCG were not predictive for preeclampsia. Also, both Spencer´s studies (Spencer et al., 2008; Spencer et al., 2005) did not report significant differences in the median free β-hCG between normal and preeclamptic groups of patients at 11 – 13+6 weeks of pregnancy. Neither a very large prospective FASTER trial did not describe any association between first trimester free β-hCG and pregnancy-induced hypertension (Dugoff et al. 2004). 1.4.2. Second Trimester Numerous studies have investigated the association between maternal serum hCG or free β-hCG levels in the second trimester and preeclampsia. Several studies concluded that hCG might be predictive for preeclampsia, while others did not sustain it.

Pregnancy Proteins as Markers for Preeclampsia

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Sorensen et al. (1993) observed that patients with elevated hCG ≥ 2.0 MoM (multiple of the median) appeared to be at higher risk of pregnancy-induced hypertension and proteinuricinduced hypertension with risk ratio 1.7 and 5.1, respectively. An analysis of more than 45 000 patients disclosed a significantly higher odds ratio for β-hCG > 2.5 MoM with pregnancy-induced hypertension (Yaron et al., 1999). Similarly women with hCG > 3 MoM for hCG had higher rates of preeclampsia however abnormal uterine artery waveform was superior for identification of patients at risk (Hershkovitz et al. 2005). Davidson et al. (2003) demonstrated a modest significant increase of hCG levels only in those women who later developed preeclampsia but not pregnancy-induced hypertension. Additionally, in a retrospective study Ashour et al. (1997) evaluated the benefit of hCG determination among multiparous and nulliparous women separately and with regard to severity of hypertension status. In this study the link of elevated second-trimester serum βhCG and the later development of hypertension in pregnancy was stronger in patients who had severe preeclampsia especially among multiparous women. A significant increasing trend between severity of preeclampsia and women with MS (maternal serum) hCG ≥ 2.0 MoM was also noted in another study (Lee et al., 2000). Women with mild preeclampsia had 2.61times greater chance while women with severe preeclampsia had a 6.13-times greater chance of having elevated MS hCG than did women with a normal pregnancy. Further the higher levels of mid-trimester hCG seemed to be more characteristic for early rather than late onset severe preeclampsia (Shenhav et al., 2002). A relationship between hCG 3 MoM or greater and preterm delivery for preeclampsia was also observed (Towner et al., 2006). In contrast to the mentioned studies, some other authors (Morssink, et al., 1997, Stamilio, et al., 2000) did not find any relationship between hypertensive disorders in pregnancy and increased second-trimester hCG. Although a weak association cannot be excluded, study of Morssink et al. (2000) did not observe any clinically important increase in risk of developing subsequent hypertensive disorders of pregnancy among women with abnormal secondtrimester levels of hCG. An efficiency of hCG measurement in preeclampsia prediction may be significant improved by use of combination of hCG with other parameters. A using of combined model composed of multiple determining factors for severe preeclampsia had strongly enhanced the sensitivity from 54 % for only hCG alone to 70 % (Lee et al., 2000). Another large FASTER trial established that preeclampsia was significantly associated with the combination of elevated hCG and AFP or inhibin A or even of all three together in contrast to if only a single marker was evaluated (Dugoff et al., 2005). The odds ratio was 7.04 for simultaneously elevated hCG, AFP and inhibin A. In a nested case-control study Wald et al. (2006) supposed that early second-trimester prenatal screening markers for Down syndrome can be used to screen for the development of preeclampsia. The evaluation of the Quadruple test markers (hCG or free β-hCG, AFP, uE3 and inhibin A) together with the history of a pregnancy with preeclampsia could identify an estimated 42 % of affected pregnancies with a 6.5 % falsepositive rate. This screening method appeared to identify early cases of preeclampsia as well as late ones. The detection rate for cases of preeclampsia diagnosed before 36 weeks of pregnancy was 47 % for a 6.5 % of false-positive rate. The Quadruple test markers yielded higher detection rates than the Triple test markers (hCG or free β-hCG, AFP, uE3) (Wald, et al 2006).

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Another series of studies focused on the examination of free β-hCG, usually in combination with other markers. Unlike the first trimester, the serum levels in the second trimester tended to be elevated or normal. It was demonstrated that free β-hCG value increased with decreasing time as early as 7 weeks before the onset of proteinuria (Wald and Morris 2001). Pouta, et al. (1998) observed that the women who later developed preeclampsia had a tendency to higher concentration of mid-trimester serum free β-hCG but not significant. Alike Luckas et al. (1998) found that second-trimester serum free β-hCG levels had some predictive value, but the authors concluded that alone measurement of free β-hCG is not clinically useful as a screening test for preeclampsia in primigravid women. Unfortunately, even several other studies showed no significant differences in free β-hCG levels between normotensive women and those in whom preeclampsia developed (Hietala et al., 2000, Raty et al., 2003, Raty et al. 1999). Similarly, Spencer, et al (2006) reported significantly elevated levels of free β-hCG together to PAPP-A, activin- A and inhibin-A, but the predicted detection rate of preeclampsia was lower than those for inhibin A, activin A and uterine artery pulsatility index. Other comparison of free β-hCG and inhibin A in screening efficiency for the subsequent development of preeclampsia also showed that inhibin A was a better marker. In this study the addition of hCG data to inhibin A ones did not improve the screening efficacy compared to inhibin A alone (Aquilina et al., 2000). No alteration in free β-hCG levels irrespective the severity of preeclampsia was found (Raty et al., 2003). Likewise in contrast to total β-hCG parity had no significant effect on the results of free βhCG levels in maternal serum (Raty et al., 1999). 1.4.3. Third Trimester Besides the older papers, only a few later studies pointed out to hCG measurement in the third trimester of pregnancy (Crosignani et al., 1974; Gurbuz et al., 2004; Kharfi et al., 2005; Said et al., 1984). Similarly as in the second trimester the third-trimester maternal serum hCG levels appeared to be elevated and might reflect the severity of the preeclampsia (Gurbuz et al., 2004; Hsu et al., 1994). The increase of hCG levels is especially significant in severe preeclampsia and superimposed preeclampsia. According to the opinion of Gurbuz et al., (2004) the differential diagnosis between chronic hypertensive and severe preeclamptic women or superimposed preeclamptic patients might be established accurately by hCG testing. High hCG levels could be a helpful tool in the maternal follow-up for patients who are candidates for expectant treatment. During the longitudinal follow up of pregnant women between 20 and 30 weeks of pregnancy the rise of β-hCG levels was shown even before the appearance of clinical signs of preeclampsia (Said et al., 1984). Interesting results demonstrated the study comparing the bioactive and immunoreactive serum hCG levels in women with severe preeclampsia (Casart, et al 2001). Serum bioactive hCG levels tended to be lower than normal while immunoactive hCG levels tended to be higher. A possible explanation for this discrepancy may be in the alteration of hCG structure, which can cause a diminished bioactivity.

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1.4.5. Evaluation of HCG Measurement in Preeclampsia It is apparent that the data from the relative extensive literature include normal or decreased values of maternal serum free β-hCG in the first trimester and normal or elevated values of hCG or free β-hCG in the second trimester of pregnancy. The tendency towards to the low concentrations of hCG in the first trimester in pregnancies resulting in preeclampsia is supposed to be the effect of impaired placentation and smaller placental mass (Ong, et al 2000). The increased hCG synthesis in the second and third trimesters can be explained by the hypoxia, which was shown to stimulate hCG in vitro production (Fox, 1970). Placental hypoxia might be a consequence of an inadequate trophoblastic remodelling of the maternal uterine vasculature with an absence of normal physiological changes in the spiral arteries (Shenhav et al., 2002). Besides, it was shown a positive correlation between serum hCG and H2O2, so that the increase of oxidative stress may trigger the secretion of hCG (Kharfi et al., 2005). Moreover, immunohistochemical studies of placentas from the third trimester showed a link between increased maternal serum hCG concentration and increased secretory activity of the syncytiotrophoblast. In placental sections from preeclamptic pregnancies the syncytiotrophoblast exhibited the intensity of hCG immunostaining higher than the one observed in placentas from normotensive pregnancies. Quantitative image analysis of the immunocytochemical staining for hCG also suggested that the production of hCG was greater in these preeclamptic placentas than in the normotensive ones (Barros et al., 2002). Although some of the clinical studies gave evidence about association between altered hCG levels in pregnancy-induced hypertension and preeclampsia modest sensitivity and positive predictive values for these tests predominated.

2. Pregnancy-Associated Plasma Protein A Pregnancy-associated plasma protein A (PAPP-A) was first described and partially purified from pregnancy plasma together with pregnancy-associated plasma protein B, C a D by Lin and his co-workers in 1974 (Lin et al., 1974). Currently maternal serum levels of PAPP-A together to free β-hCG levels and nuchal translucency are the most widely used for the prenatal screening for Down syndrome in the first trimester of pregnancy. Besides the utilization in obstetrics, a possible benefit of PAPP-A as a biomarker of acute coronary syndrome has been intensively studied (Qin et al., 2007).

2.1. Physical and Chemical Characteristics Pregnancy-associated plasma protein A is a macromolecular glycoprotein with α2electrophoretic mobility and a relatively high content of carbohydrates (13 %) (Sutcliffe et al., 1980). PAPP-A is secreted as a dimer composed of two 200-kDa subunits containing 1 547 amino acid residues (Kristensen et al., 1994) while in pregnancy blood the majority of PAPP-A circulates in the form of an approximately 500-kDa heterotetrameric complex 2:2 with the proform of eosinophil major basic protein (proMBP) joined via a number of

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disulfide bonds. In the PAPP-A/proMBP complex, proMBP acts as a physiological inhibitor of PAPP-A proteinase activity (Overgaard et al., 2000). PAPP-A belongs to the groups of metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif in the amino acid sequence. It is the first member of a new metzincin family, the pappalysins (Boldt et al., 2001; Kristensen et al., 1994).

2.2. Function For a long time until to 1999 an exact physiological role of PAPP-A had not been known. Inhibitor effects to several enzymes were mentioned. It had been considered the action on coagulation or immunosuppressive effects (Bischof 1984a). The recognition of PAPP-A as a metalloproteinases contributed to the consideration that PAPP-A could participate in the proteolytic cleavage of IGFBP. In 1999, Lawrence et al., (1999) identified PAPP-A as a metalloproteinase acting on a insulin-like growth factor binding protein-4. So far PAPP-A has been the only physiological proteinase known to degrade of IGFBP-4. Later as additional PAPP-A substrates were reported others IGF binding proteins – IGFBP-5 and IGFBP-2 (Laursen et al., 2001, Monget et al., 2003). The cleavage of IGFBP-4 by PAPP-A occurred in the absence of IGFs, but the rate of hydrolysis was very low, whereas the cleavage of IGFBP-5 did not depend on IGFs (Laursen, et al 2001). Insulin-like growth factors (IGF-I, IGF-II) is the family of polypeptides involved in the cell proliferation, migration and differentiation. They play an important role in the implantation and they are required for physiological prenatal and postnatal growth. Their actions are exerted by binding to the IGF-1 receptor. The bioactivity of IGF is regulated by their high-affinity binding to IGFBP. The IGFBPs binding to IGFs prevent their interaction with cell surface receptors and thus IGFBPs act as inhibitors of IGF function. PAPP-A may enhance the bioactivity of IGF by degrading IGFBP-4, because the cleavage of IGFBP causes the dissociation of IGF (Iwashita et al., 1992; Nayak and Giudice 2003). It seems that PAPP-A might act as a regulator of local IGF bioavailability. Because insulin-like growth factors are abundantly expressed in the placental trophoblasts (Han et al., 1996), then the proteolytic function of PAPP-A resulting into the decrease of IGFBP could play an important role at the implantation by regulating the trophoblast invasion via a paracrine or autocrine interactions (Giudice et al., 2002). The PAPP-A proteolytic activity for IGFBPs might provide one of the explanations for higher frequency of IUGR (intrauterine growth retardation) babies in preeclampsia. It can be supposed that the disturbed function of trophoblast would results in the lower production of PAPP-A. It is expected that low levels of PAPP-A could be associated with high levels of IGFBP-4 and reduced levels of free IGF. The decrease of bioavaibility of IGF might contribute to the impairment of foetal growth. However, another possibility should be mentioned. Recently, IGFBP-4 was shown to be a novel anti-angiogenic mediator secreted by glioblastoma cells (Moreno et al., 2006). If IGFBP-4 inhibited the angiogenic response, then the low PAPP-A levels found in the first trimester of preeclamptic women could have an association with the altered angiogenesis which go before the onset of preeclampsia. The

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interrelatioship among PAPP-A, system of IGFBPs – IGFs and angiogenesis seems to be highly complicated. Only further experiments could clarify some of so far unknown aspects of this problem.

2.3. Physiological Pregnancy Circulating PAPP-A levels are significantly increased over the non-pregnant values by 5 to 6 weeks after conception (Folkersen et al., 1981). During pregnancy PAPP-A levels rise up to 36 weeks and then increase more steeply with the maximum at the term (Folkersen et al., 1981; Smith et al., 1979). The PAPP-A concentrations fall more slowly than other placental proteins and steroids after delivery. The average half-life after delivery is 51 hours (Smith et al., 1979). PAPP-A is synthesised by syncytiotrophoblast and trophoblast-derived septal X cells in pregnancy (Bonno et al., 1994). However, PAPP-A was not expressed only in trophoblastic cells, but in vitro was secreted also by human decidual cells (Bischof et al., 1984).

2.4. PAPP-A in Preeclampsia Likewise hCG the potential additional benefits of PAPP-A measurement for the detection of adverse perinatal outcomes including preeclampsia have been tested for several years. 2.4.1. First Trimester The series of studies investigated the possibility of use of PAPP-A as a predictor of preeclampsia in early stage of pregnancy. The conclusions of these studies are not fully consistent. However, the studies which showed the association between lower PAPP-A levels and pregnancy complications including preeclampsia and pregnancy-induced hypertension predominated. A study of Ong et al. (2000) analysing more than 5000 pregnant women at 10 – 14 weeks of gestation demonstrated that the multiple of median MS PAPP-A was significantly lower in those pregnancies resulting in pregnancy-induced hypertension with proteinuria as well as without proteinuria whereas Yaron et al. (2002a) observed significantly higher rates only in proteinuric pregnancy-induced hypertension in patients with PAPP-A ≤ 0.25 MoM. A prospective cohort study of Smith et al. (2002) as well as a recent very large, prospectively collected patient population screening FASTER Trial also reported that women with low first-trimester PAPP-A levels were associated strongly with an increased risk of preeclampsia in later pregnancy and further adverse pregnancy outcomes (Dugoff et al., 2004). Either Spencer´s research group (Spencer et al., 2007b, Spencer et al., 2008, Spencer et al., 2005) repeatedly reported reduced first-trimester serum PAPP-A levels in women who subsequently developed preeclampsia. In the earlier study the PAPP-A MoM from 64 pregnancies with preeclampsia was 0.844 and the detection rate, for a 5 % false-positive rate, was 14.1 % (Spencer et al., 2005). It was evident that a low PAPP-A itself is not a strong

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indicator of preeclampsia. But a screening efficacy for the prediction of preeclampsia may improve the combination of second trimester uterine artery Doppler velocimetry with PAPPA testing (Spencer et al., 2008; Spencer et al., 2005). Conversely, the values of PAPP-A used together to other placental protein PP13 had no additive value for prediction of preeclampsia (Spencer et al., 2007b). Moreover, it was shown that with decreasing levels of PAPP-A the likelihood ratio for preeclampsia increased. At the 5th centile of normal PAPP-A the odds ratio for preeclampsia was increased by 3.7 fold (Spencer et al., 2008). In contrast to previous studies, a retrospective study of Tul et al., (2003) found no significant differences between PAPP-A MoM in women who developed hypertensive disorders and controls. However, in the multivariate regression model a low PAPP-A levels were significant variables for the delivery of SGA (small-for-gestational-age) babies. Similarly, Canini et al. (2008) in one of the recent study did not corroborate a significant association between PAPP-A levels and the development of hypertensive complications. Nevertheless, they observed that PAPP-A might be an independent predictor of absolute birth weight in physiological variation as well as in abnormal growth in the sense of both smallfor-gestational-age newborns and large-for-gestational-age ones. Also, Pilalis et al. (2007) summarised that the role of PAPP-A in the prediction of preeclampsia is limited; on the other hand low PAPP-A is at least as important as is abnormal Doppler in the prediction of SGA foetuses. The negative findings of these studies might be explained by a relative low number of patients with pregnancy complications – 56 women with hypertensive disorders in Tul´s study (Tul et al., 2003) or only 13 and 17 women with diagnosis of preeclampsia in study of Pilalis et al. (2007) and Canini et al. (2008), respectively. Already mentioned the much larger FASTER Trial included 764 preeclamptic women and even 1484 pregnancies with gestational hypertension. 2.4.2. Second and Third Trimester The decrease of maternal serum levels of PAPP-A in women developed preeclampsia probably continues to the early second trimester (Bersinger and Odegard, 2004). Hovewer, at 22+0 to 24+6 weeks the significant mild increase of PAPP-A or normal levels in the women with subsequent preeclampsia was observed (Bersinger and Odegard, 2004; Spencer et al., 2006). The elevation had been more pronounced in the cases with severe preeclampsia. But the 5 % predicted detection rates suggested only the minimal possibility for clinical use. The investigation of PAPP-A levels in the late pregnancy was the subject of particularly some earlier studies. In a period from 1977 to 1984 several studies about this problem had been published. Some of them reported that unlike the first trimester, the elevated maternal serum levels in the third trimester are predictive of preeclampsia and pregnancy-induced hypertension. The attempt to explore PAPP-A in the complicated pregnancy was initiated 30 years ago. Lin et al. (1977) found significantly elevated plasma PAPP-A levels in toxemic pregnancies in the last month of pregnancy. Later, these observations were sustained by others and further knowledge was added. A promising finding was that the increased PAPP-A levels at 34 weeks´ pregnancy preceded the onset of overt signs of preeclampsia (Hughes et al., 1980a). In the other study of 84 women with preeclampsia the PAPP-A levels were significantly

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elevated only in the group of severe preeclampsia, not in the cases of mild outcome (Toop and Klopper 1981). In contrast to previous studies Westergaard and Teisner (Westergaard 1982) and (Westergaard et al., 1984) did not demonstrate differences between controls and women developed pregnancy-related hypertension with and without proteinuria in whose PAPP-A were measured serially during the second and third trimester. Moreover, maternal serum levels of PAPP-A and trends of levels were irrespective of the gestation age at sampling and unrelated to the time of onset of the disease or its severity. So that an unambiguous decision about usefulness of PAPP-A measurement as a biomarker of foetal well-being in the late trimester could not be made and the further investigation was interrupted for some period. Lately, Bersinger et al. (2003) were successful in the confirmation of old reports of elevated PAPP-A levels in the third trimester of gravidity with preeclampsia. Longitudinal analysis revealed that ratios between the PAPP-A levels at 33 and 17 weeks were significantly higher in pregnancies developing preeclampsia than in controls (Bersinger and Odegard 2004). The circulating PAPP-A levels might partly reflect its placental production. The content of PAPP-A in placental extracts of preeclamptic patients at term was higher than in normal controls (Bersinger et al., 2002). In this context it was an unexpected finding that the values of circulating mRNA for PAPP-A did not differ between group of preeclampsia and controls (Farina et al., 2006). Nevertheless, not only PAPP-A produced in placenta, but also nonplacental sources would contribute to the total serum levels. 2.4.3. Evaluation of PAPP-A Measurement in Preeclampsia The time changes of maternal serum PAPP-A levels in pregnancies resulting in preeclampsia appear to be analogous to those described in hCG. The levels of PAPP-A tend to be reduced in the first and the early second trimesters. In the course of second trimester the PAPP-A levels shift from reduced values to normal or elevated ones even before preeclampsia manifestation. Decreased maternal serum PAPP-A levels in the first trimester could be explained by inadequate placentation (Ong et al., 2000). Furthermore, the low PAPP-A levels by itself could influence placental function through the action of IGFBPs and IGFs system. The altered placental function and probably reduction of synthesis of PAPP-A during the first trimester may indicate the subsequent development pregnancy at risk (Spencer et al., 2005). Elevated PAPP-A levels in the late gestation may be a result of its increased synthesis such as a compensatory mechanism on an initial abnormal situation which occurs in preeclampsia (Bersinger et al., 2002). In spite of several studies demonstrated a relationship between maternal serum PAPP-A levels and preeclampsia or pregnancy-induced hypertension the sensitivities and odds ratios seem to be relatively low to use PAPP-A determination as a predictor of preeclampsia. Moreover the present results are not uniform. Therefore more detailed investigation would be required for the decision if the PAPP-A measurement could contribute to the early diagnosis of preeclampsia.

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3. Placental Protein 13 (PP13) Placental protein 13 (PP13) is a protein which had been purified from human placenta. It had been characterised by Bohn and his co-workers as early as in 1983 (Bohn, et al 1983) but the interest about its clinical usage has been dated since about 2000.

3.1. Physical and Chemical Characteristics Placental protein 13 is a 32-kDa dimer protein composed of two identical 16-kDa subunits held together by disulfide bonds. Each subunit is formed by 139 amino acid residues. It has the lowest content of carbohydrate (0.6 %) of any known placental proteins (Bohn et al., 1983; Burger et al., 2004). The cloning and sequencing analysis revealed that the primary structure of PP13 showed 69 % homology to human eosinophil Charcot-Leyden crystal (CLC) protein (galectin-10) (Than et al., 1999). PP13 also shared a high homology to several other galectins. Therefore placental protein 13 was included into the group of galectin family as a new member. It was designed as a galectin 13 (Than et al., 1999; Visegrady et al., 2001). 3.2. Function As a member of a galectin family, PP13 was supposed to have functions similar to those of other galectins, especially in the actions requiring specifically regulated cell-matrix interaction (Visegrady et al., 2001). By this way PP13 may be involved in the processes of the normal implantation and maternal artery remodelling (Burger, et al., 2004; Than, et al., 1999; Visegrady, et al., 2001). Some mechanisms have been already investigated. PP13 possesses a single sugar binding domain, which is directed into the extracellular space. Through this domain, the secreted PP13 bound with high affinity to sugar residues of extracellular matrix molecules and created a “molecular bridge” between the placenta and the endometrium (Than et al., 2004). A mild lysophospholipase-A activity which was described for PP13 can lead to the liberation of fatty acid constituents of the plasma membrane (Burger, et al 2004; Than et al., 2004). Via this action PP13 may increase the production of prostaglandins, especially prostacyclin, which participates in trophoblast-stimulated remodelling in the maternal spiral arteries in the early development of placenta. Furthermore, PP13 may be involved in the migration of trophoblasts towards to placental bed by binding to β- and γ-actin (Than et al., 2004). Some experiments showed that the biological functions of PP13 could be regulated by phosphorylation of protein or by dimerization by disulfide bonds. The later mechanism might be influenced upon oxygenation changes in the low blood flow organ placenta (Than et al., 2004). It is speculated about other biological effects of PP13 such as cell growth regulation or immunomodulation especially at feto-maternal interfaces (Than et al., 2004, Visegrady et al., 2001).

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3.3. Physiological Pregnancy PP13 is highly expressed in human placental tissues from which was isolated and cloned. An average content of PP13 in human term placenta is 3.7 mg (Bohn et al., 1983). Besides placenta, its expression has been also found in human fetal liver and spleen as well as in some tumorous tissues (Than et al., 1999). By different immunological techniques PP13 was detected predominantly in the brush border membrane of the syncytiotrophoblasts lining at the common feto-maternal blood spaces of the placenta. Perinuclear localization proved its expression in these cells. PP13 produced in the trophoblast is packed into endosomes and then secreted by the ectocytosis to the cell surface through “coated pits” (Than et al., 2004). The serum PP13 levels in gestation start to increase at 6 to 10 weeks with the value 150 pg/mL. During second trimester the concentrations slowly continuously rise reaching at the end of the third trimester two-fold first-trimester values of about 300 pg/mL (Burger et al., 2004).

3.4. PP13 in Preeclampsia In contrast to hCG and PAPP-A, PP13 is a protein which has been explored as a biochemical marker in various pregnancy pathologies for a relative short time. Several clinical studies suggested the possible association between PP13 levels and placental dysfunction. 3.4.1. First Trimester Burger and his co-workers (Burger et al., 2004) belong to the first research group that has noticed the abnormal low maternal serum levels of PP13 in the first trimester in women suffering from preeclampsia. The decrease of PP13 was more expressive for early preeclampsia developed at ≤ 34 week. Likewise, Nicolaides and his co-workers (Nicolaides et al., 2006) in their prospective, nested-case control study found a significant decrease of PP13 levels in a small series of 10 women whose pregnancies ended in preeclampsia requiring delivery before 34 week of gestation. For a 10 % false-positive rate, the detection rates of preeclampsia were 80 % and when first-trimester uterine artery PI pulsatility index) was taken into account the detection rates rose to 90 %. The subsequent studies carried out with larger groups of patients confirmed a possible benefit of PP13 measurement in the first trimester screening for preeclampsia. Repeatedly maternal serum PP13 levels have been demonstrated to be decreased in pregnancies resulting in preeclampsia especially in early onset although the extent of decrease differed among the studies. Chafetz et al. (2007) showed significant reduction of PP13 levels to 0.2 MoM in the women who experienced preeclampsia. At a 90 % specificity rate, the sensitivity was 79 %. Another study analysing 44 women with early preeclampsia where delivery was induced prior 35 week and further 44 cases of preeclampsia in which delivery was not induced before term also reported significant lower maternal serum PP13 levels but the decrease was expressed less intensively. However, the reduction of the PP13 levels were more pronounced

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in early onset of preeclampsia. It appeared that first-trimester PP13 measurement could identify 50 % of cases of early preeclampsia at 80 % specificity and 44 % of all preeclamptic women. Combining of first-trimester PP-13 testing with second trimester uterine artery Doppler velocimetry may improve only marginally the detection rate obtained with Doppler alone (Spencer et al., 2007b). Similarly, a combination of PP13 with PAPP-A did not result in any benefit for the prediction of preeclampsia. 3.4.2. Second and Third Trimester The investigation of the second-trimester maternal serum PP13 levels seems to be important for the women who missed the first-trimester screening (Spencer et al., 2007a). In contrast to the first trimester, Burger et al. (2004) observed the PP13 levels at the second and third trimesters tend to be higher in comparison with normal pregnancies. Spencer et al., (2007a) showed that late-second trimester PP13 levels appeared to have a limited value in prediction of pregnancies with preeclampsia. But if only the early onset cases of preeclampsia were judged, PP13 could have some benefits. The medians for PP13 were more reduced in early preeclampsia than in all patients with preeclampsia. The comparison of PP13 serum levels changes between first and second trimester revealed an interesting phenomenon. In the early preeclampsia the PP13 levels remained low in the both trimesters of pregnancy. However, when whole group of women with both late and early preeclampsia were evaluated the levels of PP13 in the second trimester increase comparing to the first trimester. The combination of PP13 with uterine artery Doppler in the second trimester did not improve the detection of preeclampsia compared with Doppler examination alone. In a multi-centre study of Gonen (2007) a longitudinal follow-up of PP13 during pregnancy at 6 – 10, 16 – 20 and 24 – 28 weeks of gestation was performed in 20 women developed preeclampsia. The results proved the significant lower levels of PP13 in the first trimester, whereas at the other two time periods PP13 values were not significantly different between the preeclampsia and unaffected groups. However, if the PP13 levels in 6 – 10 and 16 – 20 gestational weeks were judged together, it was found that the lower the 6 – 10 weeks PP13 MoM the higher the 16 – 20 weeks PP13 MoM, yielding a median slope of 14, 5 %. By the evaluation of PP13 slope as a second test only in the subjects screened positive by a low PP13 MoM in the first trimester 80 % sensitivity at 92 % specificity was achieved. 3.4.3. Evaluation of PP13 Measurement in Preeclampsia The clinical studies of PP13 serum levels in pregnancy suggest that measurement of PP13 might be useful as a marker for selected pregnancy pathologies where the abnormal development of placenta is involved. It was supposed that the reduction of PP13 levels in the first trimester may be a consequence of either decreased transcription or increased catabolism (Chafetz et al., 2007). In vitro experiments reported that the gene for PP13 is down-regulated in women whose pregnancies were complicated by early preeclampsia (Tarsa et al., 2004). A PP13 shortage could contribute to impairment of critical functions that are required for normal implantation and maternal vascular remodelling (Chafetz et al., 2007). The temporal pattern in PP13 levels in preeclamptic patients between trimesters observed also in hCG as well as in PAPP-A might be explained by a feedback process (Chafetz et al., 2007). It is possible that the placenta compensates itself for PP13 deficiency by late

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overproduction or that a pathological process could be a cause of accumulation of PP13 in affected areas from which PP13 may be lately released externally (Burger et al., 2004). In fact a dramatic increase in PP13 release from villous explants of third trimester preeclamptic placentas in vitro was observed (Huppertz et al., 2005). Therefore, the detailed study of temporal changes of PP13 between the first and the second trimester might show the usefulness a repeat measurement of PP13.

4. Pregnancy-Specific β1-Glycoprotein (PSβG/SP1) In 1970, a new β-globulin present in the sera of pregnant women was described by Tatarinov and Masyukevich (Tatarinov and Masyukevich 1970). A year later the same protein was isolated from the human placenta and characterised by Bohn et al. (1971). Lin et al. (1974) also described this protein as one of four pregnancy-associated plasma proteins. Because several research groups participated in the identification of new “pregnancy-related” protein various synonyms were used for its designation – Schwangersfats protein 1 (SP1) (Bohn 1971), pregnancy-associated plasma protein C (PAPP-C) (Lin et al., 1974), pregnancyspecific β1-glycoprotein (PSβG) (Towler et al., 1976) and trophoblast-specific β1-globulin (Tatarinov and Sokolov 1977).

4.1. Physical and Chemical Characteristics Originally PSβG/SP1 isolated from the placenta was characterised as a 90-kDa glycoprotein with a carbohydrate content of 29 % (Bohn 1971). A slight heterogeneity of PSβG/SP1 in electrophoretic mobility and isoelectric point were noticed (Sorensen 1982). Later experiments showed that pregnancy-specific β1-glycoprotein consists of a group of highly similar proteins. They are a subfamily of the carcinoembryonic antigen (CEA), which belongs to the immunoglobulin (Ig) superfamily. PSβG/SP1s are encoded by at least 11 different genes clustered on chromosome 19q13.2 along with the carcinoembryonic antigen gene family (Khan et al., 1992; Teglund et al., 1994).

4.2. Function PSβG/SP1 proteins are secreted by placental trophoblast into the maternal circulation in a large amount. From this reason, it was anticipated that their biological effects may be essential for the physiological course of pregnancy. Nevertheless, PSβG/SP1s have been studied for decades. Their biological effects remain to be defined more exactly. It was supposed that PSβG/SP1 function might be associated with maternal immune system; especially the immunosuppresive properties protecting the foetus from being rejected by the mother had been judged. Some older clinical studies supported this hypothesis. Besides lower maternal serum levels of PSβG/SP1 in spontaneous abortions (Hertz and

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Schultz-Larsen 1983; Jouppila et al., 1980), it was observed that PSβG/SP1 may suppress T cells in purulent septic complications of abortion (Repina et al., 1989). In our study of pregnant women suffering from rheumatoid arthritis we found that patients with elevated PSβG/SP1 levels improved the disease, while the women with normal or reduced PSβG/SP1 levels deteriorated or developed the disease during pregnancy (Fialova, et al 1991). Also, in vitro experiments showed that PSβG/SP1 could block differentiation of lymphocytes into lymphoblasts induced by phytohemagglutinin (Cerni et al., 1977; Harris et al., 1984). Newer studies have searched the possible immunological activities of PSβG/SP1 more detailed. It was shown that PSβG/SP1 glycoproteins might modulate the macrophage activity. A peptide derived from N-terminal domain of human PSβG/SP1 interacted to human peripheral monocytes receptor through RGD motif which posses some of PSβG/SP1 glycoproteins. No binding was observed to T and B cell (Rutherfurd et al., 1995). Moreover, Snyder et al. (2001) demonstrated that PSβG/SP1s may induce the secretion of antiinflammatory cytokines such as interleukin IL-10 and transforming growth factor β1 by human monocytes. Through PSβG/SP1 synthesis by trophoblast the antiinflammatory uterine enviroment in pregnancy which is important for the maintenance of a physiological pregnancy may be established. It was proposed that via the ability to induce IL-10, PSβG/SP1s could participate in the suppression of Th1 type responses and/or shift of the maternal cell immunity toward a Th2 phenotype to assure successful pregnancy (Motran, et al 2002, Wessells et al., 2000). PSβG/SP1s influenced not only the production of certain cytokines, but were also able to modify metabolism of monocytes/macrophages by downregulation of iNOS and up-regulation of arginase (Motran et al., 2002). The actions of PSβG/SP1s need not be limited to only monocytes but also the cells from fetal-maternal interface may be influenced. The evolutionary relationship between CEA (carcinoembryonic antigen) and PSβG points to a possible common function in the control of cell invasion of trophoblast into the pregnant endometrium, or in the coordination of the interactions between cells during embryogenesis (Rooney et al., 1988; Streydio et al., 1988). Wessells et al. (2000) hypothesised that PSβG/SP1s might bind to cytotrophoblasts cells inducing them to produce IL-10. Moreover, IL-10 and TGF β1 could act in the control of placental invasion of the uterine wall (Snyder, et al 2001). Because the cytotrophoblasts invasion is disturbed in the preeclampsia the altered synthesis of PSβG/SP1s might be one of the pathophysiological mechanisms in the development of preeclampsia. Also the induction of another interleukin – IL-6 by PSβG/SP1 may have further consequences because IL-6 enhanced the release of human chorionic gonadotrophin by trophoblast and hCG supports the cytotrophoblast growth (Nishino et al., 1990).

4.3. Physiological Pregnancy PSβG/SP1s are major proteins in the serum of pregnant women produced by placental syncytiotrophoblast. PSβG/SP1 is released to the maternal circulation where may be detectable as early as 7 days after ovulation (Grudzinskas et al., 1977). Serum concentration of PSβG/SP1 increases gradually during pregnancy and tend to reach a plateau near the term with very high values ranging about 100 – 400 mg/l (Fialova et al., 1984, Gordon et al.,

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1977). After parturition PSβG/SP1 is cleared from the circulation with a half-life between 24 to 48 hours (Lin et al., 1976). The content of PSβG/SP1s in human term placenta is about 30 mg (Bohn 1972). Besides placenta, PSβG/SP1 was found in tumours of various origins (Sorensen 1984).

4.3.1. PSβG/SP1 in Preeclampsia PSβG/SP1 research in monitoring pregnancies at risk was performed particularly in the second and third trimesters while the first trimester did not attention the clinicians. Several studies about PSβG/SP1 in preeclampsia were performed, but their evaluation did not provide uniform conclusion. Elevated, normal and decreased PSβG/SP1 serum levels had been reported. 4.3.2. Second and Third Trimester Some of the earlier studies described the low third-trimester PSβG/SP1 levels in the cases of toxemic pregnancies associated with severe foetal growth retardation (Bischof 1984b, Towler et al., 1977). Heikinheimo and Unnerus (1979) had examined 169 women with toxemic pregnancies from 29 week. Similarly as previous authors they found PSβG/SP1 levels lower than in normal pregnancies, especially in those complicated by severe IUGR. The predictive value was 29 % for PSβG/SP1 ≤ 2.5 percentile. Weber et al. (1980) observed that the decrease of PSβG/SP1 third-trimester serum levels depended on the severity of preeclampsia; more than 50 % women with severe preeclampsia showed extreme decrease. Different results brought the larger study of Gemmell (1982). In the mild and severe pregnancy hypertension it was demonstrated increasing maternal serum levels of PSβG/SP1 in the third trimester in dependence on the severity of the disease. Twenty-seven percent of PSβG/SP1 results were above the normal 90 th percentile and 17 % below the normal range in the mild form, while in the cases of severe hypertension the number of elevated results rose to 40 % and only 11 % of results fell below the normal range. In pregnancies where severe pregnancy hypertension was combined with IUGR one half of PSβG/SP1 levels were normal and the remaining half included the decreased levels. An elevated concentration of PSβG/SP1 at 34 week in preeclamptic women was also shown by (Hughes et al., 1980b). Similarly, in our study we observed elevated PSβG/SP1 in even 68 % of women with severe preeclampsia in comparison to 10 % with the mild form (Fuchs et al., 1985). A prospective study of Westergaard et al. (1984) in which 37 women who developed pregnancy-related hypertension with or without proteinuria after 28 weeks gestation were serially followed during the second and third trimester reported the normal PSβG/SP1 levels. No alteration in PSβG/SP1 levels in women with slight and moderate preeclampsia or in pregnancies complicated by hypertension without retarded intrauterine growth was observed in other studies (Chapman and Jones 1978; Pluta et al., 1979). One of the sporadic later studies compared maternal serum levels of PSβG/SP1 in the 17, 25 and 33 weeks of gestation developing preeclampsia. At 17 week gestation PSβG/SP1 levels were reduced, whereas they did not distinguish between uneventful and subsequently preeclamptic groups at 25 and 33 weeks (Bersinger and Odegard 2004). Not even in the

Lenka Fialová and Ivan Matouš-Malbohan

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retrospective study of Bersinger et al. (2003) the alteration in third-trimester PSβG/SP1 levels in women with preeclampsia was seen The findings of normal PSβG/SP1 levels in maternal serum are consistent with the experiment exploring the PSβG/SP1 content in placentas from preeclamptic pregnancies but without infants with birth weight small-for-gestation-age. No differences in PSβG/SP1 content between normal and preeclamptic placentas were observed (Bersinger et al., 2002). 4.3.3. Evaluation of PSβG/SP1 Measurement in Preeclampsia Unfortunately, the evaluation of results of previous studies showed some discrepancies and therefore determination of PSβG/SP1 in maternal serum in the late pregnancies remained only at an experimental stage. For clinicians the findings did not provide substantial information which could be use in clinical practice. However, these studies might contribute to the understanding of the biological role of PSβG/SP1 and the pathophysiological mechanism of preeclampsia. Table 1. Levels of placental proteins in pregnancies with preeclampsia or pregnancy-induced hypertension

Placental protein Human chorionic gonadotrophin (hCG) Pregnancy-associated plasma protein A (PAPP-A) Placental protein 13 (PP13) Pregnancy-specific β1-glycoprotein (PSβG/SP1)

First trimester

Second trimester

Third trimester

↓ or N*

↑ or N

↑

↓ or N ↓

↓** ↑ or N ↓ ↑ or N

−

↓ or N

↑ or N ↑ or N ↓*** or ↑ or N

*

↓ Decreased levels; ↑ increased levels; N normal levels. ** Early second trimester. *** Predominant pregnancies with IUGR foetuses.

Conclusion The identification of women likely to develop preeclampsia should allow improving their antenatal surveillance including the administration of prophylactic therapy to the women at high risk. Besides non-biochemical tests such as a Doppler ultrasonography for assessing the velocity of uterine-artery blood flow a large number of biochemical markers have been evaluated in association with their possible use for early screening of preeclampsia. A suitable screening test should fulfil the conditions such as a high sensitivity and specificity with high predictive values. An optimal resolution would be if acceptable biomarkers could be included into the prenatal screening for trisomy 21. The analysis of many studies showed that placental proteins tend either decrease their levels in the first trimester or increase those in the second and third trimester (Table 1). Most authors concluded that the measurement of individual serum markers was not enough sensitive to provide as a screening tools for

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preeclampsia (Conde-Agudelo et al., 2004; Davidson et al., 2003; Dugoff et al., 2005). Some findings suggested that PP13 and PAPP-A testing might contribute to the early prediction of preeclampsia.

