Early Placentation Research Articles

Uterine influences on embryogenesis and early placentation in the horse revealed by transfer of day 10 embryos to day 3 recipient mares

Eight day 10 horse embryos were transferred non-surgically to recipient mares that had ovulated 7 days after the donors. The embryonic vesicle was seen ultrasonographically in all eight recipients, and three out of eight (38%) of the vesicles developed an embryo proper with a beating heart. Conceptus expansion was initially slower than that in control mares but continued until day 22 (recipient day 15). Time of fixation of the vesicle was related to its diameter, rather than uterine stage. Although the embryo proper first appeared ultrasonographically on day 22, as normal, it grew more slowly and the allantois expanded more slowly than that in control mares with normal pregnancies. The development of endometrial cups and their secretion of equine chorionic gonadotropin in the two mares allowed to remain pregnant to >50 days occurred at a conceptus age approximately 7 days later than that in the control mares. The results demonstrated the uniqueness of the horse conceptus in being able to overcome a 7-day asynchrony with the uterus, and also highlighted the overriding influence of the uterine environment on conceptus development in the mare.

Localization of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors for MMPs (TIMPs) in uterine natural killer cells in early human pregnancy

Invasion by extravillous trophoblast into uterine decidua and myometrium with remodeling of spiral arteries is essential for normal human pregnancy and is tightly regulated. Uterine natural killer (uNK) cells appear to be a major maternal regulator of placentation through the secretion of growth factors, cytokines and proteinases. Matrix metalloproteinase (MMP)-2 and MMP-9 activity in placental bed biopsies was studied using in situ gelatin zymography. Expression by uNK cells of MMP-2, MMP-9 and their tissue inhibitors, TIMP-1, TIMP-2 and TIMP-3, was localized in the placental bed by immunohistochemistry. Levels of MMP-2, MMP-9, TIMP-1, TIMP-2 and TIMP-3 secreted into 24 h cell culture supernatants of isolated uNK and unseparated (total) decidual cells (8-10 and 12-14 weeks gestation, n = 10 each group) were determined by gelatin gel zymography or western blot as appropriate. Gelatinase activity in situ appeared greater in decidua than myometrium. uNK cells showed strong immunostaining for MMP-2 and TIMP-2. MMP-9 activity was lower in uNK cells than total decidual supernatants (8-10 weeks