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Reisinger, K., Baal, N., McKinnon, T., Munstedt, K. and Zygmunt, M. (2007) The gonadotropins: tissue-specific angiogenic factors? (a review). Mol. Cell Endocrinol., 269, 65-80. Repina, M.A., Blagoslovenskiĭ, G.S., Gnilevskaia, Z.U., Ivanova, L.V. [The effect of specific trophoblastic beta 1-glycoprotein on changes in the cellular link of immunity in infected abortion] Akush Ginekol (Mosk), 47-50. Rooney, B.C., Horne, C.H. and Hardman, N. (1988) Molecular cloning of a cDNA for human pregnancy-specific beta 1-glycoprotein:homology with human carcinoembryonic antigen and related proteins. Gene, 71, 439-449. Rutherfurd, K.J., Chou, J.Y. and Mansfield, B.C. (1995) A motif in PSG11s mediates binding to a receptor on the surface of the promonocyte cell line THP-1. Mol. Endocrinol., 9, 1297-1305. Said, M.E., Campbell, D.M., Azzam, M.E. and MacGillivray, I. (1984) Beta-human chorionic gonadotrophin levels before and after the development of pre-eclampsia. Br. J. Obstet. Gynaecol., 91, 772-775. Shah, D. M. (2007). Preeclampsia: new insights (review). Curr Opin Nephrol Hypertens 16, 213-20. Shenhav, S., Gemer, O., Sassoon, E., Volodarsky, M., Peled, R. and Segal, S. (2002) Midtrimester triple test levels in early and late onset severe pre-eclampsia. Prenat Diagn, 22, 579-582. Sibai, B., Dekker, G. and Kupferminc, M. (2005) Pre-eclampsia (a review). Lancet, 365, 785799. Smith, G.C., Stenhouse, E.J., Crossley, J.A., Aitken, D.A., Cameron, A.D. and Connor, J.M. (2002) Early pregnancy levels of pregnancy-associated plasma protein a and the risk of intrauterine growth restriction, premature birth, preeclampsia, and stillbirth. J. Clin. Endocrinol Metab., 87, 1762-1767. Smith, R., Bischof, P., Hughes, G. and Klopper, A. (1979) Studies on pregnancy-associated plasma protein A in the third trimester of pregnancy. Br. J. Obstet. Gynaecol., 86, 882887. Snyder, S.K., Wessner, D.H., Wessells, J.L., Waterhouse, R.M., Wahl, L.M., Zimmermann, W. and Dveksler, G.S. (2001) Pregnancy-specific glycoproteins function as immunomodulators by inducing secretion of IL-10, IL-6 and TGF-beta1 by human monocytes. Am J. Reprod. Immunol., 45, 205-216. Sorensen, S. (1982) Heterogeneity of the pregnancy-specific beta 1-glycoprotein (SP1). Clin. Chim. Acta., 121, 199-208. Sorensen, S. (1984) Pregnancy-"specific" beta 1-glycoprotein (SP1): purification, characterization, quantification and clinical application in malignancies (a review). Tumour Biol, 5, 275-302. Sorensen, T.K., Williams, M.A., Zingheim, R.W., Clement, S.J. and Hickok, D.E. (1993) Elevated second-trimester human chorionic gonadotropin and subsequent pregnancyinduced hypertension. Am. J. Obstet. Gynecol., 169, 834-838. Spencer, K., Cowans, N. J., Chefetz, I., Tal, J., Kuhnreich, I. and Meiri, H. (2007a). Secondtrimester uterine artery Doppler pulsatility index and maternal serum PP13 as markers of pre-eclampsia. Prenat Diagn, 27, 258-263.

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Spencer, K., Cowans, N. J., Chefetz, I., Tal, J. and Meiri, H. (2007b). First-trimester maternal serum PP-13, PAPP-A and second-trimester uterine artery Doppler pulsatility index as markers of pre-eclampsia. Ultrasound Obstet. Gynecol 29, 128-34. Spencer, K., Cowans, N.J. and Nicolaides, K.H. (2008) Low levels of maternal serum PAPPA in the first trimester and the risk of pre-eclampsia. Prenat Diagn, 28, 7-10. Spencer, K., Yu, C.K., Cowans, N.J., Otigbah, C. and Nicolaides, K.H. (2005) Prediction of pregnancy complications by first-trimester maternal serum PAPP-A and free beta-hCG and with second-trimester uterine artery Doppler. Prenat Diagn, 25, 949-953. Spencer, K., Yu, C. K., Savvidou, M., Papageorghiou, A. T. and Nicolaides, K. H. (2006). Prediction of pre-eclampsia by uterine artery Doppler ultrasonography and maternal serum pregnancy-associated plasma protein-A, free beta-human chorionic gonadotropin, activin A and inhibin A at 22 + 0 to 24 + 6 weeks' gestation. Ultrasound Obstet. Gynecol 27, 658-63. Stamilio, D.M., Sehdev, H.M., Morgan, M.A., Propert, K. and Macones, G.A. (2000) Can antenatal clinical and biochemical markers predict the development of severe preeclampsia? Am. J. Obstet. Gynecol., 182, 589-594. Stenman, U.H., Tiitinen, A., Alfthan, H. and Valmu, L. (2006) The classification, functions and clinical use of different isoforms of HCG (a review). Hum. Reprod. Update, 12, 769784. Streydio, C., Lacka, K., Swillens, S. and Vassart, G. (1988) The human pregnancy-specific beta 1-glycoprotein (PS beta G) and the carcinoembryonic antigen (CEA)-related proteins are members of the same multigene family. Biochem. Biophys. Res. Commun., 154, 130-137. Sutcliffe, R.G., Kukulska-Langlands, B.M., Coggins, J.R., Hunter, J.B. and Gore, C.H. (1980) Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with alpha 2-macroglobulin. Biochem. J., 191, 799-809. Tarsa, M., Hull, A.D., Moore, T.R. and Bogic, L.V. (2004) Gene expression profiles suggest pro-inflammatory predominance at the maternal-fetal interface in severe preeclampsia at preterm (sPPE). In C.A. Insely and L. Myatt (Eds.), Signaling and the Placenta; Proceeding of the Placenta Association of the America Conference, September 25 - 29 Asilomar. (p. A.52). Tatarinov, Y.S. and Masyukevich, V.N. (1970) Immunochemical identification of new beta1-globulin in the blood serum of pregnant women. Byull. Eksp. Biol. Med., 69, 66 - 68 Tatarinov, Y.S. and Sokolov, A.V. (1977) Development of radioimmunoassay for pregnancyspecific beta1-globulin and its measurement in serum of patients with trophoblastic and non-trophoblastic tumours. Int. J. Cancer, 19, 161-166. Teglund, S., Olsen, A., Khan, W.N., Frangsmyr, L. and Hammarstrom, S. (1994) The pregnancy-specific glycoprotein (PSG) gene cluster on human chromosome 19: fine structure of the 11 PSG genes and identification of 6 new genes forming a third subgroup within the carcinoembryonic antigen (CEA) family. Genomics, 23, 669-684. Than, N.G., Pick, E., Bellyei, S., Szigeti, A., Burger, O., Berente, Z., Janaky, T., Boronkai, A., Kliman, H., Meiri, H., Bohn, H., Than, G.N. and Sumegi, B. (2004) Functional analyses of placental protein 13/galectin-13. Eur. J. Biochem., 271, 1065-1078.

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Than, N.G., Sumegi, B., Than, G.N., Berente, Z. and Bohn, H. (1999) Isolation and sequence analysis of a cDNA encoding human placental tissue protein 13 (PP13), a new lysophospholipase, homologue of human eosinophil Charcot-Leyden Crystal protein. Placenta, 20, 703-710. Toop, K. and Klopper, A. (1981) Concentration of pregnancy-associated plasma protein A (PAPP-A) in patients with pre-eclamptic toxaemia. Placenta Suppl, 3, 167-173. Towler, C.M., Horne, C.H., Jandial, V., Campbell, D.M. and MacGillivray, I. (1976) Plasma levels of pregnancy-specific beta1-glycoprotein in normal pregnancy. Br. J. Obstet. Gynaecol., 83, 775-779. Towler, C.M., Horne, C.H., Jandial, V., Campbell, D.M. and MacGillivray, I. (1977) Plasma levels of pregnancy-specific beta 1-glycoprotein in complicated pregnancies. Br. J. Obstet. Gynaecol., 84, 258-263. Towner, D., Gandhi, S. and El Kady, D. (2006) Obstetric outcomes in women with elevated maternal serum human chorionic gonadotropin. Am. J. Obstet. Gynecol., 194, 1676-1681. Tul, N., Pusenjak, S., Osredkar, J., Spencer, K. and Novak-Antolic, Z. (2003) Predicting complications of pregnancy with first-trimester maternal serum free-betahCG, PAPP-A and inhibin-A. Prenat Diagn, 23, 990-996. Visegrady, B., Than, N.G., Kilar, F., Sumegi, B., Than, G.N. and Bohn, H. (2001) Homology modelling and molecular dynamics studies of human placental tissue protein 13 (galectin-13). Protein Eng, 14, 875-880. Wald, N.J. and Morris, J.K. (2001) Multiple marker second trimester serum screening for preeclampsia. J. Med. Screen, 8, 65-68. Wald, N.J., Morris, J.K., Ibison, J., Wu, T. and George, L.M. (2006) Screening in early pregnancy for pre-eclampsia using Down syndrome quadruple test markers. Prenat Diagn, 26, 559-564. Wald, N.J., Rodeck, C., Hackshaw, A.K. and Rudnicka, A. (2005) SURUSS in perspective. Semin. Perinatol, 29, 225-235. Weber, H., Heller, S. and Seulen, P. (1980) [Pre-eclampsia and placental hormones (author's transl)]. Geburtshilfe Frauenheilkd, 40, 339-343. Wessells, J., Wessner, D., Parsells, R., White, K., Finkenzeller, D., Zimmermann, W. and Dveksler, G. (2000) Pregnancy specific glycoprotein 18 induces IL-10 expression in murine macrophages. Eur. J. Immunol., 30, 1830-1840. Westergaard, J.G., Teisner, B., Hau, J. and Grudzinskas, J.G. (1984) Placental protein measurements in complicated pregnancies. II. Pregnancy-related hypertension. Br. J. Obstet. Gynaecol., 91, 1224-1229. Westergaard, J.G., Teisner, B., (1982) Pregnancy-associated plasma protein A in normal and abnormal late pregnancy. In J.G.T. Grudzinskas, B. Teisner, M. Seppala (Eds.), Pregnancy proteins (pp. 345 – 354). Sydney: Academic Press. Yaron, Y., Heifetz, S., Ochshorn, Y., Lehavi, O. and Orr-Urtreger, A. (2002a) Decreased first trimester PAPP-A is a predictor of adverse pregnancy outcome. Prenat Diagn, 22, 778782. Yaron, Y., Cherry, M., Kramer, R.L., O'Brien, J.E., Hallak, M., Johnson, M.P. and Evans, M.I. (1999) Second-trimester maternal serum marker screening: maternal serum alpha-

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fetoprotein, beta-human chorionic gonadotropin, estriol, and their various combinations as predictors of pregnancy outcome. Am. J. Obstet. Gynecol., 181, 968-974. Yaron, Y., Ochshorn, Y., Heifetz, S., Lehavi, O., Sapir, Y., Orr-Urtreger, A. (2002b) First trimester maternal serum free human chorionic gonadotropin as a predictor of adverse pregnancy outcome.Fetal Diagn Ther. 17, 352-356. Zygmunt, M., Herr, F., Keller-Schoenwetter, S., Kunzi-Rapp, K., Munstedt, K., Rao, C.V., Lang, U. and Preissner, K.T. (2002) Characterization of human chorionic gonadotropin as a novel angiogenic factor. J. Clin. Endocrinol Metab., 87, 5290-5296.

In: Pregnancy Protein Research Editor: Marie O’Leary and John Arnett

ISBN 978-1-60692-396-2 © 2009 Nova Science Publishers, Inc.

Chapter II

Pregnancy in Buffalo Cows G. Campanile1, G. Neglia1, D. Vecchio1, M. Russo2 and L. Zicarelli1 1

DISCIZIA, Faculty of Veterinary Medicine, Via F. Delpino 1, Federico II University, 80137 Naples, Italy. 2 Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, Via F. Delpino 1, Federico II University, 80137 Naples, Italy

Abstract Immediately after parturition, buffaloes show several physiological modifications which are fundamental to sustain the new pregnancy. The first step is the resumption of ovarian cycle which is blocked during pregnancy by the effect of progesterone that exerts its action in avoiding other ovulations and maintaining hypotonic the uterus. In buffalo species, the resumption of ovarian activity is affected by the calving season and climate variation. Embryo development is faster in buffalo than in bovine. Therefore, the maintenance of pregnancy is due to either the embryo capacity of signalling its presence or the mother capacity of recognizing these signals and maintaining an adequate uterine environment. Embryo implantation commences around Day 30 in cattle and is presumed to be similar in buffalo. The advent of ultrasonography has contributed in the field of buffaloes reproduction, leading to new information on follicular development, pregnancy diagnosis, fetal sex determination, folliculocenteses, diagnosis of abnormalities of the reproductive organs, monitoring of treatment of ovarian cysts, monitoring of postpartum genital resumption, ultrasound-guided centesis and male genital ultrasonography. Recent applications include the use of doppler ultrasonography for ovarian, uterine and mammarian blood flow ultrasonography. In particular, the evaluation of early pregnancy, allowed it to establish an incidence of embryonic mortality of 20-40% between 28-60 days of gestation in buffaloes that conceive during increasing daylight length. A reduced capacity to secrete progesterone seems to explain in part this embryonic mortality, but other unidentified factors contribute between 40-50% to the embryonic losses. Treatments with hCG, GnRH agonist or progesterone on Days 5 after AI not always reduce embryonic mortality in buffalo species. Embryonic mortality in buffaloes appears to occur later (Day 25-40) than in cattle and P4 treatments should perhaps be applied later. Furthermore, few studies have demonstrated that new methods can be used for

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G. Campanile, G. Neglia, D. Vecchio et al. evaluating the pregnancy and, in particular, the presence of a live conceptus. In fact, also in buffalo species some pregnancy-associated glycoproteins (PAG) have been isolated and purified. PAG concentrations in pregnant buffaloes were almost 2 to 3 times higher than those observed in dairy cows from Day 30 till 60 after breeding. This rapid increase, associated with high maternal concentrations at early pregnancy period, is characteristic of caprine and ovine species and different from cattle, in which concentrations increase slowly and remain at low levels during early pregnancy gestation. Currently, strong efforts are made to develop accurate ELISA kits for PAG determination. In this review several aspects regarding buffalo reproduction will be analysed.

Introduction The optimization of production in animal depends on the quick restoration of the reproductive activity immediately after calving and/or on the anticipation of puberty in subjects destined to the culling. These conditions help to reduce the herds’ unproductive period. Moreover, the decrease of the puberty age shortens the generational interval and favours genetic improvement. Several factors influence reproductive activity, such as pathologies, farm management, climatic conditions, space availability and nutrition. They may play a direct or an indirect role but they always modify the reproductive characteristics. The reactions of each single species to the above mentioned factors are always very evident, and whereas environment has a greater influence on the photoperiodic species, nutrition plays a very important role in the species with a high productive specialization, for which it has become more and more difficult to meet the productive requirements and reduce the energetic gap at the beginning of lactation. Anyway, the factors listed above interact and strengthen the negative effects that influence the productive and reproductive activity of the subjects. The buffalo is an animal species that lives in regions found between 31°North to 2° South parallel. Currently, the distribution of the buffalo population covers the following major climatic regions of the lower latitudes (Tropical zone) and middle latitudes (Temperate zone). This geographic origin and distribution logically suggests that buffaloes are adapted to hot, humid macro- or microclimates. The buffalo is a photoperiodic species. Like sheep, buffaloes have to be considered a "short day" species. They have heats throughout the year but tend to prove more fertile when daylight hours decrease. According to Zicarelli [Zicarelli, 1995], this characteristic is due to their tropical origins. In fact, they originate from North equatorial areas, where the availability of forage coincides with the period in which dark hours increase. Therefore, it has been supposed that animals which calve in the most suitable period for survival of the offspring were selected. It seems that they have retained this characteristic even when they were transferred to places where forage is always (Italy) or less (Sud Equator areas like Sao Paulo, Br) available [Zicarelli, 1995]. Buffalo reproduction is characterized by delayed puberty, silent oestrus, long postpartum ovarian inactivity, and, on the whole, poor fertility [Singh, 1988; Madan, 1988; Singla et al., 1996]. Most of these problems result from the use of the “out of breeding season mating” technique [Zicarelli, 1997; Gasparrini B., 2002]. In fact, if buffalo are bred without modification of their natural seasonality and without controlled breeding, an inter-calving period of less than 400 days and a culling rate of less than 12% has been observed in Italy,

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Brazil, Venezuela, and Argentina [Zicarelli et al., 1994b]. Poor fertility has also been observed when biotechnologies are applied to reproduction. Immediately after parturition buffaloes show several physiological modifications, which are fundamental to sustain the new pregnancy. The first step is the resumption of ovarian cycle. This is blocked during pregnancy by progesterone, which avoids other ovulations and maintains hypotonic the uterus.

Reproductive Seasonality in Buffalo Species Wild animals are generally seasonal, although this characteristic was gradually diminishing with domestication. However, it is still present in some genetic types of the bovine (Bos taurus taurus) species bred freely such as the Podolica, the Sarda, the Maremmana and the Highland bovine and in zebu’ (Bos taurus indicus). As regard the horse, sheep, goat and the buffalo itself reproductive seasonality has only been partially influenced by domestication and the change of breeding area. The fact that selection and improved of nutritive requirements have decoupled the dairy cow from strict reproductive seasonality does not mean that this is or will be just as easily achieved in other species. For example, the egglaying hen must be conditioned by pre-established light programmes so as to ensure constant egg production throughout the year although there is a shorter interval between the generations - and hence more favourable for fixing traits over a shorter period of time - and that its nutritional requirements are satisfied better than it is currently possible in highproduction dairy cow. A comparison between the two species raises serious questions from a physiological, ethological and genetic point of view. The place of origin and the duration of gestation influenced the reproductive seasonality. In fact, the natural necessity to coincide calving and weaning with the most suitable parts of season in order to satisfy the reproduction and nutritive requirements of the offspring through a period in which etiologic agents (infectious and parasites) are less aggressive and/or present [Zicarelli et al., 1988c] represents one of the causes of this 'adaptation' process [Maeda and Lincoln, 1990]. Those subjects acquired under the most favourable conditions, have brought about natural selection of subjects endowed with a more ideal reproductive seasonality towards the survival of the species. Their reproductive characteristics have probably been determined by receptive stimulus of the central nervous system (CNS) during gestation or the early days of life. Spring calving (March-May), which guarantees the calf good availability of forage in temperate zones north of the Equator, is verified when reproduction takes place in autumn (September-November) in the case of five month gestation (sheep and goats) or the previous spring 11 and 12 months (horses and donkeys). Therefore, the same calving period is conditioned by the neuroendocrine system sensitivity, for the re-activation of the reproductive cycle, short day breeder (negative photoperiod) or long breeder (positive photoperiod) as regards the length of gestation. In some tropical zones (North of Equator), forage availability is usually found after the rainy season which begins usually in (August-December) or is conditioned by the offering of better roughage in flooded areas [Vale et al., 1996]. In water buffalo, whose pregnancy lasts

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approximately 10 months, the sensibility to decreasing of light stimulus and the reproductive season, coincides in the northern hemisphere to the period between September and January. Nevertheless, with the passing of generations, light stimulus was the phenomenon, which prevails over reproductive condition of photo-sensitive species, even after being moved from the place of origin. Meanwhile, when some animal species are transferred to zones near of equator, sensitivity to light stimulus no more influences reproductive activity due to the constant length of the light/dark ratio throughout the year. In such case, optimisation of nutritional requirements for species like buffalo, equine and ovine in the Northern areas of South America like Amazon Valley, for example prevails over light stimulus [Vale et al., 1996]. Thus, it may be predict that there is a tendency towards seasonality as far way is the buffalo species from the equator. Whatever type of photoperiod an animal shows sensitivity to, the length and density of the light source influence the hypothalamic-hypophyseal-gonadal axis, transmitted via a multi-step neural pathway which involves the retina, the suprachiasmatic nucleus (SCN), the superior cervical ganglia (SCG) and finally the pineal gland. Light regulates the melatonin rhythm in two ways: first by synchronizing a circadian pacemaker in the suprachiasmatic nucleus (SCN) that controls the activity of the pineal gland and second by an acute inhibition of melatonin production. The duration of melatonin secretion then provides an endocrine index of night length and thus day length [Lincoln et al., 2005]. Seasonal or annual photoperiodic cycles are regulated by the actions of melatonin on its target tissues. Long duration melatonin signals promote a winter physiology and short duration signals support a summer physiology. This phenomenon has been demonstrated by some studies performed in pinealectomized sheeps and hamsters, in which the duration of exogenous melatonin administration by infusions pumps, induced the activation of the two types of seasonal physiology [Bartness et al., 1993]. It is likely that the effects of photoperiod/melatonin on diverse aspects of seasonal physiology (e.g., reproduction, body weight, etc.) are mediated by melatonin receptors expressed in a range of different neural, pituitary, and possibly peripheral target cells [Lincoln et al., 2003]. Some studies carried out on ovine have shown that the SCN contains an internal biological clock which regulates the endogenous circadian rhythms [Lincoln, 1989]. The stimuli processed there are conveyed by means of the SCG to the pineal gland which operates as a transducer converting neuronal information arising from the alternating light/dark ratio. In the pineal gland, the neuronal information stimulates the rhythms of melatonin secretion that regulates hypothalamohypophyseal activity complex mechanisms and therefore gonad function [Lincoln, 1992]. The administration of melatonin decreases tyrosine hydroxilase activity and increases LH secretion; this modulation is independent of the action of the oestradiol. It is interesting to note that the inhibition of the enzyme tirosine hydroxilase activity, through a systemic injection of a dopamine antagonist (pimozide) during the anoestrous season, evokes a temporary increase in LH secretion. It is also known that PRL levels are high during anoestrous. In other species high PRL levels decrease upon administration of dopamine. Thus, the decrease of PRL is fundamental for the resumption or regularisation of the ovarian cycle in many domestic species like ovine, rodents, swine, as well as in the

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human beings. This leads to a hypothesis that the role of dopamine varies among animal species and probably with their sensitivity to the short/long day. The precise role carried out by PRL and therefore by dopamine, whose action decreases LH secretion in sheep, is difficult to define. For example, it has been observed that high PRL levels and low thyroid hormonal (Triiodothironine and Tyroxine) levels in the spring/summer months do not interfere with the oestrous cycle or with buffalo heifer conception [Avallone et al., 1994]. On the other hand, Indian authors [Sheth et al., 1978] have stated that high PRL levels are present in anoestrus buffaloes and that they decrease upon administration of bromocriptine [Madan, 1988]. Authors in different studies [Avallone et al., 1994; Campanile et al., 1994; Borady et al., 1985] founded high PRL levels that followed a lowering of thyroid hormones. Low levels of these hormones are physiologic in spring and summer and evoke an increase of TRH that is reported to increase PRL levels. There is evidence for a serotoninergic inhibition of LH secretion during the anoestrous season; however the chemical structures of the substance involved have not yet been identified [Sivaiah et al., 1987].

Figure 1. Blood plasma levels of melatonin in female buffaloes which are readily and laboriously driven out of breeding season, measured at daylight (two hours before sunset - light), and two (2s), 4 (4s), 6 (6s) hours after sunset, during the four season.

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Figure 2. Daylight length (▪▪▪▪▪ hours), and percentage of calvings at each month in different Countries.

Thus, light suppresses and dark favours melatonin secretion. In the water buffalo melatonin is the endocrine signal that marks the light-dark alternation of the day [Morgan and Williams, 1989]. Plasma values of this hormone were kept at high levels during the dark hours and particularly for a larger number of hours in autumn and winter compared to spring and summer seasons (Figure 1). Moreover, this pattern turned out to be different when measured in animals reared on farms where the out-of-breeding-season-mating (OBSM) technique (see below) has been used for many years with success (more than 80% of the animals calves during the first seven months of the year) versus animals reared on farms where this technique induces a significant decrease in reproductive efficiency. The circadian melatonin pattern is different in animals which usually calve during spring or autumn season. In fact, during winter and especially in spring, two hours after sunset, the buffaloes more sensitive to photoperiodic stimuli, show higher melatonin plasma values (Figure 2) than animals which are less sensitive to the photoperiod effect [Di Palo et al., 1993; Parmeggiani and Di Palo, 1994]. Farm management and breeding area do not influence the melatonin plasma levels although animals are moved to another zona [Di Palo et al., 1997]. Melatonin plasma levels measured in samples taken two hours after sunset, show a repeatability value of 0.733 [Di Palo et al., 1993]. The high repeatability found in buffalo

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species, allows the use of melatonin plasma levels as a tool capable of marking an animal's sensitivity to seasonal effects [Zicarelli, 1994a]. If the heredity of this character turns out to be high, such phenomenon will be involved in genetic selection programmes of this species. That being so, the nature determination of this characteristic has great economic importance, especially in Italy and in other countries where buffalo production system has seasonal involvement. Furthermore, even for other geographical regions such procedure may be profitable for breeding non seasonal sensitive buffalo females. The hypothesis of a genetic involvement of melatonin secretion pattern, or in other words, difference of sensitivity to light stimulation, has not been excluded. In ovine (for example the Romanov 58°N, the Karakul 41°N and the White-faced 51°N, sheep breeds) a continuous cyclic activity throughout the year is observed, even if living at latitudes where other genotypes are sensitive to light/dark ratio. Moreover, it can also be underlined that buffaloes that calve through the spring time, which show a lower secretion of melatonin values after sunset are those more adaptable towards the OBSM method [Di Palo et al., 1993; Parmeggiani and Di Palo, 1994]. The heifers buffalo are less sensible to seasonality than adults and show a lower melatonin plasma levels after sunset [Borghese et al., 1995]. The daylight length influences calving period throughout the year in buffalo species. In natural condition mating increases during a period in which the daylight length decreases. In fact, in Italy (Figure 2) in buffalo farms where the out-of-breeding-season-mating (OBSM) technique is not used the resumption of the reproductive activity begins from September (decreasing daylight length period) until January (increasing daylight length period) [Zicarelli et al., 1997]. This phenomenon has been observed both within free-grazing herds as well as with stabled herds pasture is fairly poor from September until February, although good pasture can occasionally be found after a wet end of summer. Usually, after the middle of June, pasture is poor and the nutritional condition of the herds in autumn at the start of lactation and ovarian cycle resumption are general poor. On the other hand in tropical areas, during the same months, generally forage is in abundance [Shah, 1990]. Sensitivity to the negative photoperiod is also found on farms where a constant balanced diet that met buffalo requirements is administered throughout the year [Zicarelli et al., 1988a; Zicarelli et al., 1988b, Zicarelli, 1992 and Zicarelli, 1994a]. This type of seasonality, where reproductive events are not synchronised with forage availability, indicates that the buffaloes bred in Italy are not autochthonous. In fact, in this area buffaloes calve in periods in which there is low forage availability and low temperature. This condition does not meet calf survival requirements, since weaning takes place between late winter/early spring, and increases sensitivity to Pasteurella bubaliseptica, an etiologic agent of a disease called "barbone" which seriously affects buffalo calves. Indeed, a similar seasonality (Figure 2) to that found in Italy [Zicarelli et al., 1977] and Asian tropical areas (India and Pakistan) is found also in Venezuela [Zicarelli, 1994a], Egypt, Argentina and Brazil [Baruselli et al., 2001]. In the equatorial zone of Brazil the calving period [Vale et al., 1996] is, however, concentrated in different months (Figure 2) and follows a rainy/wet season (NovemberApril), that coincides with a good forage availability.

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In particular, seasonality is affected by latitude (Figure 2), and it is accentuated moving from North (0–8 degrees) to South (24–32 degrees) (Figure 3). These observations have been confirmed by recent studies performed in Brazil [Lamberson et al., 2007 and Nascimiento et al., 2007], since the seasonality of buffaloes bred in Southern and South-eastern Brazil is more accentuated than that of animals bred in Goias, which is located in the North of Brazil.

Figure 3. Trimestral distribution of buffalo calvings at different latitude in Brazil. [From Baruselli et al., 2001].

Although the modification of several management conditions, the photoperiod has not been affected throughout the last 30 years [Zicarelli et al., 1977], excluding those farms, that apply the OBSM technique for long period. In this case, the animals are less sensible to the daylight length. This is a further confirmation that the character “photoperiod” will be involved in genetic selection programmes of this species. From these findings, it can be stated that: a) Although the domestic buffalo shows reproductive activity throughout the year, there is greater tendency to concentrate this physiological phenomenon in months of decreasing daylight length; b) Wherever the light/dark ratio is constant throughout the year even if a tendency towards seasonality does not exist, however a concentration of calving is found which varies from area and from year to year. It seems, in practice, that the beginning of the reproductive activity requires particular environmental conditions (good forage availability) or coral or group sexual behaviour. The last aspect, which has been noted in Amazon areas, is also present in Italy, where phases of intense reproductive activity (independently of season) are alternated with periods of sexual stasis. This particular behaviour causes a high concentration of births the following year. Usually, this is due to different environmental conditions. In fact, it is known that

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buffalo is extremely sensitive to climatic variations [Campanile et al., 1988; Roy et al., 1968; Vale et al., 1994; Singh and Desai, 1979]. However, even if the same macroclimate is present, other factors may affect the reproductive activity in different buffalo farms, such as the rearing methods, management, nutrition, hygiene and veterinary practices, etc. Finally, it has to be considered that buffalo can not be considered a species with a "lactiferus habitus" and therefore a "catabolicus habitus". In fact, the low milk yield and, probably, its lipid metabolism, whose storage ability helps to prepare for periods of scarce forage availability, (typical of animals which originate from excessively hot or cold climates), avoid that buffalo species shows characteristics similar to high production bovine milk breeds, which use their reserves to compensate for energy and protein deficiency during the first phases of lactation.

Anoestrus Buffalo seasonality is highlighted by the concentration of calving in few months (5-7) throughout the year. However, when the calving occurs in different periods from the physiological ones, a late ovarian resumption and conception are observed. This leads to periods of anoestrus with variable length and the resumption of ovarian activity during the subsequent physiological reproductive season. In bovine the phenomenon of anoestrus is often attributed to nutritional factors. In buffalo, however, nutrition is just one of the causes; other factors, such as the sensitivity to the photoperiod and the environment need to be considered. Together with environmental factors, endometritis play a main role by reducing pregnancy condition and causing acyclia. In this case the resumption of ovarian activity occurs when daylight length decreases. Buffalo shows a lower number [Borady et al., 1985] of follicular waves (2 vs. 3-4), antral [Roy et al., 1968] and non-atresic follicles, ovarian weight (4 grams vs. 8,5 grams) and volume (mean length of 2.5 cm vs. 3.7 cm) compared to cattle. This difference is observed at birth, when a buffalo calf shows 1/5 of oocytes number than a bovine calf with the same age [Roy et al., 1968]. These factors cause a lower number of oestrus cycles after calving and ovarian resumption. If the conception does not occur, the phenomenon of anoestrus will establish. However, it would be interesting to know if the lower number of follicles on buffalo ovary is the cause of its seasonality or, because of its seasonality, a lower number of follicles is required. In buffalo species it is possible to distinguish a "temporary anoestrus" (<150 days open) and a "deep anoestrus" (>150 days open). Furthermore, we can observe two different conditions of anoestrus: 1) after calving, for the lack of resumption of ovarian activity; 2) after a short interval of cyclic ovarian activity. Anoestrus depends on a lowered hypothalamus-pituitary activity that consequently influences ovarian activity [Kaker et al., 1981]. In fact, a lower LH pulsatility occurs after an evident oestrus cycle in subjects that become acyclic and do not conceive [Radzan et al.,

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1981; Zicarelli et al., 1997]. The acyclic buffaloes show low levels of FSH, progesterone [Esposito et al., 1992; Radzan et al., 1981; Sheth et al., 1978; Vale et al., 1994; Zicarelli et al., 1988a; Zicarelli, 1992; Zicarelli et al., 1997] and oestradiol-17β [Sheth et al., 1978]. Furthermore, high PRL [Sheth et al., 1978] and low thyroid hormones [Borady et al., 1985; Karsh et al., 1995] levels are found in the spring months when the incidence of acyclic subjects is highest. The interruption of cyclic activity is usually preceded by normal oestrus which is followed by an inadequate luteal secretion. This phenomenon has been found upon the onset of anoestrus season in sheep, upon the first ovulation post-partum in bovine and during the pre-pubertal phase in both species. These reproductive disorders may be subdivided into two main causes: a short luteal phase and a normal luteal phase with low progesterone production [Garverickh et al., 1992]. The causes of anoestrus are different in Italy and in other countries that breed buffalo. In Italy, market requirements cause the modification of the calving season, whereas in developing countries natural mating period is not modified. In the last case the causes of anoestrus are almost attributed to nutritional factors. In Italy, the block of ovarian resumption after a variable period is particularly evident when the herd is subjected to OBSM and concerns above all buffaloes which calve in the first two months of the year or in the first five months in cold climates. In Southern Italy, it has been verified that at 70 days from delivery the 50.3 per cent of primiparous and 24.0 per cent of pluriparous are acyclic. The calving-conception period is shorter in the subjects that calve after March because no more than 40 days last between the first fertile cycle (60 days after delivery) and the beginning of the short-day period. Buffaloes that calve in the first two months of the year, or in the first five months in cold zones, go into a "deep anoestrus" if they do not become pregnant within 70 days from delivery. Changes in the endocrine pattern may cause a delayed resumption of ovarian cyclic activity and this could have a negative influence even when the buffaloes become cyclic. This "deep anoestrus" condition ends after 200 days having been exposed to at least two months of the short-day period [Zicarelli, 1994b]. Some animals with delayed conceptions (5.3%), interrupt pregnancy between 40 and 60 days after conception. Furthermore subjects with temporary anoestrus show an embryonic mortality rate of 16% [Zicarelli, 1994b]. This also explains why the reliability of milk progesterone dosage for pregnancy diagnosis is less accurate in the spring than in the autumn (64.3% vs. 93.6%), due to a higher number of anomalous cycles and/or embryonic mortality, which occur in spring time [Campanile et al., 2005; Campanile et al., 2007a]. Age, micro and macroclimate influence buffalo endocrine patterns and increase the rate of acyclic subjects. Primiparous, and old buffaloes (>10 years old), more frequently show anoestrus. Their calving-conception interval is generally longer (about 60 days) than that of pluriparous animals. The poor management during their early years, the stress of first lactation, gestation and first post-partum period or puerperium affect anoestrus in primiparous animals that tend to be less sensitive to the photoperiod. On the contrary, old buffaloes are influenced by calving season. In fact, these subjects that calve more than 5 times (due to the longevity of the buffalo) and represent between 20 - 55% of the herd, usually, resolve their reproductive problems in autumn, making a valuable contribution to spring anoestrus. Even subjects more than 10 years old, remain in optimum health, although reproductive efficiency pertains only

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to autumn. It has been recently demonstrated that primiparous buffaloes which receive a diet characterized by high energy and starch content show higher fertility than pluriparous animals. However, this rationing schedule negatively affects the fertility in pluriparous buffaloes [Vecchio et al., 2007c]. Different techniques regarding weaning, growing subjects together with diet supplied during puerperium could be contributing factors.

The Out-of-Breeding-Season-Mating (OBSM) Method In Southern Italy the mating period of Mediterranean buffaloes is restricted to the March to September period (spring–summer), so that subsequent milk production coincides with the time of peak demand for the manufacture of mozzarella cheese [Zicarelli, 1997]. There is a requirement in the Mediterranean region for mating programs (OBSM) in buffaloes to be conducted during the seasonal trough in reproduction so that calving coincides with the yearly peak in demand for buffalo milk. This creates a potential conflict between the seasonal nadir in reproduction and the need to establish pregnancies (Figure 4). The application of OBSM technique, usually, is gradual. During the first year the mating period is limited between January and October, while in the third year between March and September. In this way a gradual change in the delivery pattern of the farm can be obtained, and the economical burden is spread. The efficacy of the OBSM technique application depends on the grade of herd seasonality, reproductive disorders and heifers availability; these latter subjects are less sensitive to the light stimulus. The OBSM technique application increases the incidence of acyclic subjects and hormonal treatments can be used to reduce the phenomenon of anoestrus [Zicarelli, 1994b].

Figure 4. Milk market requirement throughout the year, calving % and milk production in Italy without the application of the OBSM technique.

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The true economic gain resulting from pharmacological treatments derives from a lower percentage of non pregnant buffaloes culled at the end of lactation (18.1%) as compared to that of control subjects (31.2 per cent). When the technique is applied for long time (> 15 years) the mating period is modified in function of the productive state: a) in milking buffaloes, between February and September; b) in heifers, between April and August; c) in non lactating buffaloes, throughout the year. Furthermore, the application of the technique for a long time reduces the intercalving period because of the higher culling rate of the subjects more sensitive to the photoperiod. The selection of buffaloes less sensitive to the photoperiod and a high culling rate, must be continually utilised in order to avoid a change in the calving period, according to the buffalo reproductive seasonality within few years. In fact, buffaloes that naturally calve during the spring-summer period increase the intercalving of 30 days for the year. Therefore, if the OBSM technique is not applied more, the calving calendar becomes seasonal within 5 6 years. The technique does not influence the physiological characteristics of the buffalo but impedes calving in the most consistent months (July-December). The application of OBSM technique leads to a monthly reduction in milk yield (about 15%) between September and January, a plateau between February and April and an increase between May and August. This allows enough to supply milk market requirements for mozzarella cheese production.

Early Embryo Development All mammalian species show similar processes throughout the period between fertilization and blastocyst hatching [Wimsatt, 1975; Duc-Goiran et al., 1999]. In particular, embryo development in buffalo species is similar to that described in cattle, although some important differences have been observed. Following ovulation, the oocyte at the stage of Metaphase II (Figure 5), is released into the oviduct, where the fertilization takes place. Therefore, it is clear that the oviduct plays a fundamental role in either fertilization of the gametes and early embryo development. It is known that mammalian oviduct is a tortuous structure, that connects the anterior pole of the ovary with the correspondent uterine tube. The width and the diameter of the oviduct differ according to the species: usually it is very wide in the first tract and becomes narrow in proximity of the uterus [Hafez and Hafez, 2000]. Because of its willowy course, the oviduct shows a total length higher than the distance between ovary and uterus: in ruminants, swine and horse it is till 5-6 times longer than that distance [Barone, 1994]. From an anatomic point of view, it is possible to distinguish 3 sections into the oviduct: infundibulum, ampulla and istmus [Weeth and Herman, 1952]. Buffalo oviduct (Figure 6) is similar to that described in bovine, reaching a

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total length variable between 12.5 a 42.8 cm [Luktuke and Rao, 1962; Sane et al., 1964, 1965].

Figure 5. High quality buffalo oocytes recovered from ovaries of slaughtered animals.

Figure 6. Buffalo oviduct during surgery.

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However, it is more coarse and shows short and less extensible fimbriae [Taneja et al., 1988]. The three sections described in bovine, have been distinguished also in buffalo species [Luktuke e Rao, 1962], with dimensions similar to those described in cattle (Table 1) (Figure 7). Table 1. Length and width (cm) of buffalo oviducts reported by several authors [From Carvalho, N.A.T., 2006, modified] Authors

Left Oviduct

Right Oviduct

Year Races Length (cm) Width (cm) Length (cm) Width (cm)

El-Sheikh and Abdelhadi 1970 Egyptian 26.3±0.3 (16-34)

Luktuke and Rao

Sane et al.

Sane et al.

Vale et al.

1962 N.D. 21.8 (16.1-31.9)

1964 Murrah 22.38 ± 0.12 (12,5-42,8)

1965 Jaffri 24.49±0.28 (13.0-36.2)

(0.2-1.1) 26.3±0.3 (17-36)

(0.24-1.00) 22.3 (17.6-32.4)

0.2 22.56 ± 0.12 (13.0-36.0)

0.2 24.41±0.27 (13.0-36.1)

(0.2-1.1)

(0.25-1.05)

0.2

0.2

1982 N.D. 19.54±5.13 (11.0-29.0) 0.22 ± 0.14 (0.2-0.5) 20.30±3.58 (11.0-31.0) 0.26 ± 0.12 (0.2-0.5)

Figure 7. Istologic section of buffalo’s oviduct. From right to left infundibulum, ampulla and istmus [From Carvalho, N.A.T., 2006].

Two major working hypothesis on the role of oviduct in early embryonic development are facing at present: on one side, it is seen as a simple container “passively” providing the best environment for fertilization and early cleavages; on the other, oviduct is thought as an “active” source of signal molecules able to sustain these processes, finally regulating the first different steps. The tubal fluid is a complex medium formed by a combination of selective transudate from the blood and secretory products from the epithelial cells [Leese, 1988; Malayer et al., 1988]. Therefore, it is worth pointing out the changes in the composition of oviductal fluid, taking into account either the permeability of the structure and the secretory capacity of the epithelium, throughout different stages of the oestrus cycle. The concentration of nutrients in tubal fluid is generally below plasma concentrations, suggesting an overall transport of nutrients across the tube, mainly by diffusion [Leese and Barton, 1984; Leese and Gray, 1985]. Components of the tubal fluid such as ions [Brunton and Brinster, 1971], albumin [Glass, 1969], immunoglobulins [Parr and Parr, 1986], glucose and pyruvate [Leese and Gray, 1985] are considered to be transferred from blood, while a number of specific tubal proteins are known to be produced by the epithelial cells [Gandolfi et al., 1993]. The amount of fluid secreted by the uterine tube increases during oestusus and decreases during diestrus

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and pregnancy. The ampulla produces approximately two thirds of the total daily secretion while the isthmus supplies the rest [Aguilar and Reyley, 2005]. Oestrogen predominantly stimulates, while progesterone inhibits tubal fluid secretion. The volume of oviductal fluid collected in buffalo, both in follicular and luteal phases of the cycle, is lower than ovine and swine [Kavanaugh et al., 1992; Wiseman et al., 1992]. A progressive increase of oviductal fluid volume has been observed till the ovulation, with a mean production of 0.39 ml, 0.79 ml, 0.96 ml and 0.61 ml, respectively in diestrus, preovulatory, ovulatory and postovulatory phases [Vecchio, 2007; Vecchio et al., 2007b]. Furthermore, a significant lower volume of oviductal fluid is recorded in acyclic vs. cyclic buffaloes, although the basal levels of oestrogens, depending by follicular dynamics, probably affect its production also in these animals. Therefore, also in buffalo species, steroid hormones affect tubal fluid production by modulating ionic transfer cross the oviductal epithelium. In fact, the tubal fluid volume is maximum during the ovulatory phase, when chlorine concentration is highest [Vecchio, 2007; Vecchio et al., 2007b]. Chlorine passage from the blood to the oviductal lumen, would increase tubal fluid volume, by osmotic action. In buffalo, potassium levels are similar throughout the different phases of the cycle and calcium levels are highest in the preovulatory phase. The concentration of sodium shows similar trend to the phosphates, with the peak at the ovulatory stage, although their levels are not different between the diestral and preovulatory phases [Vecchio, 2007; Vecchio et al., 2007b]. The main energy substrates in tubal fluid are glucose and pyruvate derived from blood, although sucrose and lactate are also present [Aguilar and Reyley, 2005]. Carbohydrates concentration in tubal fluid is lower than that recorded in plasma, suggesting a facilitate diffusion mechanism for the transportation of these substances [Leese and Gray, 1985]. Interestingly, glucose and lactate levels decrease after ovulation, whereas pyruvate shows a different trend, since similar values are observed both in luteal and oestral phases (Figure 8). However, the lactate:pyruvate ratio in the preovulatory phase results 10:1, similarly to that described in other species. The decrease of glucose and lactate may be due to the reduction of oestrogen concentration after ovulation or to the increase of progesterone [Leese et al., 2001].

Figure 8. Characteristics of the energy substrates (Glucose, Pyruvate and lactate) concentration during diestrus, pre-ovulatory, ovulatory and postovulatory phases.

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These data are only apparently in contrast with oocyte nutrition and early embryo development, since the oocyte needs around 10-12 hours for reaching the isthmic region, where the fertilization occurs [Nichol et al., 1992]. The quantity of phospholipids increases from diestrus to the ovulatory phase and significantly decreases in the postovulatory phase. The lipids in the tubal fluid may have an important role during embryo development, as demonstrated by the evidence that fatty acid are essential for in vitro development of rabbit [Kane, 1979] and mice [Quinn and Whittingham, 1982] embryos. These elements can guarantee the substrates necessary for synthesising the constituents of cellular membranes, such as phospholipids [Pratt, 1980] and sterols [Pratt, 1982]. Protein concentration in the tubal fluid is approximately 10-15% of that in serum. Albumin and immunoglobulin G, derived from the blood stream, are the most common proteins representing about 95% of the total protein of the tubal content [Aguilar and Reyley, 2005]. Tubal-specific glycoproteins produced by the epithelial cells have been identified and characterized in several species. Continuous changes that occur in the secretory patterns throughout the oestrous cycle and among the different regions of the tube indicate the existence of systemic and local controlling mechanisms of tubal fluid production, demonstrating the active and fundamental role of the oviduct throughout the early reproductive events [Aguilar and Reyley, 2005]. According to Killian et al. [Killian et al., 1989], protein concentration in tubal fluid does not differ throughout the oestrus cycle, although the total quantity is higher during the ovulatory phase, due to the highest volume of the oviductal fluid (Figure 9). However, both protein concentration and daily total quantity are lower than those recorded in bovine [Killian et al., 1989].

Figure 9. Protein concentration (P.C.) (mg/ml) and Protein daily total quantity (P.d.t.q.) (mg/24 h) Buffalo's Oviductal Fluid during oestral cicle phases.

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A specific class of 92-96 KDa, defined as specific oviductal oestrus associated glycoproteins, are present in buffalo [Vecchio, 2007], as demonstrated in other mammalian species [Aguilar and Reyley, 2005]. It has been hypothesized that these molecules are fundamental for fertilization and further embryo development, since they have the capacity of linking the carbohydrates. In fact, this linkage may improve sperm capacitation and penetration through the zona pellucida [Abe and Hoshi, 1997; Verhage et al., 1997]. The specific oviductal oestrus associated glycoproteins may increase the viscosity of the oviductal fluid and stabilize the microenvironment of the embryo, avoiding the dispersion of nutrients and rapid osmotic changes [Hunter, 1994]. Following fertilization and fusion of gametes, the zygote undergoes subsequent mitotic divisions, which determines the formation of the blastomeres (Figure 10). These cells, at least in the early stages of development, can be considered totipotent, because they have the ability of developing into two separate embryos. This characteristic has been demonstrated until the 8-cells stage in cattle [Senger, 2005], and it is thought to be similar in buffalo (Figure 10). This stage of development (8-16 cells) is fundamental. In fact, in this period the activation of the embryonic genome, that is essential for achieving implantation competency, occurs. Once the embryonic genome is activated, the embryo grows rapidly to form a blastocyst. However, if the chronological events of in vivo embryo development are well studied in bovine [Betteridge and Flechon, 1988], only few information are present for buffalo.

Figure 10. Buffalo embryos at different stages of development: tight morulae, early blastocysts, hatching and hatched blastocysts.

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In particular, some studies performed on the development of preimplantation embryos in superovulated buffalo [Chantaraprateep et al., 1989; Drost and Elsden, 1985; Anwar and Ullah, 1998; Misra et al., 1998] indicate a faster rate of development than in cattle. These results have been confirmed from further researches carried out in vitro, that demonstrate that buffalo embryos are 12 to 24 h more advanced than the bovine counterpart developing in parallel [Galli et al., 2001]. Oocytes and embryos in buffaloes remain in oviduct for a period varying from 74 and 100 hours post-fertilization [Karaivanov et al., 1987] and hence reach the uterus 4.5-5 days after fertilization. These information were extrapolated by performing flushing on oviducts and uteri of superovulated subjects at different hours post-insemination. Similarly, in Nili-Ravi buffaloes [Anwar and Ullah, 1998], at 85 hours post insemination the embryos are in the oviduct, whereas at 108 hours most of the embryos (78%) descend from the oviduct into the uterus. It seems that buffalo embryos are at morula stage (Figure 10) when they reach the uterus [Anwar and Ullah, 1998], similarly to that described in bovine at 120 hours [Betteridge and Flechon, 1988]. This would confirm that the descent of embryos into the uterus may occur a few hours earlier in buffalo than in cattle. Compact morulae are observed from 125 to 152 h post-estrus and blastocysts from 141 hours. Similar data have been reported also in Nili Ravi buffaloes, in which the recovery of compact morulae occurrs at about 132 h post oestrus [Mehmood et al., 1989]. Although there are differences in the timing of these events and where they occur in the reproductive tract of the mother, blastocyst formation is generally initiated when the conceptus reaches the uterus (Figures 10 and 11).

Figure 11. Buffalo hatching blastocyst.

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After reaching the blastocyst stage, the embryo undergoes a differentiation into two distinct cellular populations: the inner cell mass (ICM) and trophectoderm (TE). From the ICM the body of the embryo will origin, whereas the TE cells will eventually give rise to the placenta and extraembryonic membranes. Among these, the chorion will become the fetal component of the placenta. The distinction of cells into these two primary populations, TE and ICM, can be assessed by differential labeling of the nuclei with the two polynucleotidespecific fluorochromes [Handyside and Hunter, 1984]. No data are available on embryos morphology and ICM and TE cells count in buffalo species. Some experiences have been performed by observing in vitro produced embryos with or without Insulin-Like Growth Factor I (IGF-I) [Narula et al., 1996]. In fact, it is known that during embryo development several growth factors and their cognate receptors are expressed by embryos [Schultz and Heyner, 1993; Shi et al., 1995]. Insulin and IGF-I are two small mitogenic polypeptides, that have several effects on different cellular types. It has been demonstrated in some studies performed in vitro, that the addition of growth factor from the insulin gene family to the culture medium, improves cell division and causes an increase in either cell number and percentage of blastocysts that hatched from the zona pellucida [Heyner et al., 1993]. Also in buffalo species the supplementation of IGF-I to the culture medium increases of about 57% the blastocyst total number of cells and the number of ICM and TE cells. In particular, a mean total cell number of 82±9 cells has been recorded in buffalo blastocysts after 7-9 days of co-culture in vitro on buffalo oviductal epithelial cells with IGF-I supplementation [Narula et al., 1996]. The ICM is composed by 36±4 cells and the TE is reported to be 46±7 cells. However, if only the embryos of high quality, that develop at blastocyst stage within day 7 are considered, the number of cells is definitely higher, recording 107±10, 43±5 and 64±8 cells for total cell number, ICM and TE respectively [Narula et al., 1996]. In several mammalian species, and probably in buffalo, the total number of cells in in vivo produced embryos is around 15-20% higher that the correspondents in vitro [Rubio Pomara et al., 2005]. Therefore, we can suppose that the total number of cells in an embryo produced in vivo in buffalo species is around 130 cells. The rupture of zona pellucida represents the event for a new stage of the embryo. In this moment the hatched blastocyst survival is strictly dependent by the uterine environment and the conceptus exposes the other surface of trophectoderm directly to the uterine environment. Therefore, an adequate progesterone production and the responsiveness of the uterus to progesterone are considered necessary for embryo survival. Unfortunately, no data are available on embryo development from the blastocyst hatching to the implantation in buffalo species. It can be supposed that phenomena similar to those recorded in other ruminants, such as cattle and sheep, may occur [for review see Senger, 2005]. According to that described in these species, after hatching, a logarithmic growth and an elongation of the conceptus is observed [Geisert and Malayer, 2000]. For example, the bovine conceptus shows a 3 mm spherical shape on day 13 and becomes a 25 cm filamentous on day 17. The filamentous embryo is able to occupy the controlaterale horn since day 18 of pregnancy. The progressive hyperplasia and expansion of trophoblast cells, is on the basis of this event, allowing the development of extra-embryonic membranes throughout the uterus. By this mechanism, the embryo is able to block the synthesis of PGF2α and avoid the luteolysis. In fact, it is known that in cattle, the maternal recognition of

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pregnancy occurs between days 16 and 19 of post-insemination [Thatcher et al., 1995; Roberts et al., 1996], and it is probably similar in buffalo. This process is due to the intervention of several molecules. The first messenger that has to be recorded is the Interferon-Tau (IFN- τ), which is produced by the elongated conceptus. The IFN-τ, recognized for the first time in sheep with the name of trophoblastin [Martal et al., 1979], was subsequently isolated in other ruminants [for review see Spencer and Bazer, 2004a; 2004b] and plays a fundamental role in this process, by its binding to the endometrium and the inhibition of oxytocin receptors synthesis. In particular, IFN-τ is able to induce the production of several proteins, by binding to the apical portion of the uterine glands. The synthesis of these proteins improves the uterine environment and favours embryo survival [Senger, 2005]. IFN-τ is not able to act on the corpus luteum for increasing progesterone production. For this reason it can not be considered as a luteotrophic agent.

Implantation A close cross talk between the conceptus and the mother is on the basis of the implantation process. As previously mentioned, an adequate luteal activity, and consequently an adequate progesterone concentration, induces an appropriate uterine environment together with a sufficient elongated embryo that are the essential counterparts in this phenomenon. The synchronization of the embryo with the status of the uterus is critical for a successfully implantation [Dey, 1996; Paria et al., 1993; Psychoyos, 1973]. The phenomenon of implantation occurs in different modalities in mammalians. A real implantation is described only in rodents and humans, in which the embryo is able to erode the endometrium and anchor itself; in ruminants, this process is characterized by a superficial contact between the embryo and the uterine endometrium [Senger, 2005]. According to that described by Guillomot and collaborators [Guillomot et al., 1981] in sheep, three different stages are highlighted from the descent of the embryo in uterus to the formation of placenta. The first stage is defined as pre-attachment period, during which the free floating blastocyst undergoes a significant elongation as described above. The second phase, defined as transitory attachment, is considered of primary importance in ruminants. The transitory attachment occurs between 16-18 days of pregnancy until 25-30 in various ruminants. A negative role, throughout this process, is played by a transmembrane glycoprotein called Mucine-one (MUC-1). MUC-1 has been described in several mammalian species, included buffalo [Perucatti et al., 2006]. The synthesis of this protein during the nonreceptive period for the uterine epithelium is very high, whereas it shows a drastic reduction when the endometrium undergoes the action of progesterone. In fact, it has been demonstrated in cattle that the presence of progesterone for 8-10 days is able to block the receptors on the endometrium and, consequently, the endometrial cells are not yet responsive to the progesterone stimulation [Geisert and Malayer, 2000]. This process results in the block of MUC-1 synthesis for a negative feed-back mechanism. Hence, the embryo is able to attach the uterine epithelium by the interaction between some adhesive factors [Burgdardt et al., 1997; Geisert and Malayer, 2000].

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In this stage the conceptus projects developed structure like villi into the crypts of uterine glands. The role of these structures favours complete attachment progression and furnishes a temporary anchor and adsorpitive structures for the conceptus. Furthermore, these structures allow the absorpion of the endometrial glandular secrete, a complex of histotrphic substances and proteins [Spencer and Bazer, 2004b]. These growth factors, enzymes, cytokines, lymphokines, hormones, transport proteins and other substances have a key role in embryo nutrition and development, other than allowing the production of the first signals for the maternal recognition of pregnancy. Within the chorionic villi, either in buffaloes and other ruminants, it is possible to distinguish two different cellular populations, which can be identified throughout pregnancy: the mononucleate trophoblast cells and the binucleate trophoblast giant cells (BNCs) [Wimsatt, 1951; Greenstein et al., 1958]. These cellular populations have different morphology and functions. The Mononucleate Trophoblast Cells are localized at the level of the basal lamina and are characterized by the presence of one irregularly shaped nucleus with dispersed chromatin [Boshier and Halloway, 1977]. In the cytoplasm of these cells, no periodic acid-Schiff (PAS)positive granules are observed and they are in connection by strong junction, recognized as desmosomes and tight junctions, with the adjacent cells [Carvalho AF et al., 2006]. The number of mononucleate cells in buffalo, such as in other ruminants [Igwebuike, 2006], is definitely higher than that of BNCs, since they represent around 80% of the total number of trofectoderm cells [Carvalho AF et al., 2006]. However, they show cuboidal to columnar shape and smaller dimensions compared to BNCs. The main morphological characteristic of these cells, is the surface of their apical membrane, which is organized to constitute microvillar processes. The role of these villi is to get in contact with similar digitations that originate from the maternal uterine epithelial cells, constituting the attachment zones [Bjorkman, 1969]. The principle function of these cells is to guarantee nutrients exchanges between the embryo and the mother. In fact, the presence of microvilli on the apical surface of the mononucleate trophoblast cells allows to increase the area of contact between the uterine epithelium and the foetal chorioallontois [King et al., 1980], since the microvilli are morphological features common to all the cells. Furthermore, the cells are strictly bound each other by tight junctions and desmosomes [Dent, 1973]: this particular architecture of the ruminant placenta, allows to contemporary increase the absorption surface and maintain separate the circulations of fetus and mother [Igwebuike, 2006]. The placenta barrier formed by the trofoblastic cells, has different permeability for all the substances needed from the foetus, basically on the basis of their molecular size [Stacey et al., 1977]. In any case, two are the main mechanisms by which macromolecules are transported through this barrier: facilitate diffusion and endocitosis [Igwebuike, 2006]. The glucose transport, for example, occurs by facilitate diffusion, using some carriers, called Glucose Transporters 1 and 3 [Currie et al., 1997]. On the contrary, the calcium is transported against its gradient by another carrier, the 9-KDa calcium binding protein. It has been demonstrated in cattle and sheep that this molecule appears in the second half of gestation [Wooding et al., 1996; Morgan et al., 1997] and, in particular, it is localized in the mononucleate trophoblast cells of the interplacentomal

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regions (see below). The presence of this molecule in the second half of pregnancy may be explained by the calcium requirements for embryo bone formation. But the mononucleate trophoblast cells have another important mechanism for transporting molecules: the phagocytic activity. There are some specialized trophoblasts in the interplacentomal chorioallantois areas, which are specialized in phagocytosis. In fact, these cells face below with the endometrial glands, where the uterine milk is secreted and can be phagocytized by the embryo, above all during the first stages of pre-attachment [Schlafer et al., 2000]. Other trophoblast cells specialized in phagocytosis are localized at the bases of the cotyledonary villi, where some haematomas may be observed, for the presence of erythrocytes inside the trophoblast cells [Pereira et al., 2001; see below]. This peculiar aspect will be discussed later. The binucleate trophoblast giant cells (BNCs) are typical of the ruminant placenta and they probably originate from the mononucleate trophoblast cells by acytokinetic mitoses [Wooding, 1992; Klisch et al., 1999]: the mononucleate trophoblast cells undergo consecutive nuclear divisions, without the subsequent cytokinesis. They are spherical in shape and a high number of PAS-positive granules, characterized by heterogeneous dimensions and electron density, are present in their cytoplasm. These cells appear around day 17 in a particular intraepithelial position: they are localized among the mononucleate cells, but have not contact with both the basal membrane and the apical microvillar surface of the trophectoderm. In a second phase, they mature and undergo a peculiar migration through the tight junctions of the mononucleate cells, without interrupting them. This migration, that does not disrupt the junctions between the fetal and maternal comparts, is performed by the capability of these cells of extending an apical cytoplasmic process, which becomes part of the tight junctions [Wango et al., 1990]. This process has been described to occur throughout gestation in several mammalians [Wooding et al., 1986], and it is thought to be similar in buffalo. After migration, the BNCs fuse with the underlying uterine epithelium surface [Morgan and Wooding, 1983; Wooding, 1992], to form tri- or multinucleate hybrid cells and, sometimes, syncytia. These syncytia have a primary role in the immunological protection of the conceptus during the first stages of attachment, while no barrier or structural role has the subsequent migration of BNCs during pregnancy [Wooding and Wathes, 1980]. The phenomenon of migration and fusion of the BNCs is at the basis of the classification of ruminant placenta in syndesmochorial (See below), although the migration and fusion of these cells has been described also in the interplacentomal regions of the placenta [Igwebuike, 2006]. In these zones, the syncitia are quickly replaced by the endometrial epithelium, leading to the consideration that probably they have not specific structural functions. Since these cells are shorter than the endometrium, it has been hypothesized that the role of these cells is to reduce the distance between maternal and fetal capillaries [Wooding, 1984]. Similarly to that described in bovine [Wooding, 1992], the light microscopy analysis of buffalo placenta shows a great number of trinucleate cells, that derive from the fusion between the BNCs and the uterine epithelium [Carvalho AF et al., 2006]. Some larger syncytia, characterized by more than 3 nuclei, have also been observed, but their number is definitely lower than that of trinucleate cells [Carvalho AF et al., 2006]. This particular aspect demonstrates a close phylogenetic relationship between buffalo and bovine, while it

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results different from sheep and goat, in which a higher number of syncytia is present. Furthermore, as observed in other ruminants, BNCs in buffalo represent about 20% of the total trophoblast cells at the time of implantation. Interestingly, a lower number of BNCs is present in the buffalo placenta during the first months of gestation (2-3) compared to the rest of pregnancy (28% vs. 20%) [Carvalho A.F. et al., 2006]. But, why do the BNCs undergo migration? The main accredited hypothesis is that in this way they have the possibility of delivering the PAS-granules, present in their cytoplasm, closely to the maternal blood circulation. In fact, after the fusion, the granules present into the BNCs are released at the basal membrane of the uterine epithelium [Wooding and Beckers, 1987]. The granules show a high affinity with specific antisera against bovine placental lactogen, prolactin-related protein-1 and pregnancy-associated glycoproteins by immune histochemistry. Therefore, these trophoblast-derived proteins, such as placental lactogen (PL) [Duello et al., 1986], Prolactin-Related Protein-1 (PRP-I) [Kessler and Schuler, 1997] and the Pregnancy Associated Glycoproteins (PAG) are released into the maternal vascular circulation. The first two proteins are members of the prolactin/growth hormone family [Anthony et al., 1995] and have several actions. In particular, the PL seems to have mammotrophic, luteotrophic, and somatotrophic activities [Anthony et al., 1995], for its prolactin and growth hormone affinity, that gives the possibility of binding the receptors of these hormones. Furthermore, the first expression of PL allows the beginning of production of uterine milk protein by the glandular epithelium of the endometrium, and it is thought to play a primary role in the differentiation of the uterine glands throughout pregnancy [Igwebuike, 2006]. The function of PRP-I is still not known, since it does not have the capacity of linking the receptors for prolactin and growth hormone [Kessler and Schuler, 1997]. A particular inkling needs to be carried out on the PAG and their role. The PAG are a large family of proteins that are produced during pregnancy by the placenta of several mammalians [for review, see Bella et al., 2007]. Excluding the human Chorionic Gonadotrophin (hCG) and the equine Chorionic Gonadotrophin (eCG), the first gonadotrophin-like molecule was identified by Butler et al., [Butler et al., 1982] in bovine placenta in 1982. This protein with a molecular weight of 30-32 kDa, was able to inhibit the binding of pituitary LH to the CL membrane receptors, and was hence called bovine Chorionic Gonadotrophin (bCG). On the basis of this study, several researches have been performed, above all by using new techniques of molecular biology and this large family of protein has been identified and defined with several names, such as Pregnancy Associated Glycoproteins (PAG) or pregnancy-specific protein B (PSP-B). These proteins show a high sequence homology with each other and with pepsin, pepsinogen, chymosin, cathepsin D, and cathepsin E and, therefore, were ascribed to the aspartic proteinase family [Xie et al., 1991; Guruprasad et al., 1996]. It is estimated that 100 or more PAG genes are present in cattle, sheep and probably all ruminants. It has also been possible to identify some PAG molecules that are expressed only during distinct stages of pregnancy [Green et al., 2000]. They are synthesized by the binucleate trophectoderm cells (BNC), which originate from the mononucleate chorionic epithelium [Wooding, 1980] and, after the migration into the endometrial epithelium, secrete PAG continuously throughout gestation [Zoli et al., 1992; Wooding et al., 2005]. Hence, this family of proteins can be considered good indicators of a

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live conceptus [Zarrouk et al., 1999] and the detection of these placenta specific antigens in the serum may represent a reliable tool for pregnancy diagnosis. Obviously, these proteins represent also a valuable tool for detecting embryonic mortality and late pregnancy alterations. PAG have also the possibility of regulating progesterone production, by inducing the synthesis of prostaglandin E in luteal cells [Del Vecchio et al., 1995; Weems et al., 1999], and the release of granulocyte chemotactic protein-2 in the bovine endometrium [Austin et al., 1999]. This function is usually performed by the IFN-τ during the first stages of pregnancy. Therefore, it has been supposed that they can substitute this molecule during the late stages of pregnancy. Pregnancy specific protein B was detected for the first time in buffalo species in 1997 [Debenedetti et al., 1997] by an eterologous RIA test using a bovine PSPB antibodies in the serum of pregnant animals between 25 and 30 days of gestation, with values close to 4 ng/ml [Debenedetti et al., 1997]. It can be detectable in 33% of pregnant buffaloes between 20 and 25 days post-insemination and in all pregnant subjects after day 30 post-insemination with a mean value of 1.6±1.1 ng/ml [Malfatti et al., 2001]. Interestingly, its levels rapidly increases reaching 6.6±3.2 ng/ml on day 50 of gestation and no differences are present between its levels at calving stage and those detected in the late stages of pregnancy [Malfatti et al., 2001]. A lower concentration of around 45% is observed 5 days post-partum and it disappears on day 50th post calving. The lack of specific antibodies deeply influence the evaluation of PAG concentration in buffalo species. The method of assessment and the antibodies utilized in the analysis (bovine or caprine), provide different results, because of different affinity with buffalo antigens [Debenedetti et al., 2001a]. In fact, it has been demonstrated that buffalo PAG are better recognized by antisera raised against caprine PAG (caPAG55+59 kDa and caPAG55+62 kDa) compared to antisera raised against bovine PAG (boPAG67 kDa), by using RIA methods [Debenedetti et al., 2001b]. As mentioned above, the use of rabbit antisera raised against a mixture of caprine PAG (caPAG55+62 kDa) as first antibody provide more precise results on PAG concentration in buffalo. The sensitivity of the test for PAG detection results very low (11.1%) on days 19–24 post-insemination, but progressively increase to 80% on days 25–30 and 100% on days 31– 35 of gestation [Karen et al., 2007]. These differences can be easily explained by the origin of PAG secretion. In fact, as reported above, pregnancy proteins are synthesized by the binucleate cells, which arise from the trophoblast and migrate into the endometrium, when the phenomenon of attachment occurs. For this reason PAG levels are low at 19-24 days of gestation, resulting in higher frequency of false-negative diagnoses. Furthermore, the high sensitivity of caPAG55+62 kDa to buffalo antigens allows to obtain PAG concentrations very high yet on day 25-30 of gestation (from 4.48 to 6.4 ng/ml) and a significant increase on day 51-55 post insemination (28.25 -40.6 ng/ml) [Karen et al., 2007]. PAG levels decrease on Day 77 (21.37 ng/mL) and remain constant until Day 103 (20.71 ng/mL). Interestingly PAG concentrations in pregnant buffaloes are almost 2 to 3 times higher than those observed in dairy cows from Day 30 till 60 after breeding [Perenyi et al., 2002; Lopez-Gatius et al., 2007]. This rapid increase on PAG concentrations associated with high maternal concentrations at early pregnancy period are characteristic of caprine [Gonzalez et al., 2000] and ovine [Ledezma-Torres et al., 2006] species and different from cattle [Perenyi et al.,

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2002; Patel et al., 1997; Zoli et al., 1992], in which concentrations increase slowly and remain at low levels during early pregnancy gestation. This further consideration allows to confirm that buffalo species can be considered physiologically closer to sheep than cattle. In the last years, buffalo PAG have also been purified. In fact, except for a partial description of the electrophoretic characterization of the Indian buffalo placental proteins [Singh et al., 2005], buffalo PAG have been characterized by two different radioimmunoassay systems, using a highly purified boPAG-1 as both a tracer and standard, and different antisera raised against bovine PAG (boPAG67 kDa) and a mixture of caprine PAG (PAG55kDa+59kDa) [Barbato et al., 2008]. This method is based on the evidence that lectins such as the agglutinins from Vicia villosa and Dolichos biflorus bind to the N-acetyl galactosamine (GalNAc) of asparagine-linked glycans from bovine PAG [Klisch and Leiser, 2003; Klisch et al., 2005; 2006]. Therefore, it has been proposed that lectin-based affinity chromatography can be an useful tool for PAG enrichment from the placental extracts of ruminant species [Klisch et al., 2005]. Hence, it is possible to confirm that the apparent molecular masses of the immunoreactive bands from the Vicia Villosa agglutinin affinity chromatography peaks range from 59.5 to 75.8 kDa and from 57.8 to 73.3 kDa in the midpregnancy and late-pregnancy placentas, respectively. Furthermore, the amino-terminal microsequencing of the immunoreactive proteins allowed the identification of three distinct water buffalo PAG (wbPAG) sequences that have not yet described in other species [Barbato et al., 2008]. The isolation of these new molecules would be helpful in order to produce new antisera for investigating PAG secretion and for developing accurate and specific ELISA kits for pregnancy diagnosis in buffalo. The evaluation of PAG concentration may result also useful for evidencing embryonic and fetal mortality. In fact, buffaloes which show fetal mortality at different stages (around 30, 60, 90 and 180 days of gestation), are signalled by a decline of P-SPB levels, which become undetectable at successive samplings [Debenedetti et al., 1997; Malfatti et al., 2001]. Together with the secretory capacity, the BNCs have also an important role in steroid and prostaglandin metabolism. In fact, in these cells prostaglandin E can be produced from PGF2α [Gross and Williams, 1988] and progesterone is synthesized [Reimers et al., 1985].

Placenta After the transitory attachment of the embryo to the endometrium surface, migration of BNCs and formation of syncytia and trinucleate cells, the formation of placenta takes place. This is the third, final stage [Guillomot et al., 1981] that completes embryo attachment. In fact, prior to day 16 in the sheep and day 25 in cattle, the placenta is essentially diffuse [Senger, 2005]. At this time the chorion initiates attachment to the caruncules of the uterus. Placenta can be defined as a transient organ that is able to sustain metabolic and endocrine roles. Several types of placenta are distinguished in mammalians, on the basis of the anatomical features and the number of tissue layers that separate maternal and fetal blood. As mentioned above, in ruminants, in which a superficial contact between the embryo and the uterine endometrium is described, the placenta is defined Syndesmochorial, and, taking into account the anatomical characteristics, it is defined cotyledonary. In fact, it shows small areas

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of attachment that are represented by the functional unit of this type of placenta: the placentome. This particular structure of the ruminant placenta, allow to distinguish two main areas: the placentomatal regions, in which the fetus-mother exchanges occur and the interplacentomatal areas, in which the fetal membranes are only apposed on the endometrial epithelium [Davis et al., 2000]. Each placentome is composed by two components: one that originates from the fetus (fetal cotyledon) and the other that origins from the mother (caruncle). The latter is present in some particular regions of the uterus that are called caruncular regions. In this type of placenta, the maternal capillaries are in contact with the chorion villi only in some points, where the endometrium is eroded. It is classified by multiple separate placentomes like in the cow [Björkmann, 1969; Leiser et al., 1998], which display an interdigitation of the cotyledonary fetal villi with maternal septa of the complementary caruncles [Leiser and Kaufmann, 1994; Wooding and Flint, 1994]. The placentomes may vary remarkably in number, size, form and structure according to the genus and the species [Hafez, 1954; Mossman, 1987]. The number of the placentomes ranges from about 5-8 in the deer [Mossman, 1987] to about 80-140 in the cow, 100 in the sheep and 160 in the goat [Björkmann, 1954; Leiser and Kaufmann, 1994]. In buffalo the number of placentomes is about 68–100 in the horn that sustain the pregnancy compared to 50–55 in the contralateral one [Hafez, 1954; Abdel-Raouf and Badawi, 1966]. Similarly, the form of placentomes is different according to the species. In fact, if they have a mushroom-like form in bovine and cup-like in the sheep, in buffalo they show different form in early pregnancy and mid and late stages of gestation. In the first case the presence of spherical or kidney shaped placentomes has been observed, whereas elliptical or mushroom-like forms are observed in the subsequent phases [Raja and Chandra, 1984]. The fetal cotyledons display slender, highly vascular villous trees [Sharma et al., 1983; Abd-Elnaeim et al., 2003]. The collateral arterious villous trees in each cotyledon tend to repeatedly branch in smaller capillaries which penetrate into the thickness of the cotyledons. Interestingly, in buffalo the cotyledonar blood vessels show intermediate characteristics compared to sheep and bovine [Pelagalli, 1973], in relation to the dimensions of the cotyledons. Similarly to that described in ovine, the smaller cotyledons show a central arterious blood vessel that branches into several capillaries with a fan-shaped aspect, before penetrating the thickness of the cotyledons [Pelagalli, 1973] (Figure 12). In the largest cotyledons, the arterious blood vessel surrounds the base of the cotyledon, shaping a fish-net that borders the latter, as described in bovine. However, these blood vessels are smaller and more numerous than those described in cattle [Pelagalli, 1973] (Figures 13 and 14). Furthermore, the shapes of caruncles with the correspondent cotyledons, is slightly convex, without reaching the convexity described in cattle [Pelagalli, 1973] (Figure 15).

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Figure 12. Ten years old buffalo at the third month of gestation. Distribution of arterious (blue) and venous (red) blood vessels in a cotyledon. [From Pelagalli et al., 1973].

Figure 13. Seven years old buffalo at fourth month of gestation. Distribution of arterious (blue) and venous (red) blood vessels in some cotyledons. [From Pelagalli et al., 1973].

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Figure 14. Schematic representation of blood vessels distribution in an uterine caruncle in sheep (A), cow (B) and buffalo (C). [From Pelagalli et al., 1973].

Figure 15. Schematic representation of uterine caruncles (CA), cotyledons (CO) and their relationships in sheep (A), cow (B) and buffalo (C). [From Pelagalli et al., 1973].

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The measurements of caruncles in buffaloes vary according to the pregnancy status. In fact, in non pregnant water buffaloes they measure about 6.4 mm in length, 4.6 mm in breadth and 1–2 mm in height, while, if a pregnancy occurs, all these measurements gradually increase, reaching about 46 mm in length, 25 mm in breadth and 8 mm in height in proximity of the end of gestation [Abdel-Raouf and Badawi, 1966]. In buffalo cotyledons characterized by different dimensions have been observed within the uterus, although they result smaller than those reported in cattle, in which a diameter of 50 – 60 mm has been recorded [Senger, 2005]. The largest ones are present on the dorsum of the foetus in the pregnant horn [Abd-Elnaeim et al., 2003] and it has been proposed [Hafez, 1954] that this particular position has a protective function of the foetus (Figures 16 and 17). The fetal vasculature within the cotyledons, show a variable structure and tend to greatly increase from the first stages of gestation to term. In fact, a series of conical villous trees, that modify their form during pregnancy, are observed by a histological analysis of the buffalo fetal cotyledons. In particular, they are characterized by wide to slender shape and show their base directed to the fetal side [Abd-Elnaeim et al., 2003]. Furthermore, there is the development of stem villous trees and the capillary system of the terminal villi increase especially in the late stages of pregnancy. This phenomenon may be explained by the increasing substances exchanges between the foetus and the mother, for which the feto-maternal circulation need to deeply develop [Abd-Elnaeim et al., 2003]. Usually, two types of villous trees are described in buffalo placenta: a rough type, whose number is predominant and the smooth type, localized at the periphery of the fetal cotyledons [Abd-Elnaeim et al., 2003]. The number of the latter type tend to gradually decrease throughout gestation.

Figure 16. Presence of large cotyledons on the dorsum of a 6 months old buffalo fetus.

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Figure 17. Cotyledons of different form and dimensions: cotyledon in the pregnant horn (on the left) and in the controlateral (on the right) in a 6 months old buffalo fetus.

Interestingly, the uterine caruncles in buffaloes are attached to the underlying uterine mucous membrane [Badawi and Abdel-Rauf, 1970; Abd-Elnaeim et al., 2003] and are nonstalked. Hence, it has been observed that the caruncular microvasculature is obtained by two short non-spiral caruncular arteries and a single vein for each uterine caruncle. This particular structure of the caruncles in buffalo, lead to a firmer attachment of the placentomes to the uterus, compared to bovine, in which the vasculature of the maternal component is supplied by a large caruncular stalk that contains large number of spiral arteries and veins [Pfarrer et al., 2001]. It can be supposed that this firm attachment may be necessary and accounted for the longer gestation period of buffalo compared to bovine (300-330 days vs. 280-290 days, respectively). As found in other species [Myagkaya and Vreeling-Sindelarova, 1976; Murai and Yamauchi, 1986; Leiser and Enders, 1980] also in buffalo the presence of placental haematomas at 7-8 and 9-10 months of gestation is observed, exclusively at the bases of chorionic villi. These placental haematomas show irregular form and are in great number within the uterine and trophoblastic epithelium, hence at level of the fetal-maternal interface. A variable quantity of maternal red cells is present in the trophoblastic cells near to these haemorrhagic areas, leading to the hypothesis that an erythrophagocytosis process occurs at this level [Pereira et al., 2001]. The role of these placental haematomas is still unclear, but there is a strong possibility that they represent a font of iron for the foetus [Pereira et al., 2001].

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After its development, the placenta plays several roles. In particular the main two functions are 1) the warrantee of fetal-maternal substances exchange and 2) an endocrine role. In fact, throughout gestation placenta substitute the function of the gastrointestinal tract, lungs, kidneys and liver in the foetus that does not have the possibility of an autonomous life. But, as mentioned above, it is important to underline the endocrine role of the placenta. It was referred in a previous section that the granules present inside the binucleate of the trofectoderm, release trophoblast-derived proteins, such as placental lactogen (PL) [Duello et al., 1986], Prolactin-Related Protein-1 (PRP-I) [Kessler and Schuler, 1997] and the Pregnancy Associated Glycoproteins (PAG) into the maternal vascular circulation. However, the placenta is able to play a truly hormonal rule, releasing steroid hormones in either fetal and maternal circulation. In fact, although some specific enzymatic functions essential for steroidogenesis, are absent in foetus or in placenta, they, together, are able to supply these actions [Jainudeen and Hafez, 2000]. Progesterone is mainly produced by corpus luteum during the gestation. However, successfully, the placenta is able to supply progesterone production by using the circulating cholesterol. The amount of production is different in various species: some of them, such as sheep and mare, are able to synthesize a great amount of this hormone, whereas others (cow, goat and swine) are not. For this reason in some species, such as mare and ewe, the corpus luteum can be removed (by ovariectomy) in the late stages of pregnancy, without interrupting the gestation, whereas in others (cow, sow and goat) the luteolysis causes abortion [Geisert and Malayer, 2000]. Unfortunately, few studies [Arora and Pandey, 1982; Eissa et al., 1995] have been performed throughout gestation in buffalo species, showing relatively constant levels of progesterone during the first, second and the major part of the third trimester of pregnancy, with a mean value of 2,95+0,87 ng/ml [Perozo Marin et al., 2002]. Hence it is not possible to establish the specific role played by the placenta in this process. In particular, it seems that progesterone varies between 1.9 and 3.8 ng/ml (3.5 ± 0.9 ng/ml) during the first 2 months of pregnancy. A slight decline is observed around the third month (2.9 ± 0.8 ng/ml) and the levels recorded subsequently are always lower than this value, until the last week of pregnancy. In the buffalo the corpus luteum is maintained throughout gestation. This behaviour and some field experiences let us to suppose that buffalo behaviour during pregnancy is more similar to that of cattle than ewe. At the end of gestation there are conflicting data. In fact, in some cases it has been reported an increase of progesterone concentration in both heifers and pluriparous buffalo cows from day -30 to day -15 [Perera et al., 1981; Kamonpatana, 1984], while in other cases [Arora and Pandey, 1982; Prakash and Madan, 1984; El-Belely et al., 1988] a gradual progesterone decrease has been observed in the same period, with a rapidly decrease in the last week of gestation, reaching values of around 1.5–0.9 ng/ml [Perozo Marin et al., 2002; Eissa et al., 1995]. In any case, a strong decline of the concentration is reported on the day of calving and basal levels are reached between 3 and 15 days post-partum, indicating a complete regression of the corpus luteum of pregnancy [El-Wishy, 2007]. The concentrations of total oestrogen and oestradiol-17-β, slightly increase during the first 4 months of pregnancy (14.8±2.1 pg/ml) and maintain basal levels (<12 pg/ml) until around 10 days prepartum [Eissa et al., 1995]. In this last period of pregnancy a shape increase of oestradiol-17β and total oestrogens are observed, reaching the peak (82.8± 3.6pg/ml) on the day of partum. The corsticosteroid concentration is relatively constant throughout the gestation period

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(1.7±0.3 pg/ml). Similarly to that described in cattle [Senger, 2005], also in buffalo species a significant increase of corsticosteroid, which maintain baseline levels (1.7±0.3 pg/ml) throughout gestation, is observed starting on day -12 (5.3±1.8 ng/ml) before parturition [Eissa et al., 1995]. During the immediate pre-partum period, their concentration peaks to 10.8±2.4 ng/ml, 12.5±2.9 ng/ml and 16.8±3.2 ng/ml at 60, 36 and 0 hours before delivery [Eissa et al., 1995]. During the first two days post-partum the concentration is around 7 ng/ml, for reaching 3 ng/ml on day 3. Finally, similarly for that described for corticosteroid, the PGFM concentration is at basal levels (200-600 pg/ml) throughout the pregnancy and shows a progressive increase starting on day 10 pre-partum, reaching the peak (13.8 ± 2.3 ng/ml) on the day of delivery. Therefore, it is likely that phenomena similar to those described in bovine [Senger, 2005] occur also in buffalo species. In fact, it has been described in cattle [Senger, 2005], that the increase of fetal corticosteroid promotes the synthesis of three enzymes (17-αhydroxylase, 17-20 desmolase and aromatase), that are able to convert progesterone in oestradiol. Furthermore, PGF synthesis in the endometrium is also stimulated by corticosteroid, leading to the fall of progesterone levels by the regression of the corpus luteum.

Embryonic Mortality in Buffalo Cows Embryonic mortality can be defined the phenomenon for which a fertilized oocyte develops in embryo, but the latter is not able to reach the term of gestation. Embryonic loss is increased when physiological regulation of oviductal and uterine function is inadequate or when the mother is exposed to one or more of the many stresses that can compromise embryonic survival [Hansen, 2002]. Embryonic mortality usually occurs during the first phases of gestation in various species: in cattle, for example, it is evident within 40 days of pregnancy. In particular, 30-40% of embryonic losses in bovine occurs between 7 and 17 days post fertilization and, in some cases, it can take place before embryo becomes foetus [Thatcher et al., 1995]. In any case embryonic loss is a remote eventuality (around 10%) during the subsequent phases (after 42 days) of pregnancy [Vasconcelos et al., 1997]. In buffalo species embryonic mortality is considered one of the major causes of fertility loss, especially in the animals that are not mated during their reproductive period. In Italy, in fact, the application of the out of breeding season mating technique guarantees milk production in accordance with market requirements, but it forces the breeders to mate buffaloes during the less favourable periods. Embryonic mortality in buffalo species may occur in different stages of pregnancy and it may be due to several causes. On the basis of the last studies, it is possible to distinguish several types of embryonic mortality: a) an early embryonic mortality, which occurs within 20 days of pregnancy and incides for around 20%; b) a late embryonic mortality, between 25 and 45 days, which is described in around 40% of pregnant animals at day 25 post-fertilization; c) a fetal mortality, which is observed in 10% of animals between 45 and 90 days of gestation.

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These phenomena largely affect reproductive performances in buffalo species, reducing the fertility, especially in those subjects undergone artificial insemination. In fact, it can be hypothesized that oocyte fertilization occurs in around 80% of inseminated buffaloes, but only 35% of subject carry out to term the gestation, for the occurrence of pregnancy losses throughout the phases described above. The seasonality characteristic of this species, causes an increase in the phenomenon of embryonic mortality during the periods characterized by increasing daylight length. It has been observed that embryonic loss in animals mated by artificial insemination (AI) is 20-40% during seasons characterized by high number of light hours [Campanile et al., 2005; Campanile et al., 2007a; Campanile et al., 2007b], whereas values of around 7% or 20% are recorded in Brazil during decreasing light days [Baruselli et al., 1997b] or close to the equator [Vale et al., 1989] respectively. The high variability in terms of embryonic mortality may be probably ascribed to the different distance from the equator, which influences daylight length throughout the seasons. Furthermore, it is worth pointing out that other causes may be accounted for embryonic mortality. The phenomenon is not always correlated with the breeding season, but with the ovarian resumption after calving and farm reproductive management, embryonic epigenetic expression, environmental conditions (hot, cold, etc.), nutrition, specific and aspecific pathologies, and, obviously, uterine environment [Zicarelli, 1994b]. In fact, as previously reported, embryo survival depends on the physiological regulation of oviductal and uterine function due to intrinsic errors in maternal physiology or to specific environmental stresses imposed on the mother. In any case, embryo mortality in buffalo occurs, principally, later than in bovine, usually between 25 and 40 days from AI [Campanile et al., 2005]. In buffaloes naturally mated [Vecchio et al., 2007a], independently from the conception period, 8.8% and 13.4% show embryonic or foetal mortality between 28-45 days and 46-90 days of pregnancy respectively. No differences are found between the incidence of embryonic mortality in relation to the conception period, while a high incidence of foetal mortality is recorded during a period of increasing daylight length (transitional period: December-March in Italy) compared to the April-July period. It is hypothesized that this condition is due to the presence, in the transitional period, of subjects that become pregnant, even if they have a lower function of the corpus luteum because they are going into anoestrus. In the subsequent months (AprilJuly) an increased incidence of acyclic buffaloes is observed and, hence, only the subjects that are not sensitive to the photoperiod are cyclic and become pregnant. In fact, the incidence of foetal mortality in this case is similar to that observed in the decreasing daylight length period (August-November), which is the favourable period for reproductive activity. This is confirmed by Zicarelli (unpublished data) in a study performed on 3000 conceptions in which a higher incidence of embryonic mortality is reported between 30 and 90 days in buffaloes that conceive during increasing daylight length (Figure 18), although farm management and environment play a fundamental role in this phenomenon. In the countries close to the equator embryonic mortality (after 30 days from AI) is between 13.2% and 7.0% respectively in decreasing and increasing daylight length period [Baruselli, 2007 personal communication].

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Figure 18. Incidence of embryonic mortality in relation to month of conception and daylight length in two farms.

Figure 19. Embryonic loss rate during stage of gestation.

The incidence of embryonic mortality is higher between 28-60 days of gestation and lower after 71 days (Figure 19). This result is different from that reported in cattle [Silke et al., 2002], in which the embryonic loss from 28-87 days of gestation is similar. Embryonic mortality, in buffalo species, is not affected by age, parity or lactation stage and infectious agents explain only about 2-8% of the cases [Campanile et al., 2005; Campanile et al., 2007a]. Embryonic mortality in buffalo species is primarily due to a reduced secretion of progesterone (P4) by corpus luteum. This conclusion is consistent with several findings in cattle and sheep, where early embryonic mortality is associated with reduced circulating concentrations of P4 [Garret et al., 1988; Mann and Lamming, 1999; Mann and Lamming, 2001]. In buffalo preovulatory follicle and corpus luteum dimensions are larger in pregnant

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animals than in those that show embryonic mortality [Barile et al., 2007]. The pre-ovulatory size of follicle influences corpus luteum dimension and function and affects embryonic mortality [Barile et al., 2007]. In fact a reduction of P4 concentration since day 10th after insemination causes an increase of embryonic mortality between day 25 and 45 [Campanile at al., 2005; Campanile at al., 2007a]. Progesterone is the hormone of pregnancy and it is fundamental to support conceptus viability and development and its associated membranes. The actions of progesterone are mediated by the progesterone receptors and induce the production of histotroph by endometrial glands that nourish and sustain the conceptus during remodelling, adhesion, implantation and placentation [Spencer and Bazer, 2002]. In fact, the endometrial stroma is sensible to the action of progesterone throughout pregnancy for the presence of these receptors. Stromal cells produce several growth factors, progesterone-responsive and mediate epithelial-mesenchymal interactions that are crucial for support of pregnancy [Spencer and Bazer, 2002]. Impaired P4 secretion has been linked with a reduced capacity of the developing embryo to secrete interferon-tau (IFNτ) at threshold amounts necessary to prevent luteolysis [Wathes et al., 1998]. In fact, as above mentioned, the maintenance of pregnancy is due either to the maternal recognition of pregnancy and to the embryo capability of blocking luteolysis since day 16 post-AI [Mann and Lamming, 1999]. This process occurs by the production of bovine trophoblastic protein-1 [bTP-1], also called IFNτ [Roberts et al., 1992]. This protein is able to avoid corpus luteum regression by two mechanisms: a) by inhibiting oxytocin receptors (OTR) development on endometrium [Robinson et al., 1997]; b) by activating a prostaglandin inhibitor [Thatcher et al., 1995]. It has been supposed that oestradiol is another factor involved in the luteolytic process, either by promoting OTR development and by stimulating prostaglandin secretion [Wathes et al., 1998]. In fact, it has been demonstrated in ovine that the number of oestradiol receptors on endometrium is significantly lower in pregnant vs. not pregnant animals [Lamming et al., 1995; Spencer et al., 1995]. However, in buffalo species oestradiol plasma levels do not differ between pregnant, not pregnant and buffaloes undergone embryonic mortality on day 0, 10, 20 and 25 after A.I. [Spagnuolo et al., 2007]. Gametes quality is one of the main factors involved in the phenomenon of embryonic mortality in domestic animals. Oocyte quality is able to affect embryo development and interfere with the following gestation. In buffalo species this phenomenon may be more frequent during the seasonal anoestrus, which coincides with daylight length increase [Campanile et al., 2005] and, consequently, with the resumption of sexual promiscuity in the farms in which the out of breeding season mating technique is applied. Campanile et al., 2005 report that 51% of buffaloes which showed embryonic mortality had P4 concentrations on days 10 and 20 similar to those of animals which maintained pregnancy. Therefore, it is possible that other factors, rather than reduced circulating P4 concentrations, also contribute to embryonic mortality. With this regard, it is reported that oocyte quality, judged as the capacity to result in embryonic development and pregnancy, is worse in buffaloes during the

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anoestrous period [Abdoon et al., 2001], occurring when daylight length increases [Zicarelli, 1997]. Furthermore, the incidence of embryonic mortality between 40th and 60th day post AI is three times higher in acyclic buffaloes on day 70 after calving [Zicarelli, 1994b], compared to those that are cyclic. It is known that in buffalo species high incidence of atresia is present and the mean recovery of good quality oocytes per ovary is low [Gasparrini, 2002]. The maturation and the quality of oocyte depend on the function of the granulosa cells that are sensitive to oxidative stress [Dharmarajan et al., 1999]. It is worth mentioning that the antioxidant defence system plays a key role in preventing apoptosis and atresia, thus preserving steroidogenic function of granulosa cells [Cassano et al., 1999], although no significant differences in redox status between pregnant, not pregnant and cows with embryonic mortality have been observed [Spagnuolo et al.; 2007].

Treatments for Preventing Embryonic Mortality in Buffalo Species The importance of progesterone concentration during the first weeks of pregnancy for reducing embryonic mortality has been demonstrated in cattle [Mann and Lamming, 1999 Mann and Lamming, 2001]. According to some reports the presence of an early P4 peak (within 5 days after mating or AI) facilitates the elongation of the conceptus and, consequently, the secretion of adequate interferon-tau [Starbuck et al., 1999; Mann, 2002]. In cattle, interferon-tau extends the lifespan of the corpus luteum [Plante et al., 1989] by suppressing oestradiol receptor and oxytocin receptor genes [Spencer and Bazer, 1996] and by attenuating the endometrial secretion of PGF2α [Helmer et al., 1989a]. It has also been shown that interferon-tau reduces PGF2α secretion by bovine endometrial explants [Helmer et al., 1989b] and endometrial epithelial cells [Danet-Desnoyers et al., 1994]. Several approaches have been used to increase P4 concentration in blood in order to reduce the occurrence of embryonic mortality. Increased plasma P4 concentrations are achieved either by inducing increased endogenous secretion or by administering exogenous P4 [Mann and Lamming; 1999]. Studies have shown that administration of natural sequence GnRH, GnRH agonists or hCG after AI can stimulate corpus luteum function, induce accessory corpus luteum formation, increase P4, and reduce oestradiol production, with a consequent positive effect on embryonic survival [Kerbler et al., 1997; Thatcher et al., 2003; Bartolome et al., 2005]. In buffalo species there are some controversial results, regarding the best moment for hormonal treatment. This is probably due to the different moment in which embryonic mortality occurs (early and late embryonic and fetal mortality). According to some recent experiences [Neglia et al., 2008], a simple method for increasing the endogenous progesterone production during the first days post-insemination in buffalo species, is the intravenous or intramuscular administration of a luteolytic dose of Prostaglandin F2α (PGF2α). In fact, if several studies have demonstrated that an injection of PGF2α or its analogues cause corpus luteum regression after day 6 till day 15 of the oestrus cycle in both cattle and buffalo, little information is available on its administration during the first days of the cycle. In particular, it seems that the PGF2α analogous injection on the day of oestrus, is able to reduce

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the production of vasoactive molecules, such as Endothelin-1 (ET-1) [Ohtani et al., 1998] and Angiotensin-II (Ang-II) [Hayashi et al., 2001] improving the vascularization of the follicle or the synchronization of ovulation. This technique increases progesterone production by the developing corpus luteum yet on day 11 post-insemination and, hence, increases pregnancy rate of about 16% [Neglia et al., 2008]. In this case, a reduction of 10% of the early embryonic mortality is observed. The administration of exogenous P4 by an intravaginal device on day 5 after A.I [Campanile et al., 2007a] give the lowest pregnancy rate and highest incidence of embryonic mortality, suggesting that exogenous P4 can have a detrimental effect on conception. It is possible that exogenous P4 may contribute to the regulation of LH and reduce the capacity of the preformed corpus luteum to increase P4 synthesis and release. After removal of the exogenous source of P4 the corpus luteum may not be able to secrete P4 in the amount required to maintain pregnancy. The present findings are in contrast with Kumar et al. [Kumar et al., 2003] who report an increase in conception rate in buffaloes treated with 125 mg of 17-α hydroxyprogesterone caproate s.c. on Day 4 after AI. It is possible that the type and mode of exogenous P4 treatment may influence the response in buffaloes. The endogenous stimulation of P4 production presents different response depending on the incidence of the cyclicity state of the buffaloes. In fact, the injection of 12.6 μg GnRH agonist (buserelin) or 1500 I.U. of hCG on Day 5 after A.I. increase P4 concentrations 10 days after treatments, but not always they are able to reduce the incidence of embryonic mortality. It is likely that treatment efficacy depends from the follicle competence on the day of treatment and consequently on ovulation rate. In buffalo it has been found that the injection of a GnRH agonist on Day 5 after AI increased milk whey progesterone concentrations in 97% of buffaloes subsequently pregnant on Day 40, compared to 68% in the non-pregnant buffaloes. A greater proportion of the buffaloes that ovulated (96.7%), compared to buffalo that did not ovulate (68.4%) recorded a gestational chamber on Day 40 after AI and were judged to be pregnant [Campanile et al., 2007d]. In cattle, treatment with hCG on Day 5 or Day 7 after AI increases P4 concentrations by enhancing secretion from the existing corpus luteum and also by inducing ovulation and formation of an accessory corpus luteum [Kerbler et al., 1997; Schmitt et al., 1996; Santos et al., 2001]. In buffalo treatment with hCG 5 days after AI may not increase the P4 secreting capacity of the existing corpus luteum, but can induce ovulation and formation of an accessory corpus luteum which leads to increased P4 some time later. Delayed treatment of buffaloes with GnRH agonist, hCG or progesterone on Day 25 after AI reduce embryonic mortality [Campanile et al., 2008]. The present findings would tend to suggest that the delayed treatment of buffaloes with either tropic hormones or P4 is beneficial to enhancing the likelihood that embryonic development continues during the critical phase of embryonic attachment. In sheep, P4 treatment induces increased embryonic growth which results in a greater area for attachment with uterine caruncles [Khan et al., 2003; Khan et al., 2007; Nephew et al., 1994]. The lack of decrease in P4 observed in buffaloes that received a delayed treatment have similarly enhanced embryonic development which facilitated attachment. Delayed treatments are not able to reduce embryonic mortality in buffaloes with low whey progesterone concentration on Day 20 and 25. This finding suggests that buffaloes

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with reduced P4 on Day 25 already have compromised embryos and treatment with tropic hormones or P4 on Day 25 is not able to influence embryonic mortality. Delayed treatment with hCG induces ovulation in around 57% of buffaloes [Campanile et al., 2007c] and that there is a similar response to GnRH agonists, using which ovulation rates of 62% [Campanile et al., 2007d] and 68.6% [Campanile et al., 2007c] are observed, respectively after administration on day 5 or 25 post AI. The mean follicular diameter which results sensitive to the hormonal treatment is about 8.9 mm in both treatments, varying between 4.2 and 13.0 mm [Campanile et al., 2007c; Campanile et al., 2007d]. It is worth pointing out that the dimensions of the follicles recorded in buffaloes responsive to the treatments are similar to those of buffaloes in which ovulation do not occur. These data are in accordance with those reported in bibliography in cattle [Martinez et al., 1999], regarding the incidence of subjects responsive to the treatment with GnRH and the dimensions of responsive follicles. Buffaloes that ovulate in response to the treatment with a GnRH agonist show a progressive increase in milk whey progesterone concentrations on Days 10, 15 and 20, while progesterone levels remain relatively constant for buffaloes that do not ovulate. In delayed treated buffaloes ovulation after administration of GnRH agonist reduces pregnancy loss compared with buffaloes which do not ovulate after GnRH. In contrast, there is no difference in the proportion of buffaloes pregnant between those that ovulate and those that do not ovulate after treatment with hCG. We speculate that the delayed hCG treatment had a beneficial effect on the maintenance of embryonic development by two mechanisms. The first mechanism involved the induction of ovulation and formation of an accessory corpus luteum which increase plasma concentrations of P4. The second mechanism may not have involved ovulation exclusively, but also a tropic effect of hCG on the existing corpus luteum.

Buffaloes Reproductive Ultrasonography Diagnostic ultrasonography is the most profound technological advance in the field of large animal research and clinical reproduction since the introduction of transrectal palpation and radioimmunoassay of circulating hormones. It is hard to imagine that many discoveries and procedures related to ovarian, uterine and fetal function that we use today would have been considered without the development of real-time ultrasound. One of the greatest advantages of ultrasonography is that it is totally non-invasive and so repeated examinations of an animal’s reproductive tract can be performed without impairing its breeding potential or having adverse effect on the conceptus. Real-time ultrasonic examination has allowed the monitoring of individual follicles on a daily basis. An important step in dairy management is to examine for pregnancy within 45 days after breeding [Zemjanis, 1971]. The main purpose of examining buffaloes cows early is not only to identify pregnant buffaloes but also to identify with confidence open buffaloes in order to manage, treat and cull. In buffalo species an early (20-25 days after conception) pregnancy diagnosis (Figure 20) is fundamental to reduce embryonic mortality. In fact, treatments on day 25 in pregnant animals have a beneficial effect on implantation and early embryo development (see above).

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Figure 20. Ultrasound imaging of a pregnant buffalo at 25 days after mating.

Figure 21. Heartbeat image, obtained by transrectal colour Doppler sonography in a 45 days old buffalo embryo.

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In buffaloes practice, two methods allow us to immediately diagnose pregnant/non pregnant females: palpation per rectum and transrectal ultrasonography. Palpation per rectum as a direct method for pregnancy diagnosis is performed 30 days after breeding/artificial insemination/embryo transfer. However, neither critical studies in regard to its accuracy at earlier stages nor comparing it with transrectal ultrasonography are available. Transrectal ultrasonography for pregnancy diagnosis offers some advantages over palpation per rectum: earlier diagnosis of pregnancy/non-pregnancy, determination of embryo/fetus viability (Figure 21), reduction of misdiagnosis (false negatives and false positives) and reduction of potential iatrogenic embryo/fetal attrition [Romano and Magee, 2001]. The use of new protocols for oestrous synchronization with artificial insemination at fixed time requires an early and accurate method of non-pregnancy diagnosis in order to enrol these animals in a new round of oestrous synchronization. In embryo transfer programs, the accuracy of pregnancy/non pregnancy diagnosis will enable open recipients to be returned or excluded from the recipient herd as soon as possible. Buffaloes reproductive organs are most commonly scanned per rectum using a lineararray transducer specifically manufactured for transrectal use. However, specialized applications including ovum pick-up and follicle ablation involve a transvaginal approach using a sector transducer. Linear-array transducers of 5.0 and 7.5 MHz frequency ranges are most commonly used in buffaloes, and most veterinary ultrasound scanners are compatible with probes of different frequencies. Depth of tissue penetration of sound waves and image resolution is dependent upon and inversely related to the frequency of the transducer. Thus, a 5.0 MHz transducer results in greater tissue penetration and lesser image detail, whereas a 7.5 MHz transducer results in lesser tissue penetration and greater image detail. An ultrasound scanner equipped with a 5.0 MHz transducer is most useful for bovine practitioners conducting routine reproductive examinations, however, small ovarian structures such as developing follicles are best imaged with a 7.5 to 12 MHz transducer. During the procedure the operator removed the faecal material from the rectum, introduced the probe and scanned the uterine horns and ovaries. The transducer and the sleeved arm of the examiner are lubricated with an obstetrical lube to facilitate penetration through the anal sphincter and to establish good contact with the floor of the rectal mucosa. The transducer is positioned dorsal to the genital tract and slowly advanced cranially. The cervix, right uterine horn, the left uterine horn and the uterine body are examined for signs of pregnancy. In buffaloes the genital tract is smaller compared to the bovine and almost entirely lodge within the pelvic cavity due to the less extensibility of the uterine horns. [Vittoria, 1997] The ultrasonographic appearance of the ovaries in the buffaloes show a typical round to oval shape with a fine ecotexture and a discrete ecogenicity. Antral follicles of various sizes appear as nonechogenic structures which could be distinguished from blood vessels in crosssection by the elongated appearance of the latter. Ovarian ultrasonography has shown that the development of buffaloes follicles occurs in a distinct, striking regular pattern; each wave consists of the contemporaneous emergence of a group (cohort) of follicles 5 mm or more in diameter. Within several days, one follicle has grown larger than the cohort and is considered dominant. Ovulation can be depicted by the

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absence of a preovulatory follicle that is present at a previous examination and subsequently confirmed by the development of corpus luteum at the same spot. In buffalo species follicular dynamic during oestrus cycle is characterized, normally, by two waves of follicular growth, although some animals can show three or sometimes one waves [Baruselli et al., 1997a; Neglia et al., 2007]. The first wave begins on day one and the second wave appears around day 10 after ovulation. In the animals that present three waves, the third wave starts around on day 17 after ovulation. Obviously, the interval between two consecutive ovulations, and hence the length of cycle, is related to the number of waves: usually, it is around 22 and 24 days respectively in buffaloes with two or three wave cycles. This affects also the length of the luteal phase that is meanly of 10 and 13 days, respectively in the two different types of oestrus cycle (two and three waves). The corpus luteum is identified ultrasonically from 3 days after ovulation. A developing CL appears on the ultrasound image as a poorly defined, irregular, greyish-echogenic structure with a demarcation line visible between it and the ovarian stroma; sometimes a centrally located nonechogenic area surrounded by greyish echogenic luteal structure is visible. These structures represent a normal variation of CL in buffaloes. Transrectal colour Doppler sonography is being used in recent years to evaluate ovarian perfusion, especially luteal and follicular blood flow in cows. [Acosta et al., 2002, 2003, 2005; Miyamoto et al., 2005, 2006]. In buffaloes we have started to use this technique After morphological evaluation, the flow mode is activated for blood flow mapping. Colour signals is used to generate images in which blood flow is detectable within the follicle wall and developing corpus luteum (Figure 22.).

Figure 22. Luteal blood flow in corpus luteum observed by transrectal colour Doppler sonography.

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Figure 23. Uterine horns in buffalo species.

In a non pregnant, cycling buffaloes the ultrasound image of the uterus shows a distinctly echogenic structure with different layers of the uterine horn reflected by differing echotextures (Figure 23).

Figure 24. Aspect of uterine horns during oestrus in buffalo.

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The echotexture of the endometrium is characterized by the presence of non-specular reflections with dark and bright signals seen within the ultrasound image of the endometrium. The ultrasonographic appearance of the uterus is influenced by the stage of the oestrous cycle. Uterine echotexture is characteristically dark during the follicular phase (oestrus) reflecting an extensive degree of edema of the endometrium (Figure 24). The uterine horns are maximally curled during follicular dominance but are less curled during luteal dominance. In the early stage of gestation uterus is in the pelvic cavity and it is fluid filled with an anechoic fluid material. Although we have seen that an embryonic vesicle is detectable by ultrasound as early as 18 days of gestation, accuracy of detection approaches 100% after day 25 of gestation when an oblong echogenic structure is visible within the gestational sac. For practical purposes, the efficiency (i.e., speed and accuracy) of a correct diagnosis of pregnancy should be performed in females that are at least 25 days post breeding. From day 35 the embryo is organized into head, body, and limbs are detected. Around day 45 increased echogenicity in the area of suspect fetal bone formation is considered as the time of ossification. At this time is possible to measure the following fetometric parameters: crown-rump length (CRL, a straight line between the fetal crown and the origin of tail), diameter of the amniotic vesicle (AVD, the widest diameter), uterine diameter (UTD, maximum intrauterine lumen at the level of the conceptus), biparietal diameter (BPD, the widest distance between the outer borders of the cranium at an angle of 90° to the falx cerebri), chest depth (CHD, a dorso-ventral distance just caudal to the apex of the heart). In particular, the embryonic vesicle, initially spherical in shape and with a mean height of 11.7 ± 1.88 mm is observed on day 19.0 ± 2.16. It becomes elongated on day 26. The embryo proper within the vesicle is detected similarly on day 19.0 ± 1.69, with a mean length of 4.2 ± 0.89 mm, whereas the two fetal membranes, allantois and amnios, are detected the first time on day 30.0 ± 1.14 and 33.4 ± 1.64, respectively. The forelimbs, spinal cord, hindlimbs and optical area are observed on days 34.6 ± 1.34, 35.8 ± 2.52, 36.8 ± 2.34 and 38.2 ± 2.39, respectively [Pawshe et al., 1994]. Before the use of ultrasound for pregnancy diagnosis in buffaloes, practitioners were unable to accurately determine the viability or number of embryos or fetuses. In a series of 1000 ultrasonographies we found an average of 120 bpm, and is considered to be a ultrasonographic sign of foetal wellness. Because the heartbeat of a foetus can be detected at approximately 25 days of age, foetal viability can accurately be assessed. Ultrasound is therefore a valuable tool in diagnosing early embryonic loss. In particular the sonographic appearance is variable in relation to the time of diagnosis. Between the 25 and 35 days of pregnancy the sonographic signs of embryonic loss are evidenced by an increase in ecogenicity of the allontoid fluid, with the embryonic membrane floating in the gestational sac (Figure 25 A) and an absence of heartbeat, with an increase in ecogenicity of the embryo (Figure 25 B). Fetal sex determination has several implications in the animal breeding industry. Sexing of the buffalo conceptus has been described either in specimens collected from slaughterhouses [Abdel-Raouf and El-Naggar, 1970] or by using DNA amplification method [Manna et al., 2003; Hirayama et al., 2006]. The best window for ultrasonographic diagnosis of fetal sex in buffaloes is between the 10th and 18th weeks of gestation. In cattle, the

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optimum time was recorded between the 10th and 13th weeks of pregnancy [Ali, 2004]. In male fetusesl, the genital tubercle (phallus) is hyperechogenic, oval or sometimes round structure, located cranial to the hind limbs and at the abdominal attachment of the umbilical cord. In female fetuses, the genital tubercle (clitoris) was located behind the hind limbs and toward the base of tail [Ali and Fahmy, 2008].

Figure 25. Some sonographic signs of embryonic loss. increased ecogenicity of the allontoid fluid, with the embryonic membrane floating in the gestational sac (A) and absence of heartbeat and increased ecogenicity of the embryo (B).

Conclusion The reproductive problems in the buffalo are caused by several patho-physiological factors that contribute to aggravate seasonality. A good management is the main strategy to attenuate the negative implications of seasonality and to reduce fertility loss. In conclusion, the reproductive seasonality seriously affects the reproductive physiology of the species. Some important modifications of the endocrine pattern occur when buffaloes are bred in areas where mating need to be perform during daylight length. The seasonal anoestrus, the increase of early and late embryonic mortality due to difficulties in placentation, influence the intercalving period. The knowledge of hormonal mechanisms that play a main role in oestrus cycle resumption and fetal-maternal recognition of pregnancy are fundamental in a species in which few data are available and that contributes to produce interesting energy and protein forms, mainly in developing countries.

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during prostaglandin F2α induced luteolysis in the cow. Biology of Reproduction, 1998 58, 103–108. Paria, BC; Huet-Hudson, Y; Dey, SK. Blastocyst’s state of activity determines the “window” of implantation in the receptive mouse uterus. Proceedings of the National Academy of Sciences of USA, 1993 90, 10159-10162. Parmeggiani, A; Di Palo, R. Melatonina e stagionalità riproduttiva della bufala. Agricoltura e Ricerca, 1994 153, 41-48. Parr, EL; Parr, MB. Uptake of immunoglobulins and other proteins from serum into epithelial cells of the mouse uterus and oviduct. Journal of Reproductive Immunology, 1986 9, 330-354. Patel, OV; Sulon, J; Beckers, JF; Takahashi, T; Hirako, M; Sasaki, N; Domeki, I. Plasma bovine pregnancy-associated glycoprotein concentrations throughout gestation in relationship to fetal number in the cow. European Journal of Endocrinology, 1997 137, 423-428. Pawshe, CH; Appa Rao, KBC; Totey, SM. Ultrasonographic imaging to monitor early pregnancy and embryonic development in the buffalo [Bubalus bubalis]. Theriogenology, 1994 41, 697-709. Pelagalli, GV; Mastronardi, M; Potena, A. La circolazione placentare in alcuni ruminanti. Acta Medica Veterinaria, 1973 IX (I/II), 3-31. Pereira, FTV; Miglino, MA; Bevilacqua, E; de Carvalho, AF. Morphological aspects of placental haematomes of water buffalo placenta (Bubalus bubalis bubalis ¾ Linnaeus, 1758). Brazilian Journal of Veterinary Research and Animal Science, 2001 38, 151-154. Perenyi, Z; Szenci, O; Sulon, J; Drion, PV; Beckers, JF. Comparison of the ability of three radioimmunoassay to detect pregnancy-associated glycoproteins in bovine plasma. Reproduction in Domestic Animals, 2002 37, 100-104. Perera, BMAO; Abeygunawardena, H; Thamotharam, A; Kindahl, H; Edqvist, LE. Peripheral changes of estrone, progesterone and prostaglandin in the water buffalo. Theriogenology, 1981 15, 463–467. Perozo Marin, F; Goicochea, M; Montiel, J. Niveles de progesterona durante la gestacion de bufalas (Bubalus bubalis). Revista de la Facultad de Agronomía, 2002 19, 71-76. Perucatti, A; Floriot, S; Di Meo, GP; Soglia, D; Rullo, R; Maione, S; Incarnato, D; Eggen, A; Sacchi, P; Rasero, R; Iannuzzi, L. Comparative FISH mapping of mucin 1, transmembrane (MUC1) among cattle, river buffalo, sheep and goat chromosomes: Comparison between bovine chromosome 3 and human chromosome 1. Cytogenetic and Genome Research, 2006 112, 103-105. Pfarrer, C; Ebert, B; Miglino, MA; Klisch, K; Leiser, R. The three-dimensional feto-maternal vascular interrelationship during early bovine placental development: a scanning electron microscopical study. Journal of Anatomy, 2001 198, 691–702. Plante, C; Hansen, PJ; Martinod, S; Siegenthaler, B; Thatcher, WW; Pollard, JW; Leslie, MV. Effect of intrauterine and intramuscular administration of recombinant bovine interferon- alpha 1 on luteal lifespan in cattle. Journal of Dairy Science, 1989 72, 18591865.

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Reviewed by: Prof. Pietro Sampaio Baruselli – Departamento de Reprodução Animal, FMVZ, University of São Paulo, Brazil.

In: Pregnancy Protein Research Editor: Marie O’Leary and John Arnett

ISBN 978-1-60692-396-2 © 2009 Nova Science Publishers, Inc.

Chapter III

The Dialogue between Blastocyst hCG and Endometrial hCG/LH Receptor: Impact in Implantation and Placentation

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S. Perrier d’Hauterive1,2, M. Tsampalas1, S. Berndt3, C. Munaut3, J. M.Foidart2,3 and V. Geenen1

University of Liège, Center of Immunology (CIL), Institute of Pathology CHU-B23, 4000 Liège, Belgium 2 University of Liège, Department of Gynecology and Obstetrics, Center of Medical Assisted Procreation (CPMA), CHR de la Citadelle, 4000 Liège, Belgium 3 University of Liège, Laboratory of Tumor and Development Biology, Center of Experimental Cancer Research (CRCE), CHU-B23, 4000 Liège, Belgium

Abstract The success of implantation depends on a receptive endometrium, a functionally normal blastocyst, and a synchronized cross-talk between embryonic and maternal tissues. In addition to the hormonal control, a cascade of cytokines intervenes in the dialogue at the maternal-embryonic interface, which is a crucial step at the crossroad between immunology and endocrinology. This chapter relates to the very first phases of human embryo implantation, starting from the entry of the blastocyst in the uterine cavity, its arrival in the vicinity of the maternal endometrium, and the dialogue it engages with the latter according to adhesion and paracrine modes. Among the abundant and redundant mediators of the maternal-fetal cross-talk, we will focus on the specific and very early embryonic signal: human chorionic gonadotropin hormone (hCG). Data from literature show that through this signal, the embryo profoundly intervenes in its own implantation and favors immunological tolerance and active angiogenesis which are crucial prerequisites to successful implantation and placentation.

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S. Perrier d’Hauterive, M. Tsampalas, S. Berndt et al. From our previous studies, it appears that in vitro, dose-dependent hCG enhances the endometrial production of pro-implantatory leukemia inhibitory factor (LIF), proangiogenic VEGF, and reduces pro-inflammatory interleukine (IL) -6 secretion. This positive action is mediated by hCG binding to its cognate receptor (LH/hCG-R), which we have evidenced on endometrial epithelial cells. From our preliminary results, this epithelial expression on the endometrium varies along the menstrual cycle, with a higher expression at the time of implantation. It remains to be determined whether the embryo itself modifies the endometrial LH/hCG-R expression. Successful embryo development also requires an extensive endometrial angiogenesis in the proximity of the implantation site. In our work, we have also demonstrated an angiogenic effect of hCG in several in vivo (chick chorio-allantoïc membrane, matrigel plug assay, aortic ring assay) and in vitro experimental models. LH/hCG-R was detected in endothelial cells by RT-PCR and by Western blotting. In mice aortic ring assay, angiostimulation by hCG was abrogated by deletion of LH/hCG-R (LuRKO mice). Use of recombinant hCG and anti-hCG antibody further confirmed the specificity of this angiogenic activity. By using dibutyryl cAMP, adenylate cyclase or protein kinase A inhibitors, we have demonstrated that hCG-mediated angiogenesis involves adenylylcyclase–protein kinase A activation. Altogether, these data demonstrate that peritrophoblastic angiostimulation may result from a paracrine dialogue between trophoblast, epithelial and endothelial cells through hCG and VEGF. This chapter supports that, through a specific dialogue between hCG and LH/hCGR, human blastocyst actively intervenes in implantation and placentation processes. The evaluation of the uterine receptivity, the impact of the embryo at the time of implantation and the interconnections between mother and embryo through this hCG/LH-R signaling remain further challenges for clinical practice.

Introduction Implantation of the embryo into the maternal endometrium represents a crucial step in the reproductive process—naturally or medically assisted. How the embryo apposes and adheres to the maternal endometrium while dialoguing with it and the mode by which the trophoblast invades the maternal decidua—without destroying it while outmanoeuvring maternal immunological traps—remain in 2009 among the mysteries of human reproduction. Successful pregnancy requires two actors: the receptive endometrium and the functionally normal embryo, both cross-talking in the juxtacrine/paracrine style at the maternal-fetal interface. The knowledge of the early embryo’s development has broadly benefited from in vitro fertilization and embryo culture techniques: morphological criteria used to select the best choice of embryo (the ‘top embryo’), although imperfect, are more and more precise. At the maternal level, the steroid hormones estrogen and progesterone play a critical role in preparing the endometrium for the narrow time frame for receptivity—called the implantation window—while the network of redundant and interconnected molecules are the private paracrine mediators of the dialogue at the maternal-embryonic interface [1-3]. Even if indisputable progress was made these last years in assisted medical procreation (AMP), embryo implantation as the black box of the reproduction and implantation failure remains an important obstacle to the success of AMP.

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Embryo implantation is a three-step process that implies apposition, adhesion and total invasion of the conceptus into the uterine wall together with the elaboration of a vascular network helping the beneficial supply of the embryo and then the fetus and finally with the protection of the fetal allograft from the competent maternal immune system. Deciphering the complex and fine-tuned network leading to the successful collaboration between embryo and maternal tissues, at the crossroad between endocrinology and immunology, is the challenge of the actual research. The relative contribution of each one of these factors to the success of implantation is hardly determinable [4]. Slight errors in secretion or in action may seriously impair the whole reproductive process, while compensatory mechanisms may prevent the failure [5]. This chapter relates to these very first phases of human embryo implantation, starting from the entry of the blastocyst in the uterine cavity, its arrival in the vicinity of the maternal endometrium, and the dialogue it engages with the latter according to adhesion and paracrine modes. Among the abundant and redundant mediators of the maternal-fetal cross-talk, we will focus on the specific and very early embryonic signal: human chorionic gonadotropin hormone (hCG). Indeed, the only marker of a successful implantation is the implantation itself, associated with the appearance of hCG in the maternal bloodstream. HCG is the prime mediator by which the embryo announces its presence to the maternal organism, since it produces it even before its own implantation. We will review evidence for an important role of this hormone during implantation, already demonstrated in the literature. Finally, we will describe personal data, since our team is particularly interested in the role of the hCG-LH/hCGR dialogue between embryo and endometrium and its impact during the first steps of implantation and angiogenesis.

Steps of Implantation For obvious reasons, there are few observations researching the first days of embryo implantation in humans. The crucial step of implantation in humans is unreachable for researchers or physicians. Some insights could be carefully taken from different animal species but cellular interactions that take place during embryo implantation are different from one species to another. Some important steps are yet in animals that could probably be transposed to humans. Nevertheless, the output of human reproduction is remarkably low, since it is estimated that successful implantation occurs only in 30% of cases. Implantation failures are a factor in 25% of early miscarriages without any specific solution for diagnosis or treatment. Embryo implantation is a three-step process, composed of apposition, adhesion and invasion (Figure 1).

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Figure 1. The different steps of implantation and the mediators that participate to the maternal-fetal dialogue.

Apposition This step is not stable and is not characterized by physical contact between embryo and endometrium. At this stage, the blastocyst enters in the vicinity of the endometrium and lets embryonic soluble mediators such as LIF, growth factors or hCG cross-talk with the endometrium. This stage is the time for a unique network of numerous adhesion molecules to be expressed on the trophoblast and on endometrial epithelium.

Adhesion At this time, the blastocyst is composed of a 5-cell inner cell mass and surrounded by a 53-cell trophoblast hull, the trophectoderm, which is designated to differentiate into the placenta. This step implies physical connection between trophectoderm and endometrial epithelium through the adherence of their apical plasma membranes. Normally, epithelial cells do not allow adhesion of other cells to their apical surface making trophoblast-uterine epithelial adhesion a unique circumstance [6, 7]. Through its contact with endometrial epithelium, blastocyst induces a massive endometrial apoptosis reaction that allows embryo to invade endometrium. Moreover, embryo and endometrium strongly reciprocally influence gene expression from each other. This notion strengths the concept of dialogue between the two actors and clearly demonstrates the difficulty to elucidate the implantation network in

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vitro where such a collaboration is limited or early aborted [4, 8]. Adhesion phase induces the modification of underlining stromal compartment of endometrium into decidua, initiating the development of the maternal part of the placenta. Unlike other animal species, decidualization in human is embryo independent but is amplified during pregnancy. This reaction probably controls invasiveness of trophoblast through the alteration of matrix metalloproteases (MMP), cytokines, integrins and MHC expression. Trophoblast in counterpart also produces signals that modulate gene expression of decidua [9]. Invasiveness of conceptus depends on the efficiency of this decidual reaction: if it is pathological, placentation could be too important (placenta accreta) or too slight (intra uterine growth restriction). The impact of hCG which is the earliest hormone produced specifically by the embryo during the apposition and adhesion phase is poorly explored.

Trophoblast Invasion The differentiation of the trophectoderm into two separate trophoblast subsets takes place immediately prior to invasion into the decidual matrix [10]. Trophoblast cells proliferate, migrate and invade endometrium and its vessels in a manner that mimic tumor. But unlike tumoral process, implantation process is fine controlled and limited to the placental bed. During migration and invasion, trophoblast cells undergo dramatic differentiation, control and are controlled by different cytokines, growth hormones and MMP and tissue inhibitors of the MMP (TIMP). A successful invasion required a well orchestrated network of proliferation, angiogenesis, cell differentiation, immune tolerance under hormonal, cytokine, immune cells control. The specific implication of hCG during this complex phenomenon is currently studied [11, 12] but it is presumed that, through its production of hCG, the embryo will promote its invasion, to the detriment of maternal defence such as decidual reaction.

Uterine Receptivity Implantation of the embryo is a restricted phenomenon only allowed in the uterus during a short period called the implantation window [13]. Before or after this period, endometrium is not receptive or refractory to embryo implantation. Although implantation is a dynamic and reciprocal process between embryo and the mother, the preparation of endometrial tissue for the receptive phase is strictly maternal. The study of the human implantation window is a difficult challenge burdened with a serial of redundant factors co-existing at the maternalfetal interface. Several factors such as pinopods, integrins, mucins, heparin binding epidermal growth factor or steroids receptors have been evidenced and extensively studied, but no unequivocal one can be highlighted. For a prognostic and clinical value in AMP, a good receptivity marker must be evidenced with a simple and no traumatic method, in the cycle during which an embryo could implant – if possible. Moreover, it has to attest of uterine receptivity cycle after cycle. Actually, the only specific but retro-active witness of uterine receptivity is the implantation itself and the production of hCG. The receptor to the embryo

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specific hCG is expressed in human endometrium but its implication as endometrial receptivity marker is not yet evidenced.

Human Chorionic Gonadotropin (hCG) HCG belongs to the glycoprotein hormones family, such as LH, FSH and TSH. These hormones are composed of two subunits linked in a non-covalent way. The alpha subunit is common to the whole members of the family and is coded on the chromosome 6. The beta subunit, different and specific for each hormone, is coded by distinct genes on the chromosome 19 (LH, hCG and TSH) or on the chromosome 11 (FSH). HCG beta subunit is encoded by four genes (CGB, CGB5, CGB7 and CGB8) that reside in a common genome cluster with the evolutionarily ancestral LHβ and two recently duplicated gene CGB1 and CGB2 at chromosome 19q13.3 [14, 15]. The beta hCG is the biggest beta subunit because of a larger glycosylated part. This important glycosylation confers to hCG a higher stability and makes its secretion faster. The beta subunits of LH and hCG have 96% of identity that allows to these hormones to share the same receptor. But hCG binds to this receptor with an affinity 4,5 fold higher compared to LH. Unlike the other glycoproteins that are synthesized in the anterior lobe of pituitary gland, hCG is principally produced by trophoblast (and particularly syncytium), but also by some malignant tumors. In addition to its endocrine function of delaying the apoptosis of the corpus luteum during the first trimester of pregnancy, hCG has several paracrine effects essential in process of implantation, angiogenesis and development of maternal immune tolerance, as review below.

The LH/hCG Receptor (LH/hCG-R) The LH/hCG-R is one of eight members in a cluster of the rhodopsin family of the large G-protein coupled receptor superfamily that contains some 800–900 genes in the human genome [16]. LH/hCG-R form one of the three classes in this cluster and is coded on the chromosome 2. This unique gene is composed of 10 introns and 11 exons, and span approximately 80kb. Its cDNA codes a glycoprotein of 675 amino acids. This receptor associates two functional units: a large ectodomain containing leucine-rich-repeats which serves in the recognition and the specific binding of hCG (or LH) coupled with seven transmembrane domains and an intracellular segment binding to G protein. This segment allows the signal transduction generated by the hormonal binding to the extracellular domain [17, 18]. This G-protein coupled receptor activates mainly the cAMP/PKA pathway [19]. HCG and LH activities are mediated by the same receptor (LH/hCG-R). The expression of this receptor was previously thought to be restricted to gonadal tissues. Certainly, the main physiological roles of the LH/hCG-R can be attributed to its actions in the ovaries and the testis. However, recent studies have shown its presence in many other tissues throughout the reproductive organs as well as in neural retina [20], adult rat spinal cord [21] and human skin [22]. Inactivating mutations in the LH/hCG-R are described in human presenting clinically as hypergonadotrophic hypogonadism and infertility/subfertility in both sexes. These conditions

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are extremely rare. Inactivation of LH/hCG-R results in 46, XY individuals in a disturbance in male-type sexual differentiation that ranges from male pseudohermaphroditism to mild forms such as cryptorchidism and hypospadias. The phenotype of genetic females is milder, presenting mainly as anovulatory amenorrhoea and hypo-oestrogenization [23]. More recently, Bruysters et al. described a new LH/hCG-R splice mutation responsible for a infertility with regular cycles in women suggesting that LH/hCG-R mutations may represent an underestimated cause of infertility in women [24].

hCG- LH/hCGR System: An Intimate Embryo-Endometrial Dialogue Since hCG is the specific and very early embryonic signal, it appeared to us to be a ideal mediator of the embryonic-endometrial dialogue. The specific interaction of blastocystderived hCG and endometrial LH/hCG-R constitutes probably a fundamental component in implantation cascade network. The research of our team focuses on the very early phases of human embryo implantation, starting from the entry of the blastocyst in the uterine cavity, its arrival in the vicinity of the maternal endometrium, and the dialogue it engages with this latter one according to a paracrine mode. From our previous studies held at the Center of Immunology and the Laboratory of Tumor and Development Biology, it appears that the embryo itself, through its insulin-like growth factor (IGF) 2 and TGF-beta production but also and especially through its specific and very early hCG production, positively influences its implantation. In vitro, these molecules dose-dependent enhance the endometrial production of Leukemia inhibitory factor (LIF), a cytokine crucial to mice implantation. In the same experimental setting, TGF-beta and hCG inhibited the secretion of pro-inflammatory interleukin (IL) -6 by endometrial epithelium, a cytokine implicated in the immune inflammatory response [25, 26]. The positive action of hCG on embryo implantation is mediated by hCG binding to its cognate LH/hCG-R that we have evidenced on endometrial epithelium by RT-PCR. LH/hCGR mRNA was also detected in stromal and epithelial cells isolated from endometrial biopsies from fertile women. Using real-time PCR, we have quantified LH/hCG-R mRNA expression in endometrial epithelial cells isolated from biopsies during proliferative phase and secretory phase. Our preliminary results show a basal expression which increases from early proliferative phase (d3-d10) to late proliferative phase (d11-d14) and which decreases after ovulation (early secretory phase, d15-d19). From the so called ‘implantation window’ to the end of the menstrual cycle (d20-d28), LH/hCG-R transcripts re-increase (unpublished data). However, the basal expression of the receptor is weak. This kinetic of expression of the LHR mRNA suggests that this receptor could constitute a marker of endometrial receptivity, able to answer to the low hCG production by pre-implantatory embryo. The correlation between the level of expression of the LHR mRNA, protein secretion and the uterine receptivity must however still be strengthened by other experiments. It remains also to determine whether the embryo itself modifies the endometrial LH/hCG-R expression. We have also shown that fluorescein-labelled recombinant hCG was able to bind to endometrial epithelial cells. This endometrial LHR is functional since in vitro, hCG stimulates the production of LIF. HCG

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also inhibits IL-6 production, an immunoactivatory cytokine. Both LIF stimulation and IL-6 inhibition effects were inhibited by an anti-hCG antibody and were not mediated by local IL1 beta production. Only the dimeric form of the hCG fulfills these biological functions since the alpha and beta subunits separately did not induce any modulation of the endometrial cytokine production [25]. Successful embryo development also requires an extensive endometrial angiogenesis in proximity of the implantation site. In our work, we have also demonstrated an angiogenic effect of hCG in several in vivo (Chick chorio-allantoïc membrane, matrigel plug assay, aortic ring assay) and in vitro experimental models since strongly increases VEGF production by human endometrial epithelium in vitro. LH/hCG-R was detected in endothelial cells by RT-PCR and by Western blotting. In mice aortic ring assay, angiostimulation by hCG was abrogated by deletion of LH/hCG-R (LuRKO mice). Use of recombinant hCG and anti-hCG antibody further confirmed the specificity of this angiogenic activity. By using dibutyryl cAMP, adenylate cyclase or protein kinase A inhibitors, we have demonstrated that hCG-mediated angiogenesis involves adenylyl-cyclase – protein kinase A activation. Altogether, our data demonstrate that peritrophoblastic angiostimulation may result from a paracrine dialogue between trophoblast, epithelial and endothelial cells through hCG and VEGF [27]. These studies support that, through a specific dialogue between hCG and LH/hCG-R, human blastocyst actively intervenes in implantation and placentation processes (figure 2). The evaluation of the uterine receptivity, the impact of the embryo at the time of implantation and the interconnections between mother and embryo remain further challenges for clinical practice.

Figure 2. Impact of hCG on the endometrium and endothelium at the maternal-fetal interface.

The Dialogue between Blastocyst hCG and Endometrial hCG/LH Receptor… 101 Other studies describe that hCG orchestrates and fine tunes the implantation process with an impact at the different steps of the cascade at the endocrine and paracrine/local level [2830]: initiation of pregnancy, angiogenesis and immune tolerance.

The hCG – LH/hCGR System and Initiation of Pregnancy: Pro-Implantatory Roles HCG is one of the most specific and precocious molecules produced by the embryo. Indeed, ARNm of the hCG is transcribed as soon as the eight-cell stage [31]. At the blastocyst stage, transcripts of hCG are detected in the trophoblast and hCG production by the blastocyst begins even before its implantation, at low concentration. HCG production rapidly increases and significant rates of hCG can already be measured in maternal blood 10 days after ovulation. The peak of hCG production by the placenta is reached between 10th and the 11th week of gestation, then the synthesis declines as of the 12th week to remain at low rates during all pregnancy. From current studies, hCG positively influences the implantation not only by its luteotrophic role rescuing corpus luteum from apoptosis, but also via a local action at the materno-fetal interface, through specific interaction with LH/hCG-R. Several studies indicate that hCG signals also directly to endometrium [32] and modulates the stromal and epithelial cell activities, already prior to and in preparation for blastocyst implantation (for a review, see [28]). For example, injection of hCG reduces apoptosis in human endometrium [33] and a local infusion of hCG to non-pregnant baboons functionally alters the major cell types present in the baboon uterine endometrium, in a pattern like the one observed after implantation [32, 34, 35]. In the same baboon model, hCG induces alterations in the endometrial expression of genes that regulate embryo attachment, extracellular matrix remodelling and the modulation of the immune response around the implanting blastocyst [36]. During apposition and adhesion phase, trophinin mediates cell adhesion by homophilic binding at the apical surface of trophectoderm and epithelium. Sugihara et al. observed in presence of hCG together with IL-1 beta, endometrial epithelial cells acquired strong trophinin expression and the ability for apical cell adhesion with trophinin-expressing human trophoblast [37]. Later in the process of adhesion and invasion, administration of hCG by microdialysis in human uterus causes important paracrine effects on decidualization – suggesting that the embryo increases the duration of the implantation window– on tissue remodelling (increase of MMP-9), on implantation (increase of LIF, M-CSF), and on angiogenesis (increase of VEGF). Moreover, hCG as one of first hormonal signal of the embryo selectively reduces IGF-I and IGF-BP 1 implicated in decidual reaction and may therefore contribute to the modulation of endometrial receptivity and differentiation during early implantation [38, 39]. It is also shown that hCG has a regulatory role on the MMP/TIMP system at the implantation site. By increasing trophoblastic MMP 9 secretion and by reducing endometrial TIMP 1, 2, 3 expressions, hCG may be an important tool for the invading embryo to regulate the depth of its implantation [40]. The treatment of endometrial epithelial and stromal cells with hCG increases expression of COX2, via the AMPc/protein kinase A signalling system [41]. The COX-2 enzyme, which

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catalyses formation of prostaglandins (PGE2 in particular which supports the differentiation of the stroma cells in decidua), is absolutely essential for the implantation. Targeted inactivation of Cox2 in mouse leads to severe abnormalities of the reproduction like a failure of implantation [42]. Finally, by reducing gap-junction between smooth muscle cells and intracellular calcium, and by increasing progesterone receptor expression, hCG induces a reduction of smooth muscle cells contractility that allows the implantation of the blastocyst [43].

hCG and Angiogenesis Angiogenesis is a fundamental process by which new capillary blood vessels from preexisting ones are regulated by vascular endothelial-specific growth factors and inhibitors. The female reproductive system undergoes a number of physiological angiogenesis during the menstrual cycle, folliculogenesis, ovulation and corpus luteum formation and implantation, in particular during placenta formation [44]. Among angiogenic factors, VEGF is viewed as a prime regulatory factor of blood vessel growth during angiogenesis since it is a highly specific mitogen for endothelial cells and it induces angiogenesis and increases permeability of blood vessels [45]. Successful implantation, placentation and subsequent gestation require coordinated vascular development and adaptations on both sides of the maternal-fetal interface [46]. During the pregnancy, dues to high demand for increased blood supply, the vasculature of the uterus and endometrium undergoes three main adaptative changes: vasodilatation, increased permeability and development and maturation of new vessels [47, 48]. Good regulated angiogenic process is crucial for the implantation and the evolution of the whole pregnancy since disturbance in uterine blood supply or vascular remodelling is associated with higher fetal morbidity and mortality due to miscarriages, preeclampsia or intrauterine growth restriction. The physiological changes in uterine vascular remodelling are regulated by growth factors such as VEGF but also by hormonal factors from trophoblast origin. The presence of LH/hCG-R on endothelial cells of the uterine vessels was already described [49]. Toth et al. showed that the in vivo administration of hCG reduces vascular resistance in the human uterus and reduces in vitro the vasoconstrictor eicosanoids of the vascular wall [50]. These results initiated a study in a population of patients presenting signs of miscarriage. These patients were treated with magnesium or progesterone and/or hCG. The results showed that the number of patients who reached the second trimester of pregnancy was higher when hCG was included with the therapeutic protocol, in parallel with a reduction of vascular resistance. Moreover, the number of premature births or intrauterine growth retardation was weaker when hCG had been managed in the first trimester. Zygmunt et al. have recently proposed hCG as new angiogenic factor [51]. Using a system of in vitro angiogenesis in 3D, they showed that hCG is a factor promoting angiogenesis by supporting the migration and the formation of outlines of capillaries by the uterine endothelial cells. Finally, Herr et al. showed that hCG stimulates proliferation of human placental microvascular endothelial cells (HPMVEC) in a dose-dependent manner and stimulated sprout formation when compared to controls in a spheroid angiogenesis assay [52].

The Dialogue between Blastocyst hCG and Endometrial hCG/LH Receptor… 103 Role in the Maternal Tolerance of the Fetal Allograft In parallel with its direct action on the endometrium (epithelium and stroma), hCG also contributes to maternal tolerance of the embryo. This function, witness to the intimate interrelationship between the immune and endocrine systems, is shown by several studies. Kayisli et al. suggested that hCG could be a placental key factor for the development of the local immune tolerance through the cellular system of programmed death Fas/Fas-Ligand [53]. HCG also up-regulates complement C3 and C4 and increases the pool of regulatory T cells during pregnancy [54]. In a more general way, Khan et al. showed that the administration of hCG to nonobese diabetic (NOD) mice before the beginning of the clinical symptoms reduced the increase in glycaemia, reversed establishment of insulitis, and inhibited the development of autoimmune diabetes [55].

Conclusion The black box of the reproductive process remains, in 2009, the implantation cascade. Deciphering the molecular events that characterize the apposition, adhesion and invasion stages of the embryo’s establishment is the maternal tissues that will tolerate but also tightly control the blastocyst invasion: this is the challenge for the future. A fine-tuned dialogue takes place at the maternal-fetal interface, but our knowledge of the language they speak together is still lacking some fundamental key words. The progress made in medicine or research and the technologies that they use make the network every day more complex. HCG is the prime mediator by which the embryo announces its presence to the maternal organism, since it produces the hormone even before its implantation, and the endometrium is able to answer this signal since the expression profile of LH/hCG-R by the endometrial epithelium seems to correlate with the theoretical timing of the implantation window. Among all of the mediators present at the implantation site, the specific interaction of blastocystderived hCG and endometrial LH/hCG-R constitutes a fundamental component that seems to orchestrate, partly at least, the implantation symphony.

Acknowledgments These studies are supported by the National Fund of Scientific Research of Belgium (NFSR), the Léon Frédéricq Fund of Liège University Hospital, the Serono Research Fund and the European Network of Excellence EMBIC (embryonic control of implantation).

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Short Communications

In: Pregnancy Protein Research Editor: Marie O’Leary and John Arnett

ISBN 978-1-60692-396-2 © 2009 Nova Science Publishers, Inc.

Short Communication A

Gamma Interferon Production Correlates Negatively with Plasma Levels of Pregnancy-Associated Glycoprotein-1 (PAG-1) During Gestation in Dairy Cows Naturally Infected with Neospora CANINUM F. López-Gatius∗1, S. Almería2,3, J. L. Yániz3, P. Santolaria3, C. Nogareda1, M. Mezo4, M. Gonzalez-Warleta4, J. A. Castro-Hermidae, N. M. de Sousa5 and J. F. Beckers5 1

2

Department of Animal Production, University of Lleida, Spain Department of Anatomy and Animal Health, and cAnimal Health Research Center (CReSA): Autonomous University of Barcelona, Bellaterra, Spain 3 Department of Animal Production, University of Zaragoza, Huesca, Spain 4 Agrarian Research Center of Mabegondo (CIAM), La Coruña, Spain 5 Physiology of Reproduction, Faculty of Veterinary Medicine, University of Liège, Belgium

Abstract Gamma interferon (IFN-γ) production has been ascribed a role in protecting cows infected with Neospora caninum against abortion. The present study analyzes the interaction between IFN-γ production and levels of plasma pregnancy-associated glycoprotein-1 (PAG-1), as a marker of placental/fetal well-being, throughout gestation in naturally Neospora-infected dairy cows. Data were obtained from 88 pregnant Holstein-Friesian cows in two herds: 62 seropositive and 26 seronegative for the parasite. Blood samples were collected on Days 40, 90, 120, 150, 180 and 210 of gestation. ∗

Phone: 34-973-702-500. Fax: 34-973-238-264. E-mail: [email protected]

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F. López-Gatius, S. Almería, J. L. Yániz et al. Plasma was tested for antibodies against N. caninum, PAG-1 and IFN-γ. Twenty five (28.4%) pregnancies were recorded after AI using Holstein-Friesian semen (19 in seronegative and 6 in seropositive animals), and 63 (71.6%) after AI using Limousin semen (7 in seronegative and 56 in seropositive animals). Gamma interferon was detected in the plasma of 14 (22.6%) of the 62 Neospora-seropositive cows and could not be detected in any of the 26 seronegative animals. All 14 cows producing IFN-γ became pregnant using Limousin semen. Our GLM repeated measures analysis revealed no effects of herd, lactation number, milk production at the time of pregnancy diagnosis and Neospora-seropositivity on plasma PAG-1 concentrations. Significant positive effects were observed of both the day of gestation (P<0.0001) and the interaction between day of gestation and breed of sire (P = 0.001) on PAG-1 values. Cows carrying twins had higher (P = 0.002) PAG-1 concentrations throughout gestation than cows carrying singletons. Interactions between breed of sire and Neospora-seropositivity (P<0.0001), and between IFN-γ production and Neospora-seropositivity (P = 0.04) were also detected. Thus, Neospora-seronegative cows inseminated with Limousin and Neospora-seropositive cows showing no IFN-γ production, exhibited higher PAG-1 concentrations during gestation than seropositive cows inseminated with Limousin semen and producing IFN-γ, respectively. Our data indicate that the production of IFN-γ correlates negatively and the production of antibodies against N. caninum is uncorrelated with plasma PAG-1 concentrations during gestation in Neospora-infected dairy cows. Our results also suggest that augmented Th1 cell-mediated immunity is related to a lower risk of abortion and therefore higher resistance to the parasite in cows inseminated with Limousin versus Holstein-Friesian semen.

Keywords: Pregnancy associated glycoprotein; Gamma interferon; Neospora caninum; Dairy cows.

Introduction In 1953, Medawar proposed the concept of the fetal allograft and suggested that the fetus, an antigenically foreign body, survives as the result of suppressed immunological interaction between mother and fetus [1]. The mechanisms that induce this immunological tolerance of the fetus, however, are not yet fully understood, despite the identification of several features of the immunology of successful pregnancy [2-4]. The most important components of the cellular immune response during pregnancy are T helper (Th) cells, which regulate the immune response through cytokines. Cytokines are generally classified as “inflammatory cytokines” derived from Th1 cells and “pro-gestation cytokines” derived from Th2 cells. These two cytokine types respectively, stimulate cell-mediated immunity and promote the humoral response [5]. Although there is Th1/Th2 balance during the gestation period [6], there is evidence that pregnancy involves some Th2 bias [3], whereas an excessive Th1 response can induce pregnancy loss [7-9]. Thus, most pregnancies are characterized by the predominance of humoral immunity and increased total antibody production [3]. However, the question of how the conceptus avoids maternal immune attack has still not been fully resolved, and the conflict between reproductive and immune systems is even more difficult to comprehend in the presence of a parasite infection, such as bovine neosporosis.

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Neospora caninum is an obligate intracellular protozoan that was first described in the 1980s [10] and has since been identified in a wide range of warm-blooded animals [11]. Bovine neosporosis is of international concern, and has been recognized as a major cause of abortion and congenital infection in cattle worldwide. Abortion or congenital infection occur when parasites cross the placenta and infect the fetus. The incidence of N. caninumassociated abortions peaks in the 5th to 7th month of gestation [12-14]. Losses occur as a result of maternal infection during gestation but more commonly following recrudescence of chronic infection in the pregnant dam, denoted exogenous and endogenous transplacental infection, respectively [15]. Neospora-seropositive cows are more likely to abort than seronegative cows [11]. For example, in our geographical area of study, the risk of abortion is 12-19 times greater in Neospora-seropositive dairy cows than in their seronegative counterparts [16,17]. It is accepted that abortion due to neosporosis can be the outcome of a shift from the beneficial Th2-type response towards an excessive Th1-type of immune response during the gestation period targeted at N. Caninum [18,19]. Th1 type immunity involving proinflammatory cytokines such as gamma interferon (IFN-γ) is critical for the development of host protective immunity in infections caused by intracellular parasites [20]. However, although IFN-γ production is very effective in Neospora-infected non-pregnant animals, it may be involved in the pathogenesis of fetal rejection during gestation [21]. In a recent study, IFN-γ production was, nevertheless, attributed a protective effect against abortion in pregnant dairy cows naturally infected with N. caninum [22]. Based on the odds ratio, the risk of abortion was 15.6 times higher in seropositive cows not producing IFN-γ than in seronegative animals, whereas neosporosis failed to affect seropositive cows producing IFN-γ. The placental antigens, pregnancy-associated glycoproteins (PAGs), are expressed in the mononucleate and binucleate cells of the trophectoderm, from the time the placenta attaches to parturition [23,24]. Ruminant PAGs are classified as two main groups: one of ancient origin (PAG-2 subgroup), largely localized at the placental fetomaternal interface, and one produced by a more recent series of gene duplications (PAG-1 subgroup), expressed primarily in trophoblastic binucleate cells [23,24]. In cows, pregnancy-associated glycoproteins in the PAG-1 subgroup are released into the maternal circulation soon after implantation (i.e., approximately day 25), and concentrations rise until parturition. Plasma PAG-1 levels have been used for pregnancy diagnosis and as a marker of placental/fetal wellbeing [25-27]. In a recent study [27], we could not correlate the antibody response against N. caninum with plasma PAG-1 concentrations in non-aborting chronically infected cows. The purpose of the present study was to investigate the possible relationship between IFN-γ production and plasma PAG-1 concentrations in non-aborting, chronically Neospora-infected dairy cows.

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Materials and Methods Cattle and Herd Management The study population was comprised of the 88 lactating dairy cows, 62 Neosporaseropositive and 26 seronegative, delivering live calves included in the previous study mentioned above [22]. All 62 Neospora-seropositive cows showed antibodies against N. caninum throughout the gestation period. Seronegative cows were chronologically included in the study so that for every two Neospora-seropositive cows, one seronegative partner that became pregnant during the same period was added as a control. The seropositive cows were known to be Neospora-seropositive for at least one year before they became pregnant. The study was performed on two commercial Holstein-Friesian dairy herds of 160 and 570 mature animals in northeast Spain. The herds had previously confirmed cases of N. caninum infection in aborted fetuses and were free of dogs. Cows that became pregnant from March 2003 to March 2006 were included in the study. The cows, reared within the herds, calved all year round and were milked three times per day. Mean annual milk production was 10, 900 kg per cow. All animals were tuberculosis and brucellosis free, as shown by yearly tests from 1985 to 2006. Coinciding with these tests, yearly checks for neosporosis had also been undertaken from 2002 to 2006. The mean Neospora seroprevalence for both herds was 25% during the study period. Vaccination programs were undertaken for the prevention of bovine viral diarrhea (BVD) and infectious bovine rhinotracheitis (IBR). Modified live vaccines were used for animals 6 to 8 months old. Pregnant animals were given killed vaccines in the 7th month of each gestation period. Parous cows that were not pregnant on Day 150 post partum received a further killed vaccine. All animals were bred by artificial insemination using semen from five Limousin or 16 Holstein Friesian bulls of proven fertility. All these cows had been inseminated after 80 days in milk to avoid interference with PAG-1 present in the peripheral circulation after the postpartum period. The study population only included animals receiving their last vaccine four months or longer before the first blood collection. Only healthy cows free from clinical diseases during the study period (Day 40 of gestation to parturition) were included. Exclusion criteria were: mastitis, lameness and digestive disorders. Efforts were made to reduce variation in the general status of the animals so that plasma measurement changes could be attributed to factors other than the clinical condition of the cows during the study.

Pregnancy Diagnosis and Blood Sample Collection Pregnancy was diagnosed by transrectal ultrasonography on Day 40 post-insemination. The outcome of pregnancy was recorded for all animals. Blood samples were collected from each animal into heparinized vacuum tubes (BD VacutainerTM, Becton, Dickenson and Company, Plymouth, UK) on Days 40, 90, 120, 150, 180 and 210 post-insemination. These blood samples were centrifuged (10 min, 1600 x g) and the plasma stored in three tubes at – 20ºC until analysis.

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Plasma Antibodies against N. Caninum Samples were tested for antibodies against N. caninum using a commercial enzymelinked immunosorbent assay (ELISA) kit (CIVTEST® anti-Neospora; Hipra, Girona, Spain), based on the whole tachyzoite lysate of N. caninum NC-1. This test has been previously validated [16] and was performed according to the manufacturer’s instructions. A value of 6.0 or more absorbance units was taken as the threshold for seropositivity. Duplicate determinations were performed on each sample.

Plasma IFN-γ Duplicate plasma samples were tested for IFN-γ using an ELISA for bovine IFN-γ (Bovine IFN-γ EASIA, Biosource, Nivelles, Belgium). This test has been previously validated [22] and was performed according to the manufacturer’s recommendations. The assay was calibrated using standards of recombinant bovine IFN-γ (rboIFN-γ; Serotec, Oxford, UK) prepared from doubling dilutions of a known quantity of reagent. Mean optical density (OD) values were plotted against the units/ml of rboIFN-γ. A regression line was constructed (R2 = 0.97) and the quantity of IFN-γ present in each test sample was determined from the standard curve of 26.9-4370 pg/ml. The sensitivity of the assay was 20 pg/ml rboIFN-γ and results were expressed in pg/ml. PAG-1 Radioimmunoassay Pregnancy-associated glycoprotein-1 concentrations in the plasma samples were determined using a double antibody radioimmunoassay procedure (RIA-706) [28-30]. Rabbit polyclonal antiserum AS#706 was raised against caprine PAG55kDa+62kDa (accession numbers P80935 and P80933) according to the method of Vaitukaitis [31]. The minimum detection limit (MDL) of the assay was 0.26 ng/ml. Intra-assay and inter-assay CV were 3.08 (3.43 ± 0.11 ng/ml) and 10.25% (3.29 ± 0.34 ng/ml), respectively.

Statistical Analyses The effects of herd, semen providing breed (Holstein-Friesian versus Limousin), twin pregnancy, lactation number, milk production at pregnancy diagnosis, Neosporaseropositivity, IFN-γ production at some point during gestation, day of gestation (40, 90, 120, 150, 180, 210), and possible interactions of paired factors on PAG-1 concentrations were evaluated by GLM repeated measures analysis of variance using the SPSS computer package, version 14.0 (SPSS Inc., Chicago, IL, USA). Values are expressed as the mean ± standard deviation (S.D.).

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Results Herds 1 and 2 provided 21 and 67 cows, respectively. The mean lactation number (mean ± S.D.) and milk production at pregnancy diagnosis were 2.4 ± 1.4 lactations (ranges 1-7 lactations) and 39.8 ± 8.1 kg (ranges 28-62 kg), respectively. Twenty five (28.4%) pregnancies were recorded in response to AI using Holstein-Friesian semen (19 in seronegative and 6 in seropositive animals), and 63 (71.6%) to AI using Limousin semen (7 in seronegative and 56 in seropositive animals). Twin pregnancies were recorded in 8 animals (9.1%). No triplets were registered. Gamma interferon was detected in the plasma of 14 (22.6%) of the 62 Neosporaseropositive cows and could not be detected in any of the 26 seronegative animals. All 14 cows producing IFN-γ became pregnant using Limousin semen. GLM repeated measures analysis revealed no effects of herd, lactation number, milk production at pregnancy diagnosis and Neospora-seropositivity on plasma PAG-1 concentrations. Table 1 shows the variables included in the final model for factors affecting PAG-1 values. Significant positive effects were observed of both the day of gestation and the interaction between day of gestation and breed of sire on PAG-1 values. Cows carrying twins had higher PAG-1 concentrations (mean ± SEM) throughout gestation (4.3 ± 2.8 ng/ml on Day 40 to 139.6 ± 7.4 ng/ml on Day 210 of gestation) than cows carrying singletons (3.4 ± 0.3 ng/ml on Day 40 to 91.3 ± 8 ng/ml on Day 210 of gestation). Interactions between breed of sire and Neospora-seropositivity, and between IFN-γ production and Neospora-seropositivity were also detected. Thus, Neospora-seronegative cows inseminated with Limousin and Neospora-seropositive cows showing no IFN-γ production, displayed higher PAG-1 concentrations throughout gestation than seropositive cows inseminated with Limousin semen producing IFN-γ, respectively (Figure 1-A,B). Because of the interaction between day of gestation and breed of sire, differences between plasma PAG concentrations for cows impregnated by Limousin versus Holstein-Friesian semen increased significantly as gestation progressed (Figure 1-A). Table 1. Main GLM repeated measurement analysis model for factors affecting pregnancy-associated glycoprotein-1 (PAG-1) concentrations during gestation Subject effects

Factor

d.f

F

P

Within

Days of pregnancy

5

73.2

<0.0001

Days of pregnancy x breed of sire Breed of sire x Neosporaseropositivity IFN-γ production x Neospora-seropositivity Twins

5

8.96

0.001

2

9.86

<0.0001

1

4.09

0.04

1

9.91

0.002

Between

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Discussion This study was designed to investigate the possible effect of IFN-γ production on plasma PAG-1 concentrations throughout gestation in dairy cows naturally infected with N. caninum. Our main findings were: 1) plasma PAG-1 concentrations are lower in cows that produce IFN-γ than in cows not producing IFN-γ; 2) the humoral response against N. caninum does not affect plasma PAG-1 concentrations; and 3) the use of Limousin semen rather than Holstein-Friesian seems to promote Th1 cell-mediated immunity and thus confers resistance to the parasite during pregnancy. Reinforcing the results of other studies, PAG profiles could be positively correlated with the stage of gestation and fetal number [23].

A

PAG-1 values (ng/ml)

Friesian N(-) Friesian N(+)

260 240 220 200 180 160 140 120 100 80 60 40 20 0

Limousin N(-) Limousin N(+)

40

90

120

150

180

210

180

210

Days of gestation

PAG-1 values (ng/ml)

B 120

IFN-γ (-)

100

IFN-γ (+)

80 60 40 20 0 -20

40

90

120

150

Days of gestation

Figure 1. Mean plasma concentrations (±S.E.M.) of pregnancy-associated glycoprotein-1 (PAG-1) throughout gestation recorded in Neospora-seropositive and seronegative cows impregnated with Friesian or Limousin semen (A), and in Neospora-seropositive cows producing IFN-γ (B).

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Maternal-fetal immune interactions allow two genetically different individuals to survive in close association during gestation. Given PAGs have been attributed immunosuppressive properties [24], these glycoproteins could participate in the placental mechanisms needed to avoid maternal rejection. Peripartum inhibition of polymorphonuclear leukocyte function, for example, has been related to the plasma PAG-1 peak that immediately precedes parturition [32]. However, the question of how PAGs immunomodulate gestation is not yet clearly understood. A major route of N. caninum transmission is the congenital route. Thus, we could speculate that the transplacental migration of tachyzoites would injure the placenta resulting in both IFN-γ production and reduced PAG-1 secretion. This could explain the lower plasma PAG-1 concentrations observed here in cows producing IFN-γ than in the non IFN-γproducing cows. It therefore seems that the presence of IFN-γ in plasma is linked to the inflammatory processes in the placenta, although in the present study this response was insufficient to cause abortion. On the other hand, endogenous transplacental infection at midgestation is usually associated with an acute rise in maternal antibodies [33] and the humoral response against N. caninum seems not to affect PAG production. Although all the Neosporainfected cows included herein were seropositive throughout gestation, seropositivity did not affect PAG-1 levels, in agreement with the results of a previous study [27]. Further, placental lesions due to crossing of the placental barrier by tachyzoites have been noted to regenerate in experimental infections [34]. These results suggest that plasma PAG-1 concentrations decrease following a cell-mediated immune response, although they remain within their patterns besides humoral immunity. Our study does not identify the nature of IFN-γ production, but does prompt several questions. The point is made that IFN-γ production is linked to protection against abortion and lower plasma PAG-1concentrations. The question is why do some cows produce IFN-γ? Is it that in these animals the parasitemia is sufficiently intense and long-lasting to trigger an inflammatory process in the placenta, but not enough to cause abortion? Or could it be something to do with the higher PAG-1 levels in these animals related to the use of Limousin semen? Effectively, although Neospora-infection provoked lower PAG-1 levels in Limousininseminated cows compared to uninfected animals, PAG-1 levels were significantly higher in cows inseminated with Limousin semen than in those inseminated with Holstein-Friesian semen. The factors known to affect the outcome of N. caninum infection are the quantity and duration of the parasitemia, its timing and the effectiveness of the maternal immune response and ability of the fetus to respond to infection [19]. Probably, the type of immune response, humoral versus cell-mediated, is related to the number of tachyzoites present. In the Th2 response, the number of parasites would be small and thus not affect PAG-1 concentrations and placental function, yet if this number rose, this could trigger placental inflammation and the Th1 response affecting PAG-1 concentrations, as observed here. If the number of parasites acts on theTh1/Th2 balance, the next question that arises is whether PAG immunomodulation at the feto-maternal level could be more important than at the level of the general immune response? Placental function might be favored in cross-breed pregnancies. Thus, higher peripartum PAG levels have been observed in cows carrying fetuses of a different breed compared to cows bearing fetuses of their own breed [35]. Differences between plasma PAG

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concentrations for cows impregnated by Limousin versus Holstein-Friesian semen increased significantly as gestation progressed (Figure 1-A), in agreement with a previous study [36]. Similarly, domestic goats carrying Spanish ibex fetuses were found to have PAG concentrations that were ten times higher than intra-species gestations of either domestic goats or Spanish ibex [37]. Thus, a greater genetic distance between fetus and mother was associated with higher PAG concentrations. If high plasma PAG levels reflect placental/fetal well-being [25-27], cross breed pregnancies would determine a higher threshold without abortion for a given inflammation process and IFN-γ production than within-breed pregnancies such as the present Holstein-Friesian pregnancies. Indeed, the use of beef semen has been found to reduce the risk of abortion by a factor of 2.8 in Neospora-seropositive dairy cows [38]. Although only six cows of the Neospora-seropositive group became pregnant to Holstein-Friesian semen, all 14 cows producing IFN-γ became pregnant using Limousin semen. These results suggest that the use of Limousin semen could favor Th1 cellmediated immunity, and confer a low risk of abortion to Neospora-seropositive dairy cows.

Conclusion Our data indicate that whereas the production of antibodies against N. caninum shows no correlation with plasma PAG-1 concentrations during gestation in Neospora-infected dairy cows, the production of IFN-γ was negatively correlated with plasma PAG-1. Our findings also suggest that elevated Th1 cell-mediated immunity is associated with a low risk of abortion and thus with resistance to the parasite during pregnancy when Limousin semen is used to inseminate Holstein-Friesian cows.

Acknowledgments The authors thank Ana Burton for assistance with the English translation. This work was supported by the Spanish CICYT, grants AGL2007-65521-C02-01/GAN, AGL2007-65521C02-02/GAN.

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In: Pregnancy Protein Research Editor: Marie O’Leary and John Arnett

ISBN 978-1-60692-396-2 © 2009 Nova Science Publishers, Inc.

Short Communication B

Concentrations of PregnancyAssociated Glycoproteins in Water Buffaloes Females (Bubalus Bubalis) During Pregnancy and Postpartum Periods O. Barbato1, N. M. Sousa2, A. Malfatti3, A. Debenedetti1, L. Todini3, V. L. Barile4, and J. F. Beckers∗2 1

Department of Biopathological Veterinary Science, Faculty of Veterinary Medicine, University of Perugia, 06126, Italy 2 Laboratory of Animal Endocrinology and Reproduction, Faculty of Veterinary Medicine, University of Liege, 4000, Belgium 3 Department of Veterinary Science, Faculty of Veterinary Medicine, University of Camerino, 62032, Italy 4 Consiglio per la Ricerca e la Sperimentazione in Agricultura, Monterotondo, 00016, Italy

Abstract The concentrations of pregnancy-associated glycoproteins (PAG) were determined in buffalo cows (Bubalus bubalis) using three different radioimmunoassay (RIA) systems (RIA-497, RIA-706, and RIA-708). Samples (10 mL) were collected by jugular venipuncture from Week 0 until Week 28 of pregnancy (9 females), and from parturition until Week 10 postpartum (13 females). During pregnancy, PAG molecules were detectable at Week 6 using the three aforementioned RIA systems (3.9 ± 1.3 ng/mL, 9.7 ± 1.3 ng/mL, and 9.9 ± 0.7 ng/mL for RIA-497, RIA-706, and RIA-708, respectively). These concentrations increased gradually until Week 28, reaching values of 39.6 ± 4.0 ng/mL (RIA-497), 50.5 ± 11.9 ng/mL (RIA-706), and 68.2 ± 20.8 ng/mL (RIA-708). ∗

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O. Barbato, N. M. Sousa, A. Malfatti et al. PAG concentrations determined by RIA-497, RIA-706, and RIA-708 were strongly correlated throughout the entire gestation period, RIA-708 giving the higher concentrations. At parturition, the mean concentrations ranged from 34.9 ± 4.0 (RIA497) to 84.7 ± 10.6 ng/mL (RIA-708). Thereafter, the concentrations decreased steadily, reaching very low levels (< 1.0 ng/mL) at Week 8 postpartum. In conclusion, PAG concentrations measured by the above three RIA systems showed a profile similar to those previously described in bovine species, with higher concentrations being detected by RIA-706 and RIA-708. However, the patterns of PAG concentrations, studied using the three aforementioned PAG-RIA systems, differed around parturition, with very low levels being observed in the female buffaloes.

Keywords: buffalo, pregnancy-associated glycoproteins, radioimmunoassay, pregnancy, postpartum.

1. Introduction Characterized for the first time in the early eighties, the pregnancy-associated glycoproteins (PAG; also called pregnancy-specific protein B or pregnancy-specific protein 60) constitute a large family of glycoproteins expressed in the outer epithelial layer (chorion/trophectoderm) of the placenta in eutherian species [1,2,3]. They are synthesized by the mononucleate and binucleate trophoblastic cells, and some of them are secreted into the maternal blood from the moment that the conceptus becomes more closely attached to the uterine wall and placentome formation begins [4]. A few members of the PAG family have been isolated from the cotyledons of cow [5] and goat [6] by using different chromatographic procedures. Purified and semipurified preparations have also been used to immunize rabbits, and the antisera (AS) obtained have led to the development of homologous [4] and heterologous radioimmunoassay (RIA) systems [7,8]. A bovine PAG preparation with an apparent molecular mass of 67 kDa (boPAG67kDa ) has been used as the standard and tracer in most of the PAG-RIA systems developed thus far (reviewed by Sousa et al. [9]. Some years ago, Perényi et al. [7] compared the antiboPAG67kDa antiserum (AS#497) with new AS raised against the PAG molecules isolated from caprine cotyledons (PAG55kDa+62kDa and PAG55kDa+59kDa, AS#706 and AS#708, respectively). Interestingly, regardless of the use of the same boPAG67kDa preparation as the standard and tracer, the newly developed systems (RIA-706 and RIA-708) revealed much higher PAG concentrations between Days 25 and 50 after artificial insemination (AI) in pregnant cattle, suggesting that epitopes from the earlier-expressed PAG molecules can be better recognized by AS raised against PAG isolated from other species closely related to the bovine species. The present chapter describes the results from the determination of the plasma PAG profiles during both pregnancy and postpartum periods in water buffalo females by using three distinct RIA systems: RIA-497, RIA-706, and RIA-708. The correlation between the different concentrations, as measured by the aforementioned RIA systems, has also been determined.

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2. Materials and Methods 2.1. Research Site and Experimental Animals The present investigation was carried out at the Zootechnic Experimental Institute (Monterotondo, Italy). A total of 22 water buffalo cows were used during this study. The animals were fed the concentrate-mixture recommended for optimum milk production. Hay and water were offered ad libitum.

2.2. Experimental Design and Sampling of Blood AI was carried out in all the 22 female animals. Nine buffalo cows were used for determination of PAG concentrations between Week 0 (Week of AI) and Week 28 of pregnancy (a total of 55 blood samples). Thirteen cows were used for the determination of postpartum PAG concentrations (a total of 162 blood samples). In these animals, blood samples were withdrawn from Week 0 (at parturition) until Week 10 postpartum. Approximately 10 mL of blood were collected from the jugular vein into EDTA-coated tubes. Plasma was separated by centrifugation at 2,500× g for 10 minutes and stored at -20 °C until used for further assays.

2.3. RIA for PAG Estimation Three different RIA systems (RIA-497, RIA-706, and RIA-708), derived from the method previously described by Perenyi et al. [7], were used to measure the concentrations of PAG. All the assays were carried out in Tris buffer containing 1% BSA (Fraction V, ICN Biochemicals Inc., Aurora, OH, USA). Measurements were performed in polystyrene tubes and incubations were conducted at room temperature (20 to 22 °C). A preparation of bovine PAG 67-kDa protein (boPAG67kDa, accession number Q29432) was used as both the standard and tracer for all assays. Pure stock of boPAG67kDa (lyophilized powder) was diluted with the assay buffer (Tris containing 1.0 mg/mL BSA) to create standard curves for concentrations ranging from 0.2 to 25 ng/mL (preincubated system). The iodination procedure (Na-I125, Amersham Pharmacia Biotech, Uppsala, Sweden) was carried out on the basis of the Chloramine-T method [10]. The polyclonal AS anti-boPAG67kDa (AS#497; PAG accession number Q29432, [4]), anti-caPAG55+62kDa (AS#706, PAG accession numbers P80935 and P80933), and anti-caPAG55+59kDa (AS#708; PAG accession numbers P80935 and P80934, [6]) were respectively used in RIA-497, RIA-706, and RIA-708. The double-antibody precipitation system was composed of a mixture of sheep antirabbit immunoglolobulin (0.83% v:v), normal rabbit serum (0.17% v:v), polyethylene glycol-6000 (20 mg/mL; Vel, Leuven, Belgium), microcrystalline cellulose (0.05 mg/mL; Merck, Darmstad, Germany), and BSA (2 mg/mL; Fraction V, ICN Biochemicals Inc.) diluted in Tris buffer (25 mM Tris, 10 mM MgCl2, and 0.02% w/v NaN3; pH 7.5).

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Plasma samples were assayed in a preincubated system. Briefly, a 0.1 mL volume of each sample or an appropriate standard dilution was aliquoted into duplicate assay tubes and diluted respectively with 0.1 mL and 0.2 mL of Tris-BSA buffer. To minimize the nonspecific interference by plasma proteins, bovine PAG-free plasma (0.1 mL)was added to all the standard tubes. Following this, 0.1 mL of diluted antiserum (AS#497: 1/300,000; AS#706 and AS#708: 1/80,000), and the tubes were incubated overnight at room temperature. The following day, a solution of 0.1 mL radiolabelled 125I-PAG (28,000 counts per min) was added to all the tubes, and a further 4 h incubation was before addition of 1.0 mL of the double-antibody precipitation system. A further 30 min incubation took place at room temperature before addition of 2.0 mL of assay buffer. Bound (B) and free PAG were separated by centrifugation (1,500× g for 20 min). The supernatants were discarded and the radioactivity of the pellet was determined using a multigamma counter (LKB Wallac 1261; Turku, Finland). Samples with higher PAG concentrations than the estimated standard dose at which the percentage of B/B0 was 20% (ED20) were re-assayed using 50 µL or 10 µL of sample instead of 0.1 mL, and the remaining 50 µL or 90 µL were made up with buffer. Corresponding quantities of serum-free (50 or 10 µL) were added to standard curves. The minimum detection limit, calculated as the mean concentration minus twice the standard deviation [mean − (2×SD], of 20 duplicates of the zero (B0) standard [11] were 0.1 ng/mL, 0.1 ng/mL, and 0.4 ng/mL respectively for RIA-497, RIA-706, and RIA-708.

2.4. Data Analysis Data are presented as the mean ± standard error of the mean (SEM). The regressions and the analysis of variance were carried out using the SAS program [12]. Probability (P) values < 0.05 were considered significant. The concentrations of PAG in one buffalo cow during the postpartum period were considered abnormal (test of comparison of means) and were hence excluded from the general mean. The half-life of PAG was calculated as previously described [13] from the following formula: t½ = [ln (C / 0.5 C) / λ ], where C = plasma concentration of PAG at parturition, and λ = slope of the regression equation.

3. Results 3.1. Correlation between the PAG Concentrations Detected by the Three PAG-RIA Systems All the three RIA systems proved to be efficient in measuring the buffalo PAG concentrations from Week 6 after AI onward (> 3 ng/mL). The correlation coefficient (R) between RIA-497 and RIA-706 was greater (R = 0.9819) than those obtained between RIA497 and RIA-708 (R = 0.9410; Figure 1A) and between RIA-708 and RIA-706 (R = 0.9568)

Concentrations of Pregnancy-Associated Glycoproteins in Water Buffaloes… 127 systems (Figure 1B). In general, the values of RIA-497 were significantly (P < 0.01) lower than those of systems RIA-706 and RIA-708, the last one giving the highest concentrations (nearly three times more than RIA-497).

Figure 1. Correlation between the concentrations of pregnancy-associated glycoproteins measured during pregnancy and postpartum periods in the peripheral circulation of buffalo. A) Comparison of concentrations measured by RIA-497 and RIA-706 (-z-z-) and between RIA-497 and RIA-708 (--). B) Comparison of concentrations measured by RIA-706 and RIA-708 (---).

3.2. PAG Profiles Figure 2 shows the PAG concentrations during pregnancy in water buffalo females. As measured by all RIA systems, the concentrations of PAG increased from Week 6 to 28 of pregnancy. The concentrations of PAG at Week 6 of pregnancy were 3.9 ± 1.3 ng/mL, 9.7 ± 1.3 ng/mL, and 9.9 ± 0.7 ng/mL when measured by RIA-497, RIA-706, and RIA-708, respectively. Concentrations increased gradually until Week 28, reaching 39.6 ± 4.0 ng/mL (RIA-497), 50.5 ± 11.9 ng/mL (RIA-708), and 68.2 ± 20.8 ng/mL (RIA-708). The maximum mean PAG concentration was detected using RIA-708 at Week 27 (70.2 ± 15.3 ng/mL). Throughout the entire gestation period, the higher concentrations were obtained by RIA-708 whereas lower concentrations were detected by RIA-497.

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Figure 2. Mean plasma concentrations (± SEM) of pregnancy-associated glycoproteins (PAG) from the day of artificial insemination (AI) (day 0) until Week 28 of pregnancy. Concentrations were measured using three RIA systems (RIA-497, RIA-706, and RIA-708).

Figure 3 shows the PAG concentrations measured during the postpartum period in 12 water buffalo cows. One cow showing abnormal PAG concentrations during the entire postpartum period was withdrawn from the general PAG profile and the results obtained from its plasma are shown in Figure 4.

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Figure 3. Mean (± SEM) concentrations of pregnancy-associated glycoproteins (PAG) during the postpartum period. Concentrations were measured using three RIA systems (RIA-497, RIA-706, and RIA-708).

Figure 4. Mean (± SEM) concentrations of pregnancy-associated glycoproteins (PAG) during the postpartum period in a buffalo cow (number52) showing higher concentrations. The results from the plasma of this cow were withdrawn from the data for calculating the mean PAG concentrations during the postpartum period.

Mean PAG concentrations ranged from 34.9 ± 4.0 (RIA-497) to 84.7 ± 10.6 ng/mL (RIA-708) at parturition. Thereafter, the concentrations of PAG decreased gradually until Week 8 postpartum (< 1.0 ng/mL). As observed during pregnancy, the higher concentrations of PAGs were measured by RIA-708 during postpartum, whereas lower levels were obtained by RIA-497. The estimated half-life obtained from RIA-497, RIA-706, and RIA-708 were 8.2, 8.7, and 9.9 days, respectively.

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Discussion This chapter describes the pregnancy and postpartum concentrations of PAG in water buffalo for the first time. Throughout the entire pregnancy and postpartum periods, the PAG concentrations measured using RIA-706 and RIA-708 are lower than those detected using RIA-497. At Week 6, for example, the mean concentration of PAG measured by RIA-497 is 2.5 times lower than those obtained from RIA-706 and RIA-708 (3.9 ng/mL and 9.7 and 9.9 ng/mL, respectively). Higher concentrations have been recorded using anti-caPAG AS, as previously reported in Holstein Friesian cows by Perenyi et al. [7]. These authors described the mean PAG concentrations as 4.0 ng/mL (RIA-497) and 5.6 ng/mL (RIA-706 and RIA708) at Week 7 after AI. Moreover, concentrations of PAG described in cattle recipients carrying somatic clones [14] and in high-producing dairy cows [15] indicate a better recognition of PAG molecules by AS raised against PAG derived from other species closely related to the cow, such as caprine PAG. The plasma PAG profile throughout pregnancy in buffalo cows is characterized by a gradual increase of PAG concentrations from Week 6 to 28 of pregnancy. This pattern of increase is similar to those previously described in European (Bos taurus) and Zebu (Bos indicus) cattle [4,16,17]. However, it differs from those described in caprine and ovine species, in which PAG concentrations increase rapidly from Week 3 to 4 after fertilization [18–23]. In late pregnancy and at parturition, water buffalo females (n = 12) show lower PAG concentrations (< 85 ng/mL) than the European and Zebu cattle (> 1,000 to 2,000 ng/mL) [4,16,17,24]. However, the concentrations of PAGs in one single female at parturition are higher than 120, 200, and 250 ng/mL, when measured by the RIA-497, RIA-706, and RIA708 systems, respectively. In general, the pattern of secretion of PAG during this period is quite different from that of the bovine species, with no exponential increase being observed during the peripartum period. Surprisingly, concentrations of PAG during the peripartum period are relatively similar to those described in some breeds of goats [20,25] and ewes [19]. However, in the same species or even within the same breed, PAG levels also vary markedly depending on the AS used for measurement of PAG. Further investigations, including the development of specific AS raised against PAG molecules isolated from buffalo placenta, are needed to be conducted to better understand the peripartum profile in this species. Notwithstanding the low concentrations of PAG reported at parturition, the half-life of PAG is relatively long in buffalo females (8.2 to 9.9 days). Similar half-lives have been described in the European and Zebu breeds [4,13,17]. On the contrary, PAG concentrations in caprine and ovine species decrease rapidly during the postpartum period, reaching minimal levels at Week 4 postpartum [18,20,21,23]. In 2003, Sousa et al. [17] suggested that the clearance rate of PAG is probably more related to the influence of N-linked carbohydrate and sialic acid chains on the PAG structure than to the absolute concentrations reached at parturition. In the same manner, Klisch et al. [26] have reported that the magnitude of glycan content in PAG varies considerably in late pregnancy (absence of the terminal N-acetylgalactosamine moiety in binucleate cells at parturition). The result of this study corroborates the hypothesis of the regulation of serum half-life of PAG molecules by their glycan moiety.

Concentrations of Pregnancy-Associated Glycoproteins in Water Buffaloes… 131 Regarding the glycodiversity of PAG molecules, purified PAG molecules are known to have increased molecular masses than the estimated values of their protein cores (37 kDa) as deduced from the cDNA sequence. As reported by Klisch et al. [27,28] N-glycosylation posttranslational processing contributes to a significant increase in the molecular masses of PAG. A great variability in the molecular masses of PAG isolated from different ruminant species has also been reported. The molecular masses of PAG isolated from the bovine (56 to 75 kDa) [4], bison (72 to 76 kDa) [29], and water buffalo trophectoderms (58 and 76 kDa) [30] are generally larger than those isolated from caprine (55 to 62 kDa) [6] and ovine tissues (55 to 66 kDa) [31,32]. Whether the relatively long half-life of PAG in the peripheral circulation of buffalo females is mainly due to the larger glycan content in this species, remains to be confirmed in further investigations. In conclusion, peripheral concentrations of PAGs are better recognized by using AS raised against caprine PAG (AS#706 and AS#708) than by the use of those raised against the bovine form (AS#497). Moreover, PAG concentrations measured by the above-mentioned three RIA systems show profiles distinct from those previously described in bovine species, with very low peripartum levels of PAG.

Acknowledgments This review is part of a research supported by a grant from the Ministry of the Wallonne Region (DGA) (Grant number D31-1184) and the National Fund for Scientific Research (FNRS), Belgium (Grant CC 1.5.059.08) to J.F. Beckers. The authors thank Mrs. R. Fares and G. Van Diest for their editorial assistance.

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In: Pregnancy Protein Research Editor: Marie O’Leary and John Arnett

ISBN 978-1-60692-396-2 © 2009 Nova Science Publishers, Inc.

Short Communication C

The Role of Human Chorionic Gonadotropin (HCG) in Gestational Trophoblastic Disease Caroline Wilson and B. W Hancock Sheffield Gestational Trophoblastic Tumour Centre, Weston Park Hospital, Sheffield, UK

Abstract Gestational trophoblastic disease (GTD) encompasses a number of rare related tumours, including complete hydatidform mole (CHM), partial hydatidiform mole (PHM), invasive mole, choriocarcinoma and placental site trophoblastic tumour, all of which vary in their propensity for spontaneous resolution, local invasion and metastasis. The worldwide incidence of GTD varies, with an increased incidence in women from Asia, however UK incidence is around 1.5 cases/1000 live births and the incidence of choriocarcinoma is 1/50,000 live births. Persistent GTD (termed gestational trophoblastic neoplasia, GTN) commonly follows molar pregnancy, but can occur after any type of gestation. Several risk factors are recognised i.e. maternal age, previous mole and socioeconomic factors. Clinical presentation of GTN has changed over time due to earlier diagnosis and use of ultrasound, and includes abnormal bleeding during pregnancy, anaemia, hyperemesis, pre eclampsia, pain, intraperitoneal haemorrhage, symptomatic metastatic disease and rarely thyrotoxicosis due to cross reaction of a subunit of hCG and TSH. The mainstay of diagnosis involves the measurement of hCG and its variants, produced by trophoblast cells. The amount of hCG indicates the volume of tumour and therefore influences prognosis. Other diagnostic investigations include history/examination; blood screens; imaging including chest x-ray, computed tomography scan of the thorax, ultra sound scan of the pelvis, and histological review. All patients are staged/scored according to WHO and FIGO criteria. Those who are high risk on scoring, have βHCG > 50,000iu/L, or have multiple pulmonary metastasis, undergo a central nervous system evaluation. Treatment of GTD requires a team of specialists. Initial management requires blood loss replacement and usually suction curettage of molar tissue. GTD is managed with either second evacuation, hysterectomy

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Caroline Wilson and B. W Hancock if family complete or chemotherapy chosen according to risk. In the UK, less than 10% patients require chemotherapy with a cure rate of virtually 100% in low risk disease and 86% in high risk disease. Measurements of hCG are essential to monitor response to treatment; an adequate treatment response is defined by a 50% reduction in hCG on weekly serum analysis. Once hCG becomes normal treatment continues to ensure elimination of residual tumour following biochemical remission. The choice of chemotherapy depends upon risk; low risk receiving intra muscular methotrexate and if salvage is required a combination of chemotherapy is used. High risk receive a combination chemotherapy from the outset. Follow up is essential and involves serial measurements of HCG at specified intervals. The bespoke management by specialist teams ensures that GTD remains one of the most curable of all human cancers.

Introduction Gestational trophoblastic disease (GTD) encompasses a number of rare related tumours, including complete hydatidform mole (CHM), partial hydatidiform mole (PHM), invasive mole, choriocarcinoma and placental site trophoblastic tumour. The ability of a molar pregnancy to invade and metastasise results in the development of choriocarcinoma and placental site trophoblastic tumour. Several recognised factors increase the propensity for invasion including loss of normal imprinting and alteration in expression of maternally expressed genes such as IGF2, or paternally expressed genes such as H19[1]. Once invasion occurs, metastasis can be widespread particularly in lungs, brain and pelvic organs. Human chorionic gonagotropin (hCG) is pivotal at all stages of management of GTD from diagnosis through to follow up. It is a glycoprotein exocrine hormone composed of an α and β subunit, bonded by hydrophobic interaction. The α and β subinits comprise of 92 and 145 amino acids respectively and there are 8 oligosaccharide side chains. Five structural variants of hCG are commonly detected in serum; regular hCG; hyperglycosylated hCG; nicked hCG; hCG free β subunit and hCG without free β subunit c-terminal peptide[2]. All laboratory serum tests will detect regular hCG, but they vary in their propensity to detect the other variants therefore leading to discrepancy in test results which may have huge impacts on clinical management of patients with GTD.

Epidemiology and Pathophysiology The worldwide incidence of GTD varies; this is speculated to be due to the difficulty in determining incidence rates, environmental factors and genetic factors. The current UK incidence is estimated to be around 1.5 cases/1000 live births and the incidence of choriocarcinoma is 1/50,000 live births [27]. There is no doubt that there will be an impact on incidence rates if assays used to test hCG are variable in their sensitivity and specificity as discussed below. The hCG found throughout normal pregnancy is regular hCG, produced by syncytotrophoblast cells [4], and is the variant that is detected by assays marketed for pregnancy. In persistent active GTD (gestational trophoblastic neoplasia, GTN) the

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predominant form of hCG is hyperglycosylated hCG, produced by invasive cytotrophoblast cells; while its peptide structure is similar to regular hCG it varies in its oligosaccharide structure with 15 rather than 8 sugar residues [10]. Hyperglycosyated hCG appears to have a separate role to regular hCG and acts in an autocrine manner to promote invasion and growth of the cytotrophoblast cells in vitro and in vivo [14]. It also acts to inhibit TGFβ, which is a promoter of apoptosis in trophoblast cells, further increasing its predilection for promoting cell invasion.[16]; it is hypothesised that this occurs due to interaction of the β subunit of hyperglycosylated hCG with the TGFβ receptor as the two molecules have structural homology[18]. Placental site trophoblastic disease (PSTT) is a rare variant of GTN and is notoriously chemo/radio resistant. The main form of hCG in PSTT is the free β subunit, as discussed later.

Clinical Features of GTD Clinical presentation of GTD has changed over time due to earlier diagnosis and use of ultrasound. See figure I. PHM and CHM commonly present with vaginal bleeding in pregnancy, as the molar chorionic villi disturb maternal vessels; this is often heavy leading to profound anaemia. This commonly occurs prior to 20 weeks gestation. Theca lutein ovarian cysts are evidenced on ultrasound in 20-30% patients with molar pregnancies[5]; these are a consequence of hyperstimulation of the ovaries by hCG and are found in patients with a very high hCG level, rarely these can be complicated by ascites and pleural effusions.

Figure I. Choriocarcinoma invading myometrium.

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FigureII. Solitary brain metastasis from GTN.

Hyperemesis occurs in around 25% patients and is also associated with very high hCG levels. Pre-eclampsia is a recognised resenting symptom with hypertension, oedema and proteinuria. Thyroid stimulation is recognised and is thought to be due to cross reactivity of the ά subunit of hCG with TSH. Pulmonary compromise can occur in patients with a high level of hCG, post evacuation of mole, and manifests as tachypnoea, confusion, tachycardia with hypoxia and a respiratory alkalosis; cardiovascular and respiratory support is usually needed. Invasive mole manifests as ongoing vaginal bleeding post evacuation, or persistently elevated levels of hCG. Pain is often associated, and rarely bleeding from sites involved in invasion such as rectum and bladder can occur. Gestational trophoblastic neoplasia (GTN) may present with the symptoms of metastatic disease, up to a year after the antecedent pregnancy. This may manifest as lung/brain/peritoneal/vaginal metastasis (see figure II) and the symptoms associated with these i.e. breathlessness, cough, haemoptysis, pleuritic chest pain, focal neurological symptoms, seizures, headache, abdominal pain and bleeding respectively.

Investigation and Diagnosis The mainstay of diagnosis involves accurate clinical assessment and the measurement of serum/urine hCG. As mentioned prior the main form of hCG in GTN is hyperglycosylated and in PSTT is the free β subunit, however many marketed tests do not equally detect these variants. An ideal assay should be sensitive and detect all variants of hCG, even at low levels

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to avoid false negatives; it would also be specific and not detect interfering substances that may lead to false positive results. Antibodies present in serum can interact with the anti-hCG antibodies used in the hCG assay, with false positive results that can generate a βhCG level as high as 1100IU/L [19]. The interfering antibodies, however, do not cross the glomerular basement membrane and therefore parallel urine samples are negative for hCG, stressing the importance of paired urine/serum samples. The implication of false positive results is extreme with patients receiving chemotherapy or surgery unnecessarily; this can be compounded by an initial fall in hCG once treatment is implemented which may be due to immune suppression and a decrease in interfering antibodies therefore falsely reassuring clinicians of a ‘response’ to treatment. Many publications have demonstrated a link between lutanizing hormone (LH) and hCG identifying a surge in hCG at the same time as the pre ovulatory surge in LH [28]; also pituitary derived hCG is normally present along side LH in post menopausal women due to the lack of oestrogen/progesterone suppression. This may lead to post menopausal women being misdiagnosed with a malignancy when it is part of normal physiology; the use of high oestrogen pills in this patient group suppresses the hCG and can therefore resolve any confusion over diagnosis.[13] Several publications have examined the difference between assays used to assess hCG level and have found discrepancies between specificity and sensitivity; in 2005 at the XIIIth World Congress on Gestational Trophoblastic Disease the two main assays were compared, that of the single antibody based radioimmunoassay(RIA) used in the UK and the Diagnostic Products Corp.(DPC) Immulite system used in the United States; the two assays could produce differences on comparable serum of up to 2 fold[25]. It was also found that the commercial hCG pregnancy tests have a marked false negative rate in malignancy and therefore are not suitable for the detection of hCG in GTD. The accurate measurement of hCG is paramount in diagnosis of GTD and therefore clinicians must be aware of the limitations of the assay used in their centre, and this highlights the importance of patients undergoing diagnostic review at specialist centres prior to commencement or change in treatment. Other principal investigations used in diagnosis include ultrasound to assess uterine size, histopathology following uterine evacuation, baseline haematology/biochemical parameters/thyroid function and pituitary gonadotropins, chest x ray and CT thorax for review of possible metastatic spread. Patients with a very high hCG, known pulmonary metastasis or who have bulky disease also undergo assessment of the central nervous system with a lumbar puncture and CT head; if the results are equivocal then an MRI head is sought.

Staging and Scoring Systems Several prognostic factors are established and these influence the treatment given. GTD encompasses a spectrum of diseases and therefore treatment is tailored according to risk, from surgical re-evacuation to intensive chemotherapy. Several staging classification systems have been established; however there is no clear superiority of one system over another; the revised WHO prognostic scoring system and FIGO criteria[8] are detailed below (figure III

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and IV respectively). The advantages of these systems include their versatility of use, as the prognostic factors depend on patient’s history and hCG measurement, which can be performed in any centre, it is also easy for clinicians to remember and use. The more adverse prognostic indicators are given a higher numerical score with the following conferring a poor prognosis; term antecedent pregnancy, long interval between antecedent pregnancy and start of chemotherapy, high hCG levels indicating a large quantity of viable tumour, metastasis in liver and brain, large number of metastasis and ≥2 failed prior chemotherapy agents. Patients are categorised according to risk which then determines treatment. Low risk score ≤6 and high risk score ≥7 based on the FIGO scoring system. SCORE

0

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≤39 Hydatidiform mole <4

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>104-105 ≥5 Gastrointes tinal tract 5-8 Single drug

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Figure III.Revised WHO prognostic scoring system.

Stage 1 Stage II Stage III Stage IV

Disease confined to uterus GTD extends outside uterus but is limited to genital structures (adnexa, vagina, broad ligament) GTD extends to the lungs with of without genital tract involvement All other metastatic sites

Figure IV. FIGO Staging.

Treatment The need for treatment in GTD is defined as ‘evidence of persistent disease activity unlikely to resolve spontaneously’. There was noted to be a difference between different centres as to the definition of ‘persistent’ and therefore the FIGO committee recommended the following to define persistent GTD requiring treatment[8]: 1) Plateau of hCG for ≥ 4 measurements over a period of 3 weeks or longer.

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2) Rise in hCG of 3 weekly consecutive readings over a period of at least 2 weeks i.e. day 1,7,14. 3) hCG remaining elevated for 6 months or more 4) Histological diagnosis of choriocarcinoma. Once patients have been staged and scored they begin treatment tailored to risk which may involve surgery, single agent chemotherapy or combination chemotherapy as detailed below.

Low Risk Patients The majority of patients fall into the low risk group and have small volume disease, which can be cured with methotrexate and folinic acid alone. The schedule is well tolerated and the majority of side effects can be ameliorated by good hydration and folinic acid rescue i.e. mucositis and conjunctivitis. Occasionally patients can develop methotrexate induced hepatic damage requiring a change in treatment. Pleurisy and peritonism can be a concern with methotrexate and can be severe requiring a change in treatment despite adequate tumour response. The advantages to methotrexate include good long term safety profile with no alopecia. The survival in this group with methotrexate is 99.7%[6]

Low Risk Salvage 20% of patients will require additional chemotherapy due to a plateau in hCG secondary to methotrexate resistance. The regimen comprises dactinomycin and etoposide. The response rate of low risk relapsed disease is virtually 100%[9], however it is imperative that these patients are identified early to prevent emergence of multiple drug resistance, therefore the close monitoring of hCG is essential. The common problems encountered include emesis, alopecia and bone marrow suppression.

High Risk Treatment in this group of patient’s aims to facilitate the delivery of the three drugs mentioned above proven to have the highest efficacy in GTN. Etoposide was found to be active against GTN in the 1990s and therefore was been incorporated into the high risk regimen [11-12]. The problems with such regimens include; bone marrow suppression often requiring GSCF support to maintain dose schedule intensity, MTX toxicity due to the increased dose requiring alkalinisation of the urine and adequate hydration and folinic acid rescue. One major problem is pulmonary toxicity seen in patients with large volume disease in the lungs; they have stiff non compliant lungs, which are already compromised, and following

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chemotherapy can haemorrhage leading to pulmonary distress requiring ventilatory support with continuous positive airways pressure(CPAP). See Figure V

Figure V. Pulmonary metastasis in a GTN patient.

High Risk Salvage There is no chemotherapyy with a guaranteed outcome in these patients. Cisplatin has been shown to be active [17], and salvage surgery plays a role to remove the foci of resistant tumour. Placental site trophoblastic tumour (PSTT) is a rare variant of GTN and, when metastatic, is notoriously chemo/radio resistant and therefore a high percentage of these patients require high risk salvage and surgery.[15] It is a malignancy of non-villous trophoblasts, often with marked necrosis due to the lack of early and widespread vascular invasion. It usually presents with vaginal bleeding following a normal pregnancy, spontaneous abortion or hydatidiform mole. It is derived from intermediate trophoblast cells and is associated with significantly lower hCG levels which fail to rise sharply with time, when compared with choriocarcinoma. The level of hCG is often <200 and is commonly in the form of free β subunit hCG [20], as the hCG subunits produced in PSTT appear not to be in high enough concentrations to form α and β subunits. It also produces human placental lactogen(hPL) and pregnancy associated basic protein; however hPL cannot be used as a tumour marker as its level does not accurately reflect disease activity. It appears that free β subunit may represent an accurate way of distinguishing PSTT from other forms of GTD, but there needs also to be caution when using free β subunit hCG as a tumour marker as this is also produced in nontrophoblastic malignancies[21]. Patients presenting with low levels of hCG should be investigated for other malignancies along side PSTT, especially if there is no history of an antecedent pregnancy. Response to treatment is monitored by serial hCG measurements. GTN tumours never stop producing hCG and therefore it can be used as a reliable marker of residual disease. An

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adequate treatment response is defined by a 50% reduction in hCG on weekly serum analysis, therefore identifying drug resistance and the need to change chemotherapy. The level of hCG may normalise when there is still residual tumour burden, therefore all treatment regimens mentioned above continue for six weeks following biochemical remission. See figures VI and VII.

Figure VI. Fall in hCG, with time, during treatment with chemotherapy.

Figure VII. Showing a plateau in the fall of HCG therefore requiring treatment change.

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Follow Up Once treatment has begun both normal and hyperglycosylated hCG become nicked, thus leading to rapid dissociation of the subunits. In GTD, once hCG falls below 100IU/L the major variants seen are nicked hCG or free β subunit, thus assays must be sensitive to both these forms to prevent false negative results and undetected recurrent disease. Cases have been reported of missed recurrence due to use of an assay which does not detect nicked hCG[22] and therefore it is imperative that clinicians are aware of the sensitivity and specificity of the assays available to them when interpreting hCG during follow up. During follow up patients are advised not to become pregnant for a minimum of 6 months following normalisation of hCG and ideally not for 1 year from completion of chemotherapy, in order to minimise the potential teratogenic effects of the chemotherapy on the foetus, and avoiding the time span when the recurrence risk of GTD is at its highest [23]. With effective registration, appropriate treatment and follow up of these patients the overall long term survival exceeds 95% [26]. An area of concern exists in patients whom hCG persists at a low real level with no evidence of tumour or active disease. When this occurs with a history of appropriate pregnancy event, it is termed quiescent GTD and hCG may persist at hCG levels up to 250 with no obvious increasing trend for several months to occasionally years. It seems likely that it represents slow growing well differentiated syncytiotrophoblast tissue rather than invasive trophoblastic disease. Hyperglysosylated hCG can be of use in this situation as its absence indicates non invasive quiescent GTD since its production is dependent on the presence of cytotrophoblast cells. Clinically quiescent GTD does not require treatment, however there are reported cases of inappropriate treatment either surgical or chemotherapy, which tragically in some cases lead to death due to toxicity of the received treatment[24]. This emphasises the need to ensure if there is any doubt in the diagnosis that results are reviewed in expert centres prior to initiation of treatment. However, quiescent GTD must be considered as a pre-malignant disease with 1 in 5 cases transforming to choriocarcinoma, and serial hyperglysosylated hCG measurement over time may allow early diagnosis of its transformation to a more aggressive, invasive form of GTN and allow initiation of treatment early. There is estimated to be an increased risk of recurrent disease of 40% in patients with quiescent GTD following a previous molar pregnancy [24]. As demonstrated above, several possibilities must be considered in non-pregnant women with a persistent low level elevation of hCG. This needs to be taken in clinical context with attention given to details such as age and medical history. In summary the raised hCG may be due to the following; 1. 2. 3. 4. 5.

GTD which may be quiescent Non trophoblastic malignancy False positive immunoradiometric assay Normal, eg pituitary hCG produced around menopause Unascertained

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Current guidance for such cases is to exclude ‘false’ hCG, investigate thoroughly, avoid immediate invasive treatment and monitor closely. As an example in Figure VIII, this young patient with persistent low level HCG elevation of unascertained origin had a normal pregnancy!

Figure VIII.

The Future The success of GTD treatment has, in part, been due to the coordinated management by specialist centres. The multi disciplinary approach to diagnosis, treatment and follow up has ensured the high survival figures. The future would aim to ensure that countries with the resources ensure this coordinated approach to management. As emphasised above, the role of hCG and its varients is pivotal in this disease, and as our understanding of this molecule and its degradation products increases, we may be able to tailor the measurement of these to each variety of GTD.

References [1] [2]

[3]

Hancock B.W, Newlands E.S, Berkowitz R.A et al. Gestational Trophoblastic Disease, 2003; 2nd Edition. Cole L.A, Shahabi S, Butler S.A et al. Utility of commonly used commercial hCG immunoassays in the diagnosis and management of trophoblastic diseases. Clinical Chemistry, 2001;47:308-315. Bagshaw K.D, Lawler S.D et al. Cancer epidemiology and Prevention, 1982; 909-924.

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[7]

[8] [9]

[10]

[11]

[12]

[13]

[14]

[15] [16]

[17]

Caroline Wilson and B. W Hancock Cole L.A, Dai D, Leslie K.K et al. Gestational Trophoblastic diseases; Pathology of hyperglycosylated hCG-regulated neoplasia. Gynaecological Oncolgy 2006;102:114149. Kohorn E.I. Molar pregnancy; presentation and diagnosis. Clinical Obstetrics and Gynacology; 27:181-191. Bagshaw K.D, Dent J, Newlands E.S et al. The role of low dose methotrexate and folinic acid in gestational trophoblastic tumours. British Journal of Obstetrics and Gynaecology;96:795-802. Kohorn E.I, Goldstein D.P, Hancock B.W et al. Combining the staging system on the International Federation of Gynecology and Obstetrics with the scoring system of the World Health Organisation for trophoblastic neoplasia. Report of the working committee of the International Society for the study of Trophoblastic Disease and the International Gynaecological Cancer Society. International Journal of Gynacological Cancer;10:84-88. FIGO Oncology Committee. FIGO staging for gestational Trophoblastic neoplasia. International Journal Obstetrics and Gynaecology;77:285-287. McNeish I.A, Strickland S, Holden L et al. Low risk persistent gestational trophoblastic disease;outcome after initial treatment with low dose methotrexate and folinic acid. Journal of Clinical Oncology 2002;20:1838-1844. Elliot M, Kardana A, Lustbader et al. Carbohydrate and peptide structure of the α and β subunits of human Chorionic Gonagotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine Journal, 1997:7:15-32. Newlands E.S, Bagshawe K.D, Begent R.H.J et al. Results with the EMA/CO regime in high risk gestational trophoblastic tumours, 1979-1989. British Journal of Obstetrics and Gynacology 1991;98:550-557. Berkowitz R.S, Goldstein D.P, Bernstein M.R et al. Modified Triple chemotherapy in the management of high-risk metastatic gestational trophoblastic tumours. Gynaecoligical Oncology 1984;19:173-181. Huang S.C, Chen H.C, Chen R.J et al. The secretion of human chorionic gonadotropinlike substance in women employing contraceptive measures. Journal of Clinical Endocrinology Metabolism 1984;58:646-653. Cole L.A, Khanlian S.A, Riely J.M et al. Hyperglycosylated hCG in gestational implantation, and in choriocarcinoma and testicular germ cell malignancy tumorigenesis. Journal of Reproductive medicine 2006;51: 919-929. Gillespie A.M, Liyim D, Goepel J.R, et al. Placental site trophoblastic tumour;a rare but potentially curable cancer. British journal of Cancer 2000;82:1186-1190. Liu Y.X, Gao F, Wei P et al. Involvement of molecules related to angiogenesis, proteolysis and apoptosis in implantation in rhesus monkey and mouse. Contraception 2005;71:249-262. Newlands E.S, Mulholand P.J, Holden L et al. Etoposide and cisplatin/etoposide, methotrexate and actinomycin D (EMA) chemotherapy for patients with high risk gestational trophoblastic tumours refractory to EMA/cyclophosphamide and vincristine chemotherapy and patients presenting with metastatic placental site tropoblastic tumours. Journal of Clinical Oncology 2000;18:854-859.

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[18] Lapthorn A.J, Harris D.C, Littlejohn A et al. Crystal structure of human chorionic gonadotropin. Nature 1994; 369:455-461. [19] Cole L.A, Khanlian S.A, Giddings A et al. Gestational Trophoblastic diseases;4. Presentation with persistant low positive human chorionic gonadotropin test results. Gynaecological Oncology 2006; 102:165-172. [20] Rinne K, Shahabi S, Cole L.A et al. Following metastatic placemtal site trophoblastic tumour with urine β core fragment. Gynaecological Oncology 1999;74:302-303. [21] Cole L.A, Khanlian S.A, Muller C.Y et al. Gestational Trophoblastic diseases: 3. Human Chorionic Gonadotropin Free subunit. A reliable marker of Placental Site Trophoblastic Tumours. Gynacological Oncology 2006;102:159-163. [22] Cole L.A, Kohorn E.I, Kim G.S. Detecting and monitoring trophoblast disease; New perspectives in measuring human chorionic gonadotropin levels. Journal of Reproductive Medicine 1994;39:193-200. [23] Hancock B.W, Newlands E.S and Berkowitz R.S. Gestational Trophoblastic Disease ed 2. pp143-156. [24] Cole L.A. Khanlian S.A. Inapproriate management of women with persistent low hCG results. Journal of Reproductive Medicine 2006;49:423-432. [25] Mitchell H, Bagshaw K.D, Newlands E.S et al. Importance of Accurate Human Chorionic Gonadotropin Measurement in the Treatment of Gestational Trophoblastic Disease and Testicular Cancer. Journal of Reproductive Medicine 2006;11:868-870. [26] Hancock B.W, Newlands E.S and Berkowitz R.S. Gestational Trophoblastic Disease ed 2. pp211-216. [27] Bagshaw K.D, Dent J, Webb J. Hydatidiform moles in England and Wales 1973-1983. Lancet 1986;11:673-677. [28] Synder J.A, Haymond S, Parvin C.A et al. Diagnostic considerations in the measurement of human chorionic gonadotropin in aging women. Clinical Chemistry 2005;51:1830-1835.

In: Pregnancy Protein Research Editor: Marie O’Leary and John Arnett

ISBN 978-1-60692-396-2 © 2009 Nova Science Publishers, Inc.

Short Communication D

Fusogenic Syncytin-1 and Transcription Factor Glial Cells Missing-A: Presumed Regulators in Human Placental Physiology and Pathophysiology

1

Christina Wich1, Said Hashemolhosseini2 and Ina Knerr∗3

Department of Medicine, University of Erlangen-Nuremberg, Erlangen, Germany 2 Institute of Biochemistry, University of Erlangen-Nuremberg, Germany 3 Children’s and Adolescents’ Hospital, University of Erlangen-Nuremberg, Erlangen, Germany

Abstract The differentiation process and cell-cell fusion of human trophoblasts in the human placenta are controlled by a variety of regulatory genes and key molecules. In this article, we focus on the fusogenic glycoprotein syncytin-1, which originally derived from the human endogenous retrovirus HERV-W. In addition, we look at its transcription factor GCMa (glial cells missing-a or Gcm1). We propose that GCMa-driven syncytin-1 expression is a key mechanism for syncytiotrophoblast formation in the human placenta. Besides the physiological significance of syncytin-1 and GCMa, we discuss their pathophysiological role in preeclampsia and intrauterine growth restriction (IUGR). In addition, we focus on the effects of hypoxia on the expression of syncytin-1 and GCMa in trophoblastic cells because changing oxygen availability contributes to abnormal placental development. Basically, any alteration of the cAMP signaling cascade involving protein kinase A (PKA), GCMa and syncytin-1 can be considered a major risk factor for diminished trophoblast differentiation and impaired syncytiotrophoblast formation, followed by placental dysfunction, for example, in the course of hypoxia. Furthermore, hypoxia∗

Tel: ++49 9131 85-33118; Fax: ++49 9131 85-33113; Email: [email protected]

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Christina Wich, Said Hashemolhosseini and Ina Knerr related down-regulation of syncytin-1 can, to a great extent, be compensated by stimulating the cAMP-driven PKA pathway. Considering that pre-eclampsia is unique to humans and that syncytin-1 is derived from the HERV-W family exclusively found in humans and in higher primates, syncytin-1 is an interesting candidate for research into human placental physiology and altered placental function. Similarly, we describe a putative mode of action at the cellular level. Using a cell culture model of syncytin-1 overexpressing cells it has been shown that syncytin-1 is capable of anti-apoptotic functions. A lower apoptotic response, such as a lower level of caspase 3 along with higher amounts of anti-apoptotic Bcl-2 have been found in syncytin-1 transfected cells compared with controls. In conclusion, we propose that fusogenic syncytin-1 may function as an antiapoptotic glycoprotein during cell-cell fusion processes. Conversely, alterations in the syncytin-1/GCMa system may, in certain circumstances, be followed by placental disturbances and disorders of pregnancy such as pre-eclampsia and IUGR.

Introduction The human placenta is essential for the intrauterine development of the fetus, but is also involved in programming health in later life (Plagemann et al., 2008). This organ is an interesting tool for studying the fundamental processes of cell differentiation and cell-cell fusion controlled by a variety of regulatory genes and key molecules. In this review we focus on the fusogenic glycoprotein syncytin-1, which originally derived from the human endogenous retrovirus HERV-W, and its transcription factor GCMa (glial cells missing-a or Gcm1). We discuss their putative role in normal human placentogenesis and in hypertensive disorders of pregnancy such as pre-eclampsia and intrauterine growth restriction (IUGR).

Syncytin-1, a Placental Fusogenic Protein Syncytin-1, the envelope (Env) protein of the human endogenous retrovirus HERV-W was first described in 2000 (Mi et al., 2000, Blond et al. 2000). It has been shown that this endogenous retroviral particle was involved in the formation of cell syncytia, which is an essential mechanism during placentogenesis. The application of antisense oligonucleotides against syncytin-1 was followed by an inhibition of cell-cell fusion and impaired trophoblast cell differentiation (Frendo et al., 2003b). Accordingly, fusogenic syncytin-1 is considered to be a key player in normal human placentogenesis (Frendo et al., 2003b, Knerr et al., 2004). The syncytin-1 gene maps to chromosome 7q21-7q22 (OMIM 604659). More recently, other HERV families have been described, and the Env protein of one of them, HERV-FRD, was named syncytin-2 (Blaise et al. 2003). However, the physiological role of syncytin-2 is, as yet, unknown. Syncytin-1 is predominantly found in the syncytiotrophoblast of the human placenta. Its localization seems to vary during pregnancy. In detail, syncytin-1 is expressed at the apical membrane site during the 1st trimenon and in term placental tissue, and at the basal site of the

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syncytiotrophoblast in the 2nd and 3rd trimenon placenta (Frendo et al., 2003b, Blond et al., 2000, Lee et al. 2001). The syncytiotrophoblast forms a selective maternal-fetal barrier in the human placenta and this essential cell layer is formed by the cell-cell fusion of villous cytotrophoblasts. Moreover, it provides the transport interface between the mother and fetus regulating oxygen exchange, metabolic supply and endocrine functions such as the production of steroid hormones, human chorionic gonadotropin (hCG) and human placental lactogen (hPL) (Bischof et al., 2005). In principle, fusion processes between trophoblastic cells can be stimulated by endothelial growth factor (EGF), hCG, estrogen and dexamethason. Moreover, an upregulation of trophoblastic cell function occurs via the cyclic adenosine monophosphate (cAMP), cAMP-dependent protein kinase A (PKA) pathway (Kudo et al., 2002; Knerr et al., 2005). The placental differentiation markers hCG and hPL are increased following syncytial differentiation, and syncytin-1 mRNA and glycoprotein expression are colinear with cytotrophoblast differentiation and hCG expression (Frendo et al., 2003b). Following stimulation with cAMP agonists or the adenylate cyclase activator forskolin, human trophoblasts exhibit a rapid increase of syncytin-1 transcripts within the first 24h of culture along with its transcription factor GCMa (Figure 1). In addition, syncytin-1 transcript levels in placental villi specimens are positively correlated with gestational age (Knerr et al., 2004).

mRNA amounts [relative units]

10.0

*** ***

7.5

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Syncytin-1/ß-actin

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l 24 h

tro co n

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l 24 h

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co

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Figure 1. Gene expression of GCMa, syncytin-1 and connexin43 (Cx43) normalized to ß-actin in primary term human trophoblasts after incubation at normoxia (21%O2) with 100 µM forskolin for different incubation periods (24h, 48h and 72h). p<0.001 (***) vs. unstimulated controls. Methodological details are reported elsewhere (Knerr et al., 2005). Following an early increase, we observed a progressive decrease of syncytin-1 and GCMa transcripts after 48h of cell culture, indicative of a regulatory limitation of the fusion process. Peak levels of Cx43 mRNA were found after 24h of culture which is related to an initiation of syncytialisation and activation of gap junctions.

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The syncytiotrophoblast forms a selective maternal-fetal barrier in the human placenta and this essential cell layer is formed by the cell-cell fusion of villous cytotrophoblasts. Moreover, it provides the transport interface between the mother and fetus regulating oxygen exchange, metabolic supply and endocrine functions such as the production of steroid hormones, human chorionic gonadotropin (hCG) and human placental lactogen (hPL) (Bischof et al., 2005). In principle, fusion processes between trophoblastic cells can be stimulated by endothelial growth factor (EGF), hCG, estrogen and dexamethason. Moreover, an upregulation of trophoblastic cell function occurs via the cyclic adenosine monophosphate (cAMP), cAMP-dependent protein kinase A (PKA) pathway (Kudo et al., 2002; Knerr et al., 2005). The placental differentiation markers hCG and hPL are increased following syncytial differentiation, and syncytin-1 mRNA and glycoprotein expression are colinear with cytotrophoblast differentiation and hCG expression (Frendo et al., 2003b). Following stimulation with cAMP agonists or the adenylate cyclase activator forskolin, human trophoblasts exhibit a rapid increase of syncytin-1 transcripts within the first 24h of culture along with its transcription factor GCMa (Figure 1). In addition, syncytin-1 transcript levels in placental villi specimens are positively correlated with gestational age (Knerr et al., 2004).

Transcription Factor GCMa (Glial Cells Missing A) Glial cells missing a (GCMa), discovered in Drosophila, is a member of a transcription factor family characterized by a zinc-containing DNA binding domain at its aminoterminus (Cohen et al., 2003). Two different family members are known in mammals: GCMa is found in syncytiotrophoblast, thymus, kidneys and astrocytes, GCMb in parathyroid tissue (Hashemolhosseini and Wegner, 2004). Two GCMa binding sites have been identified in the syncytin-1 promoter region (Janatpour et al., 1999) which supports the assumption that syncytin-1 is a target gene of GCMa (Prudhomme et al., 2004, Yu et al., 2002). We have described in detail the cAMPdriven PKA-dependent/GCMa/syncytin-1 signaling pathway (Knerr et al., 2005; Figure 2). Recently, we reported that the cAMP-PKA pathway activates bZIP-type transcription factors CREB (cAMP response–element binding protein) and OASIS (old astrocyte specificallyinduced substance), thereby stimulating GCMa gene transcription (Schubert et al., 2008). Moreover, there are other posttranslational events that modulate GCMa activity, like CBP (CREB binding protein)-mediated acetylation (Chang et al., 2005).

Connexin 43, a Putative Effector Molecule At the cellular level, syncytialisation requires an up-regulation of cell-cell communication and contact. Cellular interaction is enabled by gap junctions localized in the

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Stimulus: Forskolin

ATP Adenylatcyclase cAMP

PKA

Nucleus CREB

Nucleocytoplasmatic shuttling

Cytosol

Interaction TORC1

Target genes: Aromatase

Interaction Pitx2 (Modulation) CBP (Acetylation)

Effect: Formation of placental syncytiotrophoblast

Figure 2. Model demonstrating the known mechanisms of regulation and target genes of syncytin-1 in trophoblastic cells. One of the master regulators of placental cell fusion is the transcription factor GCMa which is regulated at different levels within a network of other transcription factors. The cAMPdriven protein kinase A signaling pathway is fundamental for the up-regulation of GCMa and syncytin1. Further details are presented elsewhere (Schubert el., 2008).

plasma membrane. They allow the exchange of metabolites and signaling molecules such as Ca2+ and cAMP. Gap junctions are constructed of eight membrane proteins called connexins (Cx). Several different Cx families can be found in the placenta. It appears that Cx43 is required for the fusion of cytotrophoblasts to build the syncytiotrophoblast, whereas Cx40 may play a role in the switch from a proliferative to an invasive phenotype of cytotrophoblast (Cronier et al., 2003, Knerr et al., 2005, Malassiné and Cronier, 2005). It has been shown that a stimulation of the cAMP/PKA signaling pathway leads to a CREB / OASIS / GCMadependent increased expression of Cx43 and the simultaneous loss of adherence markers such as desmosomal desmoplakin (Douglas et al., 1990; Knerr et al., 2005, Schubert et al., 2008). Direct interaction of Cx43 in cell-cell fusion has been demonstrated using antisense oligonucleotides which was followed by a reduced fusion rate and altered expression of syncytin-1 and hCG (Frendo et al., 2003a). Conversely, higher amounts of GCMa and syncytin mRNA are followed by higher Cx43 expression levels in human trophoblasts (Figure 1).

Human Endogenous Retroviruses (HERVs) In the 1970s, investigations on baboon placentas using electron microscopy revealed the existence of HERVs. Distinctive particles, having strong similarities to retroviruses, were punched of the syncytiotrophoblast (Ryan et al., 2004, Harris et al., 1998). About 5-8% of the

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human genome consists of retroviral genome sequences derived from the typical retroviral gene sequence 5’-LTR-gag-pol-env-LTR-3’ (Gifford et al., 2003; Medstrand et al., 2002, Bannert et al., 2004). However, during the past million years, open reading frames are rarely kept intact and genetic information has been lost due to mutations. The origin of HERVs is still not clear and there are several different theories. HERVs could be the legacy of ancient germ cell infections by exogenous retroviruses, dating from 60 million years ago to the present (Ryan et al., 2004). In many cases, however, exogenous counterparts could not be identified, or may have disappeared (Loewer et al., 1996). It seems that HERVs could be involved in various pathologies in humans. HERV elements have been detected in malignancies such as seminoma, chorioncarcinoma, breast cancer, and renal cancer, but a direct activation of oncogenes could not be demonstrated (Gifford et al., 2003; Nelson et al., 2004; Ryan et al., 2004). Moreover, hereditary diseases have been discussed in the context of HERV-related elements, induced mutations and altered gene expression, including neurofibromatosis or Lesch Nyhan syndrome (Ryan et al., 2004). In the cerebrospinal fluid of patients with multiple sclerosis high amounts of HERV-W transcripts have been detected, which could be depressed following successful treatment with interferone ß. Conversely, interferone γ and TNF α activate macrophages which may be followed by an overexpression of HERV-W (Johnston et al., 2001; Taruscio et al., 2004). Therefore, the physiological and pathophysiological role of HERV-W during inflammation is, as yet, unclear. It is of interest to know that syncytin-1 covers a 25-amino acid sequence (residues 373 to 397) conserved in other retroviral envelope proteins which mediates immunosuppression (Mangeney et al., 2007). This may explain the immune tolerance of cells to HERV. The placenta should protect the fetus against the maternal immune system and resist infection (Muir et al., 2004). Retroviral envelopes could theoretically mediate such functions with receptor interference, and their immunosuppression and fusion properties. Conversely, dysfunction of the maternal immune system may contribute to an increased release of placental cell detritus into the maternal circulation, systemic inflammatory reaction and, eventually, clinical symptoms such as in pre-eclampsia (Sacks et al., 1998; Bachmayer et al., 2006). It may be that reduced syncytin-1 expression, together with an altered distribution in the syncytiotrophoblast, plays a pathophysiological role, especially through a diminished immunosuppressive effect in the placenta.

Clinical context: Hypertensive Disorders of Pregnancy Disorders of placental development may be followed by increased maternal and fetal morbidity, in particular hypertensive disorders of pregnancy such as pre-eclampsia or the HELLP syndrome, and fetal intrauterine growth restriction (IUGR). Pre-eclampsia is defined by the onset of arterial hypertension (blood pressure >140/90mmHg), proteinuria with renal dysfunction (> 300mg protein excretion/24h) and peripheral edema. The HELLP syndrome is characterized by hemolysis, elevated liver enzymes and low platelets.

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Disease specific incidences are 6% to 8% for hypertensive disorders in general and 3.9/1000 pregnancies for severe pre-eclampisa, 0.5/1000 for the HELLP syndrome, 0.2/1000 for eclampsia with convulsions (Waterstone et al., 2001, Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy, 2000). Etiology and pathogenesis of hypertensive disorders of pregnancy are multifactorial and still not completely understood. Essentially, maternal risk factors for endothelial and vascular diseases, such as preexisting arterial hypertension and diabetes mellitus, predispose to these disorders (Al-Muhamid et al., 2003). It is of interest that clinical symptoms occur as late as the 2nd and at 3rd trimenon of pregnancy, while placental alterations start early in placentogenesis. A possible explanation could be that disturbed placentogenesis with reduced syncytiotrophoblast formation may manifest only after placental volume and fetal demands increase. Histological investigations exhibit poor invasion of cytotrophoblasts in maternal uterine arteries along with disturbed remodelling of spiral arteries. Large intervillous lacunae in the placenta which, under normal conditions, are responsible for adequate blood supply are formed in a defective way, which may result in a chronic hypoxic state (Pijnenborg et al., 1998, Redman and Sargent, 2003). As a consequence, pre-eclampsia can be associated with IUGR. It is of interest to know that comparable processes are not well documented in other species (Redman and Sargent, 2003).

Syncytin and GCMa: Alterations Inin PreEclampsia Andand UUnder Hypoxia Syncytin-1 gene expression is reduced in placentas of women with pre-eclampsia or HELLP syndrome (Knerr et al., 2002; Chen et al., 2006). Additionally, altered protein localization in the apex of the syncytiotrophoblast was observed (Lee et al., 2001). Considering that pre-eclampsia is typically associated with local hypoxia, it was of interest to focus on syncytin-1 expression under experimental hypoxia. A decrease of syncytin-1 and GCMa expression could be demonstrated (Knerr et al., 2005, Kudo et al., 2003). Reduced oxygen availability in the placenta may lead to a reduced syncytin-1 gene expression which, in certain circumstances, may be followed by disturbed placentogenesis. Normal placental cell differentiation of cytotrophoblasts to the syncytiotrophoblast layer is reduced under hypoxia and the proliferative cell phenotype increases (Soleymanlou et al., 2005). Consequently, defective fusion processes occur and can be demonstrated, for example, by a decreased immunocytochemical detection of Cx43 and persistent desmoplakin staining. To a certain extent, stimulation of the cAMP/PKA-pathway can attenuate hypoxia-related effects and facilitate syncytialization (Knerr et al., 2005). The cellular mechanisms include many other elements and need to be further elucidated.

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Syncytin Receptor, the Amino Acid Transporter ASCT2 The first known syncytin binding site is a sodium-dependent neutral amino acid transporter at the basal membrane of syncytiotrophoblast, which transports alanine, serine, and cysteine. It is named by the acronym ASCT2 or type D retrovirus receptor, RDR (Sommerfelt et al. 1990; Rasko et al., 1999; Kudo and Boyd, 2002). Syncytin-1 is also capable of binding to the transporter ASCT1 (Marin et al., 2000). The degree of structural homology between ASCT1 and 2 is 57% (Zerangue et al., 1996, Kudo and Boyd, 2002). In transfected CHO cells, intercellular fusion of ASCT1 overexpressing cells was even higher than the fusion rate of ASCT2-transfected cells (Lavilette et al., 2002). In humans, ASCT1 is predominantly found in liver and brain. During the process of syncytialisation, syncytin-1 mRNA amounts increase in 1st trimenon placental tissue, but ASCT2 receptor expression decreases after the 13th week of gestation which could be interpreted as a limitation of cell-cell fusion (Chen et al., 2006). In placentas of women with pre-eclampsia or HELLP syndrome an almost constant ASCT2 mRNA level could be detected throughout pregnancy (Chen et al., 2006). ASCT2 gene expression is not significantly affected under hypoxic cell culture conditions (Knerr et al., 2003). Moreover, ASCT2 expression is down-regulated after stimulation of trophoblast cell fusion, and not associated with the proliferative status of trophoblasts (Hayward et al., 2007).

Interaction of Syncytin-1 with Apoptosis Elements Apoptosis, or programmed cell death, is an essential mechanism in early human development and for maintaining tissue homeostasis. Early apoptotic steps and syncytium formation share common mechanisms, such as changes in plasma membrane phospholipid orientation, particularly the phosphatidylserin-flip (Poetgens et al., 2002, Lyden et al., 1993). In trophoblasts, however, cell death seems to be deferred after fusion processes, and the apoptotic cascade initiated in the villous cytotrophoblast, in turn promotes syncytial fusion (Huppertz et al., 1998). Using a cell culture model of syncytin-1 overexpressing cells it has been shown that syncytin-1 is capable of anti-apoptotic functions. A lower apoptotic response has been found in syncytin-1 transfected cells compared with mock transfectants, in particular, a lower level of executioner caspase 3 along with higher amounts of anti-apoptotic Bcl-2 (Knerr et al. 2007).

Excursion: Mouse Placenta In the mouse placenta, fusogenic Env proteins of retroviral origin have been also detected and labeled, in analogy to syncytin-1 and 2, syncytin-A and B (Dupressoir et al., 2005). There is a high conformity in genomic sequence and protein structure between the

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human and mouse Env proteins which may be helpful for further studies of normal and abnormal pregnancies (Peng et al., 2007). Constitutive knockout mice for GCMa show embryonal death at mid gestation. Embryonic malformation was not observed, so an underlying placental pathology was suspected. In detail, GCMa knockouts are unable to develop a functional syncytiotrophoblast layer and as a consequence the embryo can not be supplied by sufficient amounts of nutrients and oxygen (Schreiber et al., 2000). Syncytin-1/A knockout mice are not available.

Conclusion Formation of the placental syncytiotrophoblast is a complex process. Its success, together with a normal development of fetus and placenta, is regulated by various factors. We propose that syncytin-1, a glycoprotein of endogenous retroviral origin, may exert fusogenic, antiapoptotic and immune modulatory properties and heretofore unknown influences on the cell membrane which together facilitate cell-cell fusion between trophoblasts. Alterations of this process, such as hypoxic conditions, may eventually lead to disorders of pregnancy such as pre-eclampsia and IUGR.

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Index A abnormalities, vii, 4, 31, 102 abortion, ix, 18, 24, 27, 61, 111, 113, 118, 119, 120, 122, 142 absorption, 51 accuracy, 70, 73 acetylation, 152, 157 acid, 5, 9, 10, 14, 22, 25, 46, 51, 80, 89, 130, 141, 146, 154, 156, 159, 160, 161 ACM, 76 ACS, 26 actin, 14, 151 activation, ix, 4, 25, 33, 34, 47, 94, 100, 105, 151, 154, 158 acute, 9, 34, 118, 121 acute coronary syndrome, 9 Adams, 83 adaptation, 5, 33 adenosine, 151, 152 adhesion, viii, 65, 93, 95, 96, 97, 101, 103, 104 administration, 20, 34, 66, 67, 68, 84, 85, 101, 102, 103 adult, 98, 105 adults, 37 aetiopathogenesis, 4 age, x, 12, 13, 20, 21, 32, 39, 64, 73, 75, 120, 135, 144, 151, 152, 160 agent, 37, 50, 141 agents, 33, 64, 140 aging, 147 agonist, viii, 31, 67, 68, 77, 87 airways, 142 alanine, 156

albumin, 44 alkalosis, 138 allantois, 73 allograft, 95, 112 alopecia, 141 alpha, 22, 23, 24, 25, 26, 28, 29, 75, 78, 85, 98, 100 alpha-fetoprotein, 22, 24, 25, 26, 30 alternative, 25 alters, 80, 88, 101, 159 Amazon, 34, 38, 88 amino, 5, 9, 10, 14, 55, 80, 98, 136, 154, 156, 159, 160, 161 amino acid, 5, 9, 10, 14, 80, 98, 136, 154, 156, 159, 160, 161 amino acids, 5, 98, 136 amniotic, 73 ampulla, 42, 44, 45 anaemia, x, 135, 137 analysis of variance, 115, 126 anatomy, 76 aneuploidy, vii, 3, 4 angiogenesis, viii, 4, 5, 10, 24, 93, 94, 95, 97, 98, 100, 101, 102, 107, 146 Angiogenesis, 102, 107 angiogenic, viii, 5, 10, 25, 27, 30, 94, 100, 102, 104, 107 angiogenic process, 102 Angiotensin, 67 animals, ix, 32, 33, 36, 38, 39, 40, 43, 45, 54, 62, 63, 65, 68, 70, 71, 76, 79, 88, 95, 112, 113, 114, 116, 118, 125 Animals, 75, 79, 80, 83, 85, 89, 125, 132, 133 anomalous, 40 antagonist, 34 anti-angiogenic, 10, 25

Index

164 anti-apoptotic, xi, 150, 156, 157, 159 antibodies, 115, 139 antibody, ix, 54, 94, 100, 112, 113, 115, 121, 125, 126, 139 antigen, 17, 18, 27, 28, 105, 133 antioxidant, 66 antisense, 150, 153 antisense oligonucleotides, 150, 153 anti-tumor, 25 apoptosis, 66, 78, 86, 96, 98, 101, 106, 107, 137, 146 apoptotic, xi, 150, 156, 157, 158, 159 APP, 11, 12, 13, 22, 28 application, 27, 41, 42, 62, 121, 131, 150 Argentina, 33, 37 arterial hypertension, 154, 155 arteries, 4, 9, 14, 60, 75, 107, 155 artery, vii, 3, 7, 8, 12, 14, 15, 16, 20, 24, 26, 27, 28 ascites, 137 Asia, x, 135 Asian, 37 assessment, 24, 54, 139 astrocyte, 152 astrocytes, 152 ATPase, 84 atresia, 66 attachment, 50, 51, 52, 54, 55, 56, 60, 67, 74, 79, 80, 101 Aurora, 125 Australia, 88 autocrine, 10, 137 autoimmune, 103, 119 autoimmune disease, 119 autoimmune diseases, 119 availability, xi, 32, 33, 37, 38, 39, 41, 149, 155

B B cell, 18 babies, 10, 12, 120 barrier, 51, 52, 104, 118, 151, 152 basal lamina, 51 basement membrane, 139 Bcl-2, xi, 150, 156 beef, 79, 83, 119, 122, 132 Belgium, 93, 103, 111, 115, 123, 125, 131 beneficial effect, 68 benefits, 11, 16 bias, 112 binding, viii, 5, 10, 14, 18, 23, 24, 25, 27, 50, 51, 53, 84, 90, 94, 97, 98, 99, 101, 106, 152, 156, 158 bioavailability, 10, 23

biochemistry, vii, 3 biological activity, 5 biomarkers, 4, 5, 6, 9, 13, 20 biometry, 75, 83, 86 biopsies, 99 biotechnologies, 33, 87 birth, 12, 20, 22, 23, 27, 39, 87 birth weight, 12, 20, 22, 23 births, x, 38, 102, 135, 136 bison, 131, 133 bladder, 138 blastocyst, viii, ix, 42, 47, 48, 49, 50, 84, 93, 94, 95, 96, 99, 100, 101, 102, 103, 104, 105 bleeding, x, 135, 137, 138, 142 blood, vii, x, 9, 14, 15, 20, 28, 31, 44, 45, 46, 53, 55, 56, 57, 58, 66, 70, 71, 75, 84, 87, 101, 102, 114, 124, 125, 135, 154, 155, 160 blood collection, 114 blood flow, vii, 14, 20, 31, 71, 75 blood plasma, 84 blood pressure, 154 blood stream, 46 blood supply, 102, 155 blood vessels, 56, 57, 58, 70, 102 bloodstream, 95 body fluid, 22 body weight, 34, 160 bonds, 10, 14 bone marrow, 141 Bose, 160 bovine, vii, x, 25, 31, 33, 39, 40, 42, 44, 46, 47, 48, 49, 52, 53, 54, 55, 56, 60, 62, 63, 65, 66, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 112, 114, 115, 120, 121, 124, 125, 126, 130, 131, 132, 133 brain, 120, 136, 138, 140, 156, 158 Brazil, 33, 37, 38, 63, 75, 76, 77, 78, 82, 84, 88, 91, 133 Brazilian, 85 breast cancer, 154 breathlessness, 138 breeder, 33, 88 breeding, viii, 32, 33, 35, 36, 37, 54, 62, 63, 65, 68, 70, 73, 82, 87 brucellosis, 114 buffalo, vii, ix, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

Index 83, 84, 85, 86, 87, 88, 89, 90, 91, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133 buffer, 125, 126 Bulgaria, 78

C calcium, 45, 51, 84, 90, 102 calf, 33, 37, 39 calving, vii, 31, 32, 33, 37, 38, 39, 40, 41, 42, 54, 61, 63, 66, 77, 80, 87, 133 cAMP, ix, xi, 25, 94, 98, 100, 149, 151, 152, 153, 155, 159 cancer, 104, 146, 154 cancer cells, 104 candidates, 8 capillary, 59, 102 caprine, viii, 32, 54, 55, 115, 124, 130, 131 carbohydrate, 14, 17, 130 carbohydrates, 5, 9, 47 carcinoembryonic antigen, 17, 18, 27, 28 carrier, 51 caspase, xi, 150, 156 catabolism, 16 cattle, vii, 31, 39, 42, 44, 47, 48, 49, 50, 51, 53, 54, 55, 56, 59, 61, 62, 64, 66, 67, 68, 73, 75, 79, 80, 81, 83, 85, 87, 88, 90, 113, 120, 121, 124, 130, 132, 133 cDNA, 25, 27, 29, 98, 131 CEA, 17, 18, 28 cell, ix, xi, 10, 14, 15, 18, 26, 27, 49, 75, 80, 81, 89, 90, 96, 97, 101, 104, 106, 112, 117, 118, 119, 120, 137, 146, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159 cell adhesion, 101 cell culture, xi, 75, 150, 151, 156 cell death, 156 cell differentiation, 97, 150, 155 cell division, 49 cell fusion, xi, 89, 149, 150, 151, 152, 153, 156, 157, 158, 159 cell growth, 14 cell invasion, 18, 137 cell line, 27 cell surface, 10, 15, 159 central nervous system, x, 33, 135, 139 cerebrospinal fluid, 154 cervical ganglia, 34 cervix, 70 cheese, 41, 42 chemical structures, 35

165

chemokine, 75 chemokines, 104 chemotherapy, x, 136, 139, 140, 141, 142, 143, 144, 146 chest, x, 73, 135, 138, 139 chloride, 76, 161 chlorine, 45 CHO cells, 156, 159 cholestasis, 26 cholesterol, 61, 81 chorioallantois, 52 choriocarcinoma, x, 135, 136, 141, 142, 144, 146, 159 chorion, 49, 55, 56, 124 chorionic gonadotropin, 21, 24, 78, 104, 105, 106, 107 chorionic villi, 51, 60, 137 chromatin, 51 chromatography, 28, 55, 75, 133 chromosome, 17, 28, 85, 98, 150, 161 chromosomes, 85 CIL, 93 circadian, 34, 36, 83 circadian rhythm, 34, 83 circadian rhythms, 34 circulation, 17, 18, 53, 59, 61, 113, 114, 127, 131, 154 cisplatin, 142, 146 classes, 98, 120 classification, 22, 28, 52, 139 cleavage, 10, 25 cleavages, 44 climatic factors, 78 clinical assessment, 138 clinical symptoms, 4, 103, 154, 155 cloning, 14, 27 CNS, 33 coagulation, 4, 10 codes, 98 cohort, 11, 70 collaboration, 95, 97 collateral, 56 College Station, 86 communication, 63, 152, 158 competence, 67 competency, 47 compilation, 159 complement, 103 complications, 11, 12, 18, 22, 24, 25, 26, 28, 29 components, 56, 112

166 composition, 44, 75, 89 computed tomography, x, 135 concentration, 6, 8, 9, 18, 19, 38, 39, 44, 45, 46, 50, 54, 55, 61, 65, 66, 67, 80, 81, 83, 101, 126, 127, 130 conception, 11, 35, 39, 40, 63, 64, 67, 68 confidence, 68 conflict, 41, 112 conformity, 156 confusion, 138, 139 conjunctivitis, 141 control, 7, 15, 18, 42, 76, 82, 86, 97, 103, 105, 114, 120, 161 conversion, 104 convex, 56 coral, 38 corpus luteum, 5, 50, 61, 63, 64, 65, 66, 67, 68, 71, 75, 76, 78, 79, 80, 84, 98, 101, 102 correlation, 99, 119, 124, 126 correlation coefficient, 126 correlations, 78 corrosion, 82 corticosteroids, 79 cotyledon, 56, 57, 60 cough, 138 covalent, 98 cows, viii, ix, 32, 54, 61, 66, 68, 71, 75, 76, 77, 78, 79, 81, 83, 84, 86, 87, 88, 89, 90, 91, 111, 112, 113, 114, 116, 117, 118, 119, 120, 121, 122, 123, 125, 128, 130, 132 COX-2, 101, 102 COX-2 enzyme, 101 CPAP, 142 cranium, 73 CREB, 152, 153, 160 crosstalk, 104 cross-talk, viii, 93, 94, 95, 96 cryptorchidism, 99 CSF, 101 culture, 23, 49, 81, 94, 151, 152 curettage, x, 135 cycles, 34, 39, 40, 71, 99, 105 cyclic AMP, 25, 157 cycling, 72, 86 cyclooxygenase, 106 cyclooxygenase-2, 106 cyclophosphamide, 146 cyst, 120 cysteine, 156 cysts, vii, 31, 137

Index cytochemistry, 89 cytokine, 97, 99, 100, 104, 112, 157 cytokines, viii, 18, 51, 93, 97, 112, 113, 120 cytokinesis, 52 cytoplasm, 51, 52, 53 Czech Republic, 3

D dairy, viii, ix, 32, 33, 54, 68, 75, 76, 83, 87, 88, 111, 113, 114, 117, 119, 120, 121, 122, 130, 132 dating, 154 death, 83, 103, 144, 156, 157 defense, 77 deficiency, 16, 39 definition, 140 degradation, 25, 145 degrading, 10 delayed puberty, 32 delivery, 11, 12, 15, 40, 41, 62, 76, 141 density, 34, 52 depressed, 154 detection, 7, 8, 11, 12, 15, 16, 22, 54, 73, 78, 88, 115, 126, 139, 155 detritus, 154 developing countries, 40, 74 diabetes, 26, 103, 107, 155 diabetes mellitus, 26, 155 diarrhea, 114 dibutyryl cAMP, ix, 94, 100 diet, 37, 41 differential diagnosis, 8, 23 differentiation, xi, 10, 18, 49, 53, 97, 99, 101, 102, 104, 105, 149, 150, 151, 152, 155, 158 diffusion, 44, 45, 51 dimer, 9, 14 dimeric, 100 dimerization, 14 direct action, 103 disease activity, 140, 142 diseases, 5, 114, 119, 139, 154 disorder, 4 dispersion, 47 dissociation, 10, 144 distress, 142 distribution, 23, 32, 38, 58, 76, 90, 121, 131, 154, 158 disulfide, 10, 14 disulfide bonds, 10, 14 diversity, 158 division, 49

Index DNA, 73, 152, 158 dogs, 114, 120 domestication, 33 dominance, 73 donors, 79 dopamine, 34 Doppler, vii, 12, 16, 20, 24, 26, 27, 28, 31, 69, 71 dosage, 40 Down syndrome, vii, 3, 4, 6, 7, 9, 29 down-regulation, xi, 5, 18, 150 Drosophila, 152 drug resistance, 141, 143 drugs, 140, 141 duration, 33, 34, 101, 118 dysregulation, 158

E eclampsia, vii, x, xi, 3, 21, 22, 25, 26, 27, 28, 29, 102, 135, 138, 150, 154, 155, 156, 157, 158, 159, 160 edema, 73, 154 education, 155, 160 egg, 33, 86 Egypt, 37 eicosanoids, 102 elaboration, 95 electron, 52, 76, 82, 85, 90, 91, 121, 131, 153 electron density, 52 electron microscopy, 82, 153 ELISA, viii, 32, 55, 115 elongation, 49, 50, 66 embryo, vii, viii, ix, 31, 42, 46, 47, 49, 50, 51, 52, 55, 62, 63, 65, 68, 69, 70, 73, 74, 77, 79, 80, 81, 83, 88, 93, 94, 95, 96, 97, 99, 100, 101, 103, 104, 157 embryogenesis, 18 embryonic development, 44, 65, 67, 68, 77, 85 embryonic stem, 87 embryonic stem cells, 87 embryos, 46, 47, 48, 49, 68, 73, 75, 76, 80, 81, 82, 83, 87 encephalomyelitis, 120 encoding, 29 endocrine, 34, 36, 40, 55, 61, 74, 81, 83, 98, 101, 103, 151, 152 endocrine system, 103 endocrinology, viii, 93, 95 endometrial glands, 52, 65 endometritis, 39

167

endometrium, viii, 14, 18, 26, 50, 52, 53, 54, 55, 62, 65, 73, 75, 78, 82, 88, 93, 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106 endothelial cells, viii, 5, 94, 100, 102 endothelial dysfunction, 4 endothelin, 67 endothelium, 100 energy, 39, 41, 45, 74, 81, 84 England, 90, 147 environment, vii, 31, 32, 39, 44, 49, 50, 63, 83, 103, 120 environmental conditions, 38, 63, 90 environmental factors, 39, 136 enzymatic, 61 enzyme-linked immunosorbent assay, 115 enzymes, 10, 51, 62, 154 epidemiology, 145 epidermal growth factor, 97 epigenetic, 63 epithelia, 106 epithelial cells, viii, 44, 46, 49, 51, 66, 75, 78, 85, 94, 96, 99, 101, 105, 106 epithelium, 44, 50, 51, 52, 53, 56, 60, 82, 84, 96, 99, 100, 101, 103, 105 epitopes, 124, 159 equilibrium, 78 erythrocytes, 52 estradiol, 65, 75, 79, 89 estriol, 30 estrogen, 88, 94, 151, 152 estrogens, 79 etiologic agent, 33, 37 evacuation, x, 135, 138, 139 evolution, 102, 104, 157, 158 ewe, 61, 80, 81, 133 examinations, 68, 70 excretion, 154 exocrine, 136 exons, 98 exploitation, 6 extracellular matrix, 14, 101

F faecal, 70 failure, 4, 79, 94, 95, 102, 120, 160 false negative, 70, 139, 144 false positive, 70, 139 family, x, xi, 10, 14, 17, 24, 28, 49, 53, 75, 76, 80, 87, 90, 98, 124, 132, 136, 150, 152, 158, 159 family members, 152

Index

168 farms, 36, 37, 38, 39, 64, 65 Fas, 103, 107 feedback, 16, 50 females, x, 37, 70, 73, 77, 99, 123, 124, 127, 130, 131 fertility, 32, 41, 62, 63, 74, 78, 81, 82, 86, 89, 90, 114 fertilization, 42, 44, 46, 47, 48, 62, 63, 94, 130 fetal, vii, viii, ix, 15, 18, 24, 26, 28, 31, 49, 52, 55, 59, 60, 61, 62, 66, 68, 70, 73, 74, 75, 83, 84, 85, 87, 88, 93, 94, 95, 96, 97, 100, 101, 102, 103, 106, 107, 111, 112, 113, 117, 118, 119, 120, 121, 132, 151, 152, 154, 155 fetal growth, 24, 26 fetomaternal, 89, 113 fetus, 51, 56, 59, 60, 70, 95, 112, 113, 118, 119, 122, 150, 151, 152, 154, 157 fetuses, 73, 74, 83, 114, 118, 121, 132 fibroblasts, 25 Finland, 126 fish, 56 FISH, 85 floating, 50, 73, 74 flow, vii, 14, 20, 31, 71, 75, 84 fluid, 44, 45, 46, 47, 73, 74, 75, 77, 81, 82, 84, 89, 154 flushing, 48 follicle, 5, 64, 67, 70, 71, 75, 76, 83, 87, 107 follicle stimulating hormone, 5 follicles, 25, 39, 68, 70, 75 follicular, vii, 31, 39, 45, 68, 71, 73, 75, 76, 77, 83 follicular fluid, 77 Fox, 9, 23 fracture, 84 FSH, 5, 40, 98 fusion, xi, 47, 52, 53, 89, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161

G G protein, 98, 104 gametes, 42, 47, 80 Gamma, v, ix, 111, 112, 116 ganglia, 34 gastrointestinal, 61 gastrointestinal tract, 61 gender, 75 gene, 16, 17, 28, 49, 75, 77, 96, 98, 104, 106, 107, 113, 150, 152, 154, 155, 156, 157, 158, 161 gene expression, 77, 96, 104, 154, 155, 156 generation, 82

genes, xi, 17, 23, 28, 53, 66, 82, 88, 98, 101, 104, 106, 136, 149, 150, 153, 158, 160 genetic factors, 136 genetic information, 154 genome, 47, 98, 154, 157, 160 genome sequences, 154 genomic, 104, 156 genotypes, 37 Germany, 83, 125, 149 gestation, viii, ix, x, 4, 6, 11, 13, 15, 16, 19, 20, 25, 26, 28, 31, 33, 40, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 65, 73, 79, 81, 85, 101, 102, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 124, 127, 132, 133, 134, 135, 137, 156, 157 gestational age, 21, 75, 151, 152 gestational diabetes, 26 gland, 34, 106 glial, xi, 149, 150, 152, 160 glial cells, xi, 149, 150, 160 glioblastoma, 10, 25 globulin, 17, 23, 28 glucose, 44, 45, 51, 82 GLUT, 77 glycans, 55, 82 glycol, 125 glycoprotein, vii, ix, xi, 3, 4, 5, 9, 17, 20, 21, 22, 23, 24, 25, 27, 28, 29, 50, 76, 78, 79, 81, 82, 83, 85, 89, 90, 91, 98, 105, 111, 112, 115, 116, 117, 121, 122, 131, 132, 133, 136, 149, 150, 151, 152, 157, 158, 159 glycoproteins, viii, ix, 18, 27, 32, 46, 47, 53, 75, 79, 80, 81, 83, 85, 90, 98, 113, 118, 121, 123, 124, 127, 128, 129, 131, 132, 133, 134 glycosylated, 5, 98 glycosylation, 81, 98, 131, 133 gonad, 34 gonadotropin, viii, 21, 22, 24, 25, 26, 27, 28, 29, 30, 76, 78, 84, 87, 93, 95, 104, 105, 106, 107, 146, 147, 151, 152 Gore, 28 G-protein, 98 grants, 119 granules, 51, 52, 53, 61 granulocyte, 54 granulosa cells, 66 grazing, 37 groups, 6, 10, 15, 16, 17, 19, 113 growth, xi, 4, 5, 10, 12, 14, 18, 19, 23, 24, 25, 26, 27, 49, 51, 53, 65, 67, 71, 75, 79, 81, 87, 88, 89,

Index 96, 97, 99, 102, 105, 106, 107, 132, 137, 149, 150, 151, 152, 154 growth factor, 5, 10, 18, 23, 24, 25, 49, 51, 65, 87, 89, 96, 97, 99, 102, 105, 106, 107, 151, 152 growth factors, 5, 10, 49, 51, 65, 87, 89, 96, 102, 105, 106 growth hormone, 53, 75, 97, 132 guidance, 145

H H19, 136 haemoptysis, 138 half-life, 11, 19, 126, 129, 130, 131 headache, 138 health, 40, 87, 150, 158, 160 heart, 73, 120 heartbeat, 73, 74 heat, 88 height, 59, 73 hematoma, 82 hemisphere, 34 heredity, 37 heterodimer, 5 heterogeneity, 17 heterogeneous, 4, 52 high risk, x, 20, 24, 135, 140, 141, 142, 146 histochemical, 76, 87, 89 histochemistry, 53, 79 histological, x, 59, 89, 135 histology, 82 histopathology, 139 homolog, 160 homology, 14, 27, 53, 137, 156 hormonal control, viii, 93 hormone, viii, 5, 36, 53, 61, 65, 75, 76, 81, 86, 87, 89, 93, 95, 97, 98, 103, 104, 105, 106, 107, 132, 136, 139 hormones, 4, 5, 29, 35, 40, 45, 51, 53, 61, 67, 68, 76, 77, 81, 86, 94, 97, 98, 119, 151, 152 horse, 33, 42 horses, 33 host, 113, 120 housing, 87 HPM, 86 human, vii, viii, ix, xi, 3, 4, 5, 11, 14, 15, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 53, 84, 85, 87, 93, 94, 95, 97, 98, 99, 100, 101, 102, 104, 105, 106, 107, 132, 136, 142, 146, 147, 149, 150, 151, 152, 153, 154, 156, 157, 158, 159, 160, 161

169

human chorionic gonadotropin, viii, 21, 22, 24, 25, 26, 27, 28, 29, 30, 84, 87, 93, 95, 105, 106, 107, 146, 147, 151, 152 human development, 156 human genome, 98, 154, 157, 160 human leukocyte antigen, 105 humans, xi, 50, 95, 150, 154, 156 humoral immunity, 112, 118 hybrid, 52 hybrid cell, 52 hybrid cells, 52 hybridization, 84 hydatidiform mole, x, 135, 136, 142 hydration, 141 hydrolysis, 10 hydrophobic, 136 hygiene, 39 hyperemesis, x, 135 hyperplasia, 49 hypertension, 4, 6, 7, 9, 11, 12, 13, 19, 20, 23, 24, 26, 27, 29, 138 hypertensive, 7, 8, 12, 25, 150, 154, 155 hypogonadism, 98, 105 hypospadias, 99 hypothalamic, 34, 82 hypothalamus, 39 hypothesis, 17, 18, 24, 35, 37, 44, 53, 60, 82, 130, 137 hypoxia, xi, 9, 23, 138, 149, 155, 159, 161 hypoxic, 155, 156, 157, 159 hysterectomy, x, 135

I iatrogenic, 70 ICM, 49 identification, 7, 17, 20, 24, 25, 26, 28, 55, 112, 120 identity, 98 IFN, ix, 50, 54, 111, 113, 115, 116, 117, 118, 119 IGF, 5, 10, 23, 24, 25, 26, 49, 84, 99, 101 IGF-1, 10 IGF-I, 10, 23, 24, 49, 84, 101 IL-1, 18, 27, 29, 100, 101, 106 IL-10, 18, 27, 29 IL-6, 18, 26, 27, 100, 105 Illinois, 81, 86 image analysis, 9 images, 71 imaging, x, 69, 84, 85, 135 immune cells, 97 immune response, 101, 112, 113, 118, 119

170 immune system, 17, 95, 112, 154 immunity, ix, 18, 27, 112, 113, 117, 119, 120 immunoassays, 145 immunogen, 121 immunoglobulin, 17, 24, 46 immunoglobulin G, 46 immunoglobulin superfamily, 24 immunoglobulins, 44, 85 immunohistochemical, 9, 120 immunological, viii, 15, 18, 52, 93, 94, 112, 120 immunology, viii, 93, 95, 112, 119, 120 immunomodulation, 14, 118 immunosuppression, 154 immunosuppressive, 10, 118, 154, 159 imprinting, 136 in situ, 80, 84 in situ hybridization, 84 in vitro, viii, 9, 11, 17, 18, 23, 24, 46, 48, 49, 78, 80, 86, 94, 97, 99, 100, 102, 107, 137, 158 in vitro fertilization, 94 in vivo, viii, 47, 49, 86, 94, 100, 102, 107, 137 inactivation, 102 inactive, 80, 90 incidence, viii, x, 31, 40, 41, 63, 64, 66, 67, 68, 86, 113, 135, 136 incubation, 126, 151 incubation period, 151 India, 37, 77, 83, 87, 88, 90, 91 Indian, 35, 55, 83, 86, 87 indicators, 53, 140 indices, 87 induction, 18, 68 industry, 73 infants, 20 infection, 112, 113, 114, 118, 120, 121, 154 infectious, 33, 64, 114 infertility, 98, 105 inflammation, 4, 118, 119, 154 inflammatory, viii, 18, 28, 94, 99, 112, 118, 154, 158, 160 inflammatory response, 99, 160 infundibulum, 42, 44 infusions, 34 inhibition, 34, 35, 50, 83, 100, 118, 150 inhibitor, 10, 23, 26, 65, 80, 106 inhibitors, ix, 10, 94, 97, 100, 102 inhibitory, viii, 94, 99, 105 initiation, 101, 144, 151 injection, 34, 66, 67, 75, 88, 101 inner cell mass, 49, 80, 96

Index inoculation, 121 iNOS, 18 insemination, 48, 50, 54, 63, 65, 66, 70, 76, 82, 88, 114, 124, 128 insight, 158 insulin, 5, 10, 23, 24, 25, 49, 87, 89, 99, 106, 160 insulin-like growth factor, 5, 10, 23, 24, 25, 89, 99, 106 integrins, 97 interaction, ix, 10, 14, 24, 50, 99, 101, 103, 111, 112, 116, 136, 137, 152, 153 interactions, 10, 18, 23, 65, 78, 95, 105, 115, 118 interface, viii, 18, 23, 28, 60, 78, 93, 94, 97, 100, 101, 102, 103, 105, 107, 113, 120, 151, 152 interference, 114, 126, 154 interferon, ix, 65, 66, 78, 81, 85, 88, 111, 112, 113, 116, 120 interferons, 82 interleukin, 18, 26, 99, 105, 106, 157 interleukin-1, 106, 157 interleukin-6, 26, 105 interleukine, viii, 94 internal biological clock, 34 interval, 32, 33, 39, 40, 71, 87, 140 intervention, 50 intramuscular, 66, 85 intraperitoneal, x, 135 intrauterine growth retardation, 10, 102 intravenous, 66 intrinsic, 63, 104 introns, 98 invasive, x, 68, 104, 135, 136, 137, 144, 145, 153 involution, 79 ionic, 45 ions, 44 iron, 60 ischemia, 4 isoelectric point, 17 isoforms, 28 isolation, 55 isothermal, 80 Israel, 158 Italy, 23, 31, 32, 37, 38, 40, 41, 62, 63, 76, 77, 78, 79, 83, 88, 89, 90, 91, 123, 125 IUGR, xi, 10, 19, 20, 149, 150, 154, 155, 157

J Japanese, 84 Jordan, 81 Jung, 159

Index

K kidney, 56, 140 kidneys, 61, 152 kinase, ix, xi, 94, 100, 101, 149, 151, 152, 153, 157 King, 51, 75, 81, 158 knockout, 157

L labeling, 49 lactate level, 45 lactating, 42, 75, 76, 87, 114 lactation, ix, 32, 37, 39, 40, 42, 64, 112, 115, 116 lamina, 51 language, 103 later life, 150 lectin, 55 lesions, 118 leucine, 98 leukemia, viii, 94, 105 leukocyte, 105, 118, 121 leukocytes, 160 liberation, 14 LIF, viii, 94, 96, 99, 101, 105 lifespan, 66, 80, 85 ligament, 140 ligand, 104, 107 ligands, 76 likelihood, 12, 67 limitation, 151, 156 limitations, 139 Lincoln, 33, 34, 82, 83, 107 linear, 70 linkage, 47 lipid, 39 Lipid, 89 lipid metabolism, 39 lipids, 46 liver, 15, 61, 120, 140, 154, 156 liver enzymes, 154 Livestock, 77 local action, 101 localization, 15, 79, 89, 150, 155, 159 location, 75 London, 21, 84, 89 long period, 38 longevity, 40 losses, 31, 62, 63 lover, 132

171

low risk, x, 119, 136, 141 lumbar, 139 lumbar puncture, 139 lumen, 45, 73 lung, 138 lungs, 61, 136, 140, 141 lutein, 137 luteinizing hormone, 86, 104, 105, 106, 107 lymphocyte, 22, 23 lymphocytes, 18, 120

M macromolecules, 51 macrophages, 18, 29, 154 magnesium, 102 maintenance, vii, 18, 31, 65, 68, 87, 88 major histocompatibility complex, 78 malignancy, 139, 142, 144, 146 malignant, 98, 144 malignant tumors, 98 mammalian, 79 mammals, 82, 152 management, x, 32, 36, 38, 39, 40, 63, 68, 74, 83, 86, 105, 135, 136, 145, 146, 147 manufacturer, 115 mapping, 71, 85 market, 40, 41, 42, 62 mastitis, 114 maternal, vii, viii, x, 3, 4, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 49, 51, 52, 53, 54, 55, 59, 60, 61, 63, 65, 74, 78, 79, 81, 82, 83, 84, 85, 93, 94, 95, 97, 98, 99, 101, 103, 107, 112, 113, 118, 120, 121, 124, 135, 137, 154, 155, 157, 160 maternal age, x, 135 maternal-embryo, viii, 93, 94, 104 matrix, 14, 77, 97, 101, 106 matrix metalloproteinase, 106 maturation, 66, 102 measurement, x, 6, 7, 8, 11, 13, 15, 16, 17, 20, 22, 28, 114, 116, 121, 130, 132, 135, 138, 139, 140, 144, 145, 147 measures, ix, 112, 115, 116, 146 median, 6, 7, 11, 16 mediators, viii, 4, 93, 94, 95, 96, 103 medicine, 103, 146 Mediterranean, 41, 77, 84, 91 melatonin, 34, 35, 36, 37, 76, 78, 83, 84 membranes, 23, 46, 49, 56, 65, 73, 84, 87, 106 menopause, 144

172 menstrual cycle, viii, 94, 99, 102 Merck, 125 messenger ribonucleic acid, 22 MET, 78 metabolic, 55, 151, 152 metabolism, 18, 39, 55 metabolite, 79 metabolites, 22, 153 metalloproteinase, 10 metalloproteinases, 10, 106 metastasis, x, 135, 136, 138, 139, 140, 142 metastatic, x, 135, 138, 139, 140, 142, 146, 147 metastatic disease, x, 135, 138 MHC, 97 mice, viii, 46, 94, 99, 100, 103, 106, 107, 157, 160 microcrystalline cellulose, 125 microdialysis, 101 microenvironment, 47, 107 microenvironments, 80 microscope, 90, 121, 131 microscopy, 52, 82, 153 microvascular, 102, 107 microvasculature, 60 migration, 10, 14, 52, 53, 55, 84, 90, 97, 102, 118 milk, ix, 39, 40, 41, 42, 52, 53, 62, 67, 68, 112, 114, 115, 116, 125 miscarriages, 95, 102 Missouri, 89 mitogen, 22, 102 mitogenic, 49 mitosis, 82 mitotic, 47 MMP, 97, 101 MMP-9, 101 mobility, 9, 17 modalities, 50 modeling, 80 models, viii, 94, 100 modulation, 34, 100, 101, 103, 104 mole, x, 135, 136, 138, 140 molecular biology, 53 molecular dynamics, 29 molecular mass, 55, 124, 131 molecular weight, 5, 53 molecules, x, xi, 14, 44, 47, 50, 52, 53, 55, 67, 94, 96, 99, 101, 123, 124, 130, 131, 137, 146, 149, 150, 153 monocytes, 18, 26, 27 morbidity, vii, 3, 4, 102, 154, 161 morphogenesis, 79, 89, 160

Index morphological, 51, 71, 89, 94, 105 morphology, 49, 51, 84 mortality, vii, viii, 3, 4, 31, 40, 54, 55, 62, 63, 64, 65, 66, 67, 68, 74, 76, 77, 80, 88, 102, 133 mortality rate, 40 morula, 48 mouse, 26, 79, 80, 82, 85, 102, 146, 156, 161 MRI, 139 mRNA, 13, 23, 84, 99, 151, 152, 153, 156 mucin, 85 mucosa, 70 mucous membrane, 60 multiple sclerosis, 154 multivariate, 12 muscle, 102 muscle cells, 102 mutation, 99, 105 mutations, 98, 154 myometrium, 106, 137

N N-acety, 55, 82, 130, 133 National Academy of Sciences, 85, 90 natural, 32, 33, 37, 40, 66, 157 natural killer, 157 natural selection, 33 necrosis, 142 neonatal, 4 neoplasia, x, 135, 136, 138, 146 nervous system, 82 network, 94, 95, 96, 97, 99, 103, 153 neuroendocrine, 33 neuroendocrine system, 33 New York, 21, 78 New Zealand, 120 Newton, 77, 80 NK-cell, 157 non invasive, 144 non-invasive, 68 normal, viii, x, 6, 7, 8, 9, 12, 13, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 29, 40, 71, 76, 79, 93, 94, 119, 120, 125, 136, 139, 142, 144, 145, 146, 150, 155, 157, 158, 159 normal conditions, 155 normal development, 157 Northeast, 78 N-terminal, 18, 26 nuclear, 52 nuclei, 49, 52, 80, 158 nucleus, 34, 51

Index nulliparous, 7 nutrients, 44, 47, 51, 157 nutrition, 32, 39, 46, 51, 63

O oat, 33 obligate, 113 observations, 12, 38, 83, 95 odds ratio, 7, 12, 13, 113 oedema, 4, 138 oestrogen, 45, 61, 86, 88, 139 oligonucleotides, 150, 153 oligosaccharide, 136, 137 oncogenes, 154 Oncology, 146, 147 oocyte, 42, 46, 62, 63, 65 oocytes, 39, 43, 66, 75 operator, 70 optical, 73, 115 optical density, 115 optimization, 32 organ, 14, 23, 55, 150 organism, 95, 103 orientation, 156 osmotic, 45, 47 ossification, 73 ovarian cysts, vii, 31, 137 ovariectomized, 89 ovariectomy, 61 ovaries, 43, 70, 98, 137 ovary, 39, 42, 66 overproduction, 17 oviduct, 42, 43, 44, 46, 48, 76, 79, 80, 81, 82, 85, 89 ovulation, 18, 40, 42, 45, 67, 68, 71, 76, 77, 88, 99, 101, 102 ovum, 70, 79 oxidative, 4, 9, 24, 66 oxidative stress, 4, 9, 24, 66 oxygen, xi, 149, 151, 152, 155, 157 oxygenation, 14 oxytocin, 50, 65, 66, 78, 82, 84, 86, 88

P pacemaker, 34 pain, x, 135, 138 Pakistan, 37, 80 palpation, 68, 70 paracrine, viii, ix, 10, 23, 93, 94, 95, 98, 99, 100, 101, 104

173

parasite, ix, 111, 112, 117, 119, 120 parasitemia, 118 parasites, 33, 113, 118 parathyroid, 152 Paris, 21, 104 Parkinson, 78 particles, 153 pasture, 37 pathogenesis, 113, 121, 155 pathology, 157 pathophysiological, xi, 18, 20, 149, 154 patho-physiological, 74 pathophysiological mechanisms, 18 pathophysiology, 4, 22 pathways, 105 patients, x, 6, 7, 8, 11, 12, 13, 15, 16, 18, 21, 26, 28, 29, 102, 135, 136, 137, 138, 139, 141, 142, 144, 146, 154 PCR, viii, 94, 99, 100 peak demand, 41 pelvic, 70, 73, 136 pelvis, x, 135 pepsin, 53 peptide, 18, 26, 136, 137, 146 percentile, 19 perfusion, 4, 71, 82 perinatal, vii, 3, 11, 157 periodic, 51 peripheral, 85, 133 peripheral blood, 160 peritoneal, 138 permeability, 44, 51, 88, 102, 107 personal communication, 63 PGE, 89 pH, 125 phagocytic, 52 phagocytosis, 52 pharmacological, 42 pharmacological treatment, 42 phenotype, 18, 99, 153, 155 Philadelphia, 78 phosphates, 45 phosphatidylserine, 159 phospholipids, 46, 81 phosphorylation, 14 photoperiod, 33, 34, 36, 37, 38, 39, 40, 42, 63 phylogenetic, 52 physicians, 95 physiological, vii, xi, 9, 10, 12, 17, 18, 23, 26, 31, 33, 38, 39, 42, 62, 63, 74, 98, 102, 149, 150, 154

174 physiological factors, 74 physiological regulation, 62, 63 physiology, vii, xi, 3, 34, 63, 74, 76, 80, 139, 150 pig, 77 pigs, 84 pineal, 34, 82 pineal gland, 34 pituitary, 34, 53, 98, 139, 144 pituitary gland, 98 placental barrier, 118 placental hormones, 29 plaque, 106 plasma, vii, ix, 3, 4, 9, 12, 14, 17, 20, 21, 22, 23, 25, 26, 27, 28, 29, 35, 36, 37, 44, 45, 65, 66, 68, 75, 78, 81, 82, 83, 84, 85, 96, 111, 113, 114, 115, 116, 117, 118, 119, 121, 122, 124, 126, 128, 129, 130, 132, 133, 153, 156 plasma levels, 35, 36, 37, 65, 78, 83, 121, 132 plasma membrane, 14, 96, 153, 156 plasma proteins, 17, 25, 126 platelets, 154 play, 10, 32, 39, 53, 61, 63, 74, 94, 153 pleural, 137 pleural effusion, 137 pleuritic chest pain, 138 polyethylene, 125 polymerase, 83 polymerase chain reaction, 83 polymorphonuclear, 118, 121 polynucleotide, 49, 80 polypeptides, 10, 49 polystyrene, 125 poor, 6, 32, 37, 40, 140, 155 population, 11, 22, 32, 102, 114 positive correlation, 9 postpartum, vii, x, 31, 79, 114, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133 postpartum period, 114, 124, 126, 127, 128, 129, 130, 132 potassium, 45 powder, 125 precipitation, 125, 126 prediction, vii, 3, 6, 7, 12, 16, 21, 25 predictors, 22, 26, 30, 161 preeclampsia, vii, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 158, 160, 161 pre-eclampsia, xi, 21, 22, 25, 26, 27, 28, 29, 102, 149, 150, 154, 155, 156, 157, 158, 159 pregnancy test, 5, 139

Index pregnant, vii, viii, ix, 3, 8, 11, 17, 18, 21, 23, 28, 32, 40, 42, 54, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 72, 78, 89, 101, 111, 113, 114, 116, 119, 120, 121, 124, 133, 144 pregnant women, vii, 3, 8, 11, 17, 18, 21, 23, 28, 144 preimplantation embryos, 48, 82 press, 75, 77, 122 pressure, 142, 154 preterm delivery, 7 prevention, 114 primate, 105, 157 primates, xi, 150 private, 94 probe, 70 procreation, 94 production, viii, ix, 5, 9, 10, 13, 14, 18, 21, 32, 33, 34, 37, 39, 40, 41, 42, 45, 46, 49, 50, 51, 53, 54, 61, 62, 65, 66, 67, 78, 79, 80, 83, 86, 89, 94, 97, 99, 100, 101, 105, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 125, 132, 144, 151, 152 progesterone, vii, 5, 31, 33, 40, 45, 49, 50, 54, 55, 61, 64, 65, 66, 67, 68, 75, 76, 77, 78, 79, 81, 82, 83, 84, 85, 87, 88, 89, 94, 102, 106, 132, 139 prognosis, x, 135, 140 prognostic factors, 139 program, 104, 126 programming, 150, 160 proinflammatory, 113 pro-inflammatory, viii, 28, 94, 99 prolactin, 53, 75, 81, 87, 106, 133 proliferation, 10, 23, 26, 97, 102 promoter, 137, 152, 160 promoter region, 152 prophylactic, 20 propositus, 105 prostaglandin, 54, 55, 65, 75, 78, 80, 84, 85 prostaglandins, 14, 78, 102 protection, 52, 95, 118 protein structure, 156 proteinase, 10, 53, 90, 121 proteins, vii, 3, 4, 3, 10, 11, 14, 17, 18, 20, 21, 24, 25, 27, 28, 29, 44, 46, 50, 51, 53, 54, 55, 61, 77, 83, 85, 126, 153, 154, 156, 158, 159 proteinuria, 4, 8, 11, 13, 19, 138, 154 proteolysis, 25, 146 protocol, 102 protocols, 70 protozoan, 113, 120 PRP, 53, 61 PSG, 28

Index PSP, 53 puberty, 32 puerperium, 40 pumps, 34 purification, 27 pyruvate, 44, 45

R radio, 137, 142 Raman, 87 range, 19, 34, 55, 113 rat, 98, 105 reactivity, 138 reading, 154 reagent, 115 reception, 76 receptors, 5, 10, 34, 49, 50, 53, 65, 76, 82, 84, 87, 97, 105, 107, 159 recognition, 10, 49, 51, 65, 74, 84, 86, 87, 88, 98, 130, 158 recovery, 48, 66 rectum, 70, 138 recurrence, 144 redox, 66, 87 refractory, 97, 146 regenerate, 118 regional, 78 regression, 12, 61, 65, 66, 115, 126 regression equation, 126 regression line, 115 regressions, 126 regular, 70, 99, 105, 136 regulation, xi, 5, 14, 18, 23, 24, 25, 62, 63, 67, 78, 79, 84, 107, 120, 130, 150, 152, 153, 158, 160 regulators, 153 rejection, 113, 118 relationship, 7, 13, 18, 23, 52, 85, 113, 120, 121, 132, 133 relationships, 58, 84 reliability, 40 remission, x, 136, 143 remodelling, 9, 14, 16, 65, 101, 102, 155 renal, 154 renal dysfunction, 154 renin, 26 repeatability, 36, 78 reproduction, vii, 31, 32, 33, 34, 41, 68, 76, 81, 83, 86, 88, 94, 95, 102, 104 reproductive activity, 32, 34, 37, 38, 63 reproductive organs, vii, 31, 70, 98

175

reserves, 39 residual disease, 142 residues, 9, 14, 137, 154 resistance, ix, 102, 105, 112, 117, 119, 141, 143 resolution, x, 20, 70, 135 resources, 145 respiratory, 138 responsiveness, 49 retardation, 19 retina, 34, 98, 105 retroviral, 154 retrovirus, xi, 149, 150, 156, 157, 158, 159, 160 retroviruses, 153, 158, 160, 161 Reynolds, 107 rheumatoid arthritis, 18, 23 rhodopsin, 98 rhythm, 34 rhythms, 34 ribonucleic acid, 22 risk, vii, ix, x, xi, 3, 7, 11, 13, 19, 20, 24, 27, 28, 112, 113, 119, 122, 135, 139, 140, 141, 142, 144, 146, 149, 155, 157 risk factors, x, 135, 155, 157 rodents, 34, 50 room temperature, 125, 126 ruminant, 51, 52, 55, 56, 75, 80, 84, 90, 121, 131 rural, 87

S safety, 141 sample, 115, 126 sampling, 13 SCN, 34 seasonal effects, 37 seasonality, 32, 33, 34, 37, 38, 39, 41, 42, 63, 74, 90 secrete, viii, 31, 51, 53, 65, 67 secretion, viii, 5, 9, 18, 27, 34, 35, 36, 37, 40, 45, 54, 55, 64, 65, 66, 67, 75, 78, 80, 94, 95, 98, 99, 101, 105, 118, 130, 132, 146 seizures, 138 SEM, 116, 126, 128, 129 semen, ix, 112, 114, 115, 116, 117, 118, 119, 122 sensitivity, vii, 3, 7, 9, 15, 16, 20, 33, 34, 35, 37, 39, 54, 115, 136, 139, 144 sepsis, 160 sequencing, 14 series, 8, 11, 15, 59, 73, 113 serine, 156 serum, vii, x, 3, 4, 6, 7, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

176 46, 54, 85, 87, 121, 125, 126, 130, 131, 132, 133, 136, 138, 139, 140, 143, 157 severity, 7, 8, 13, 19, 120 sex, vii, 31, 73, 75, 87, 119 sex hormones, 119 sex ratio, 87 sexual behaviour, 38 SGA, 12 shape, 49, 51, 52, 59, 61, 70, 73 shaping, 56 sheep, 32, 33, 35, 37, 40, 49, 50, 51, 53, 55, 56, 58, 61, 64, 67, 76, 82, 83, 84, 85, 88, 89, 90, 125, 132, 133 short period, 97 shortage, 16 sialic acid, 130 side effects, 141 sign, 73 signal transduction, 98 signaling, ix, xi, 76, 94, 149, 152, 153, 157, 159 signaling pathway, 152, 153 signalling, vii, 31, 101, 104 signals, vii, 31, 34, 51, 71, 73, 88, 97, 101, 103, 104 signs, 8, 12, 70, 73, 74, 102 sites, 138, 140, 152 skin, 98, 105 smooth muscle, 102 smooth muscle cells, 102 socioeconomic, x, 135 sodium, 45, 156 South America, 34 Spain, 111, 114, 115, 120 specialization, 32 species, vii, x, 31, 32, 33, 34, 37, 38, 39, 40, 42, 44, 45, 46, 47, 49, 50, 54, 55, 56, 60, 61, 62, 63, 64, 65, 66, 68, 71, 72, 74, 79, 86, 95, 97, 119, 124, 130, 131, 155 specificity, vii, ix, 3, 15, 16, 20, 94, 100, 121, 136, 139, 144 spectrum, 139 speed, 73 sperm, 47, 105 sphincter, 70 spinal cord, 73, 98, 105 spleen, 15 spontaneous abortion, 17, 120, 142 sporadic, 19, 120 SPSS, 115 stability, 98 stabilize, 47

Index stages, 44, 47, 50, 52, 53, 54, 55, 56, 59, 61, 62, 70, 74, 79, 84, 103, 133, 136 standard deviation, 115, 126 standard error, 126 standards, 115 starch, 41 stasis, 38 Statistical Analysis System (SAS), 126, 132 staurosporine, 159 stem cells, 87 steroid, 45, 55, 61, 94, 119, 151, 152 steroid hormones, 45, 61, 94, 151, 152 steroidogenesis, 61 steroids, 11, 97 sterols, 46 stillbirth, 27 stimulus, 33, 34, 41 stock, 125 storage, 39 strategies, 120 stress, 4, 9, 24, 40, 66, 88 stroma, 23, 65, 71, 102, 103 stromal, 78, 97, 99, 101, 104, 105, 106 stromal cells, 78, 101, 105, 106 substances, 45, 51, 59, 61, 139 substrates, 10, 45, 46, 84 sucrose, 45 suffering, 15, 18 sugar, 14, 137 sulphate, 79 summer, 34, 35, 36, 37, 41, 42, 86 superiority, 139 superoxide, 78 superoxide dismutase, 78 supply, 42, 61, 95, 102, 151, 152 suppression, 18, 82, 120, 139, 141 suprachiasmatic, 34 suprachiasmatic nucleus, 34 surgery, 43, 78, 81, 139, 141, 142 surgical, 139, 144 surveillance, 20 survival, 32, 33, 37, 49, 50, 62, 63, 66, 88, 141, 144, 145 Sweden, 125 symbiotic, 160 symptom, 138 symptoms, 4, 103, 138, 154, 155 synchronization, 50, 67, 70, 76 syncytium, 98, 156 syndrome, 5, 6, 9, 154, 155, 156, 158

Index synthesis, 5, 6, 9, 13, 18, 49, 50, 54, 62, 67, 80, 81, 101 systems, ix, 55, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 139

T T cell, 18, 26, 103, 107, 120 tachycardia, 138 TAL, 79 tau, 65, 66, 81, 88 temperate zone, 33 temperature, 37, 126 temporal, 16 teratogenic, 144 testis, 98 TGF, 18, 27, 99 TGFβ, 137 Thai, 77 therapy, 20 thorax, x, 135, 139 threatened, 24 threatened abortion, 24 three-dimensional, 85 threshold, 65, 115, 119 thymus, 152 thyroid, 5, 35, 40, 76, 77, 81, 139 thyroid stimulating hormone (TSH), x, 5, 98, 135, 138 thyrotoxicosis, x, 135 tight junction, 51, 52 time, viii, ix, x, 8, 10, 13, 15, 16, 33, 37, 40, 41, 42, 50, 53, 54, 55, 67, 68, 70, 73, 74, 80, 82, 84, 94, 96, 99, 100, 112, 113, 124, 130, 135, 137, 139, 142, 143, 144 time frame, 94 time periods, 16 timing, 48, 103, 118 TIMP, 97, 101 tissue, x, 21, 27, 29, 55, 70, 97, 101, 106, 135, 144, 150, 152, 156 tissue homeostasis, 156 tissue remodelling, 101 TNF, 154 Tokyo, 21 tolerance, viii, 93, 97, 98, 101, 103, 112, 120, 154 totipotent, 47 toxicity, 141, 144 traits, 33 trans, 86 transcript, 151, 152

177

transcription, xi, 16, 88, 104, 149, 150, 151, 152, 153, 158, 160 transcription factor, xi, 149, 150, 151, 152, 153, 158, 160 transcriptional, 157 transcripts, 99, 101, 151, 152, 154 transducer, 34, 70, 104 transduction, 105 transfer, 45, 70, 77, 88 transformation, 22, 106, 144 transforming growth factor, 18, 105 transmembrane, 50, 85, 98, 104 transmembrane glycoprotein, 50 transmission, 118 transport, 44, 51, 76, 83, 90, 151, 152, 159 transportation, 45 trees, 56, 59 trial, 6, 7 trisomy, 20, 21 trophoblast, ix, x, xi, 4, 10, 11, 14, 15, 17, 18, 21, 23, 49, 51, 52, 53, 54, 61, 76, 80, 81, 82, 84, 94, 96, 97, 98, 100, 101, 102, 104, 131, 133, 135, 137, 142, 147, 149, 150, 156, 158, 159, 160 tropical areas, 37 Tropical zone, 32 tuberculosis, 114 tumor, 97, 107 tumorigenesis, 146 tumorigenic, 25 tumors, 98 tumour, x, 135, 136, 140, 141, 142, 143, 144, 146, 147 tumours, x, 5, 19, 28, 135, 136, 142, 146 Turku, 126 twins, ix, 112, 116, 132 tyrosine, 34

U ultrasonography, vii, 3, 20, 28, 31, 68, 70, 81, 86, 114 ultrasound, vii, x, 26, 31, 68, 70, 71, 72, 73, 83, 135, 137, 139 umbilical cord, 74 uniform, 13, 19 urine, 138, 141, 147 uterus, vii, 31, 33, 42, 48, 49, 50, 55, 56, 59, 60, 72, 73, 85, 86, 89, 97, 101, 102, 107, 120, 140

Index

178

V vaccination, 114 vaccine, 114, 120 vacuum, 114 vagina, 140 values, ix, x, 9, 11, 12, 13, 15, 16, 18, 20, 23, 36, 37, 45, 54, 61, 63, 112, 115, 116, 123, 126, 127, 131 variability, 63, 131 variables, 12, 116 variance, 115, 126 variation, vii, 12, 31, 71, 76, 114 vascular disease, 155 vascular endothelial growth factor (VEGF), viii, ix, 5, 94, 100, 101, 102, 106, 107 vascular wall, 102 vascularization, 67 vasculature, 5, 9, 59, 60, 102 vasculogenesis, 5 vasoconstrictor, 102 vasodilatation, 102 vein, 60, 125 velocity, 20

Venezuela, 33, 37, 76 venipuncture, x, 123 versatility, 140 vesicle, 73 vessels, 97, 102, 137 virus, 160 viruses, 159 viscosity, 47 visible, 71, 73

W water, 33, 36, 55, 59, 74, 75, 76, 77, 78, 80, 84, 85, 87, 124, 125, 127, 128, 130, 131, 133 well-being, ix, 13, 24, 111, 113, 119 wellness, 73 whey, 67, 68 winter, 34, 36, 37 women, vii, x, 3, 4, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 23, 28, 29, 99, 135, 139, 144, 146, 147, 155, 156 workers, 9, 14, 15 World Health Organization (WHO), x, 22, 135, 139, 140, 146

Y yang, 120 yield, 39, 42, 75 yin, 120

Z zinc, 10, 152 zygote, 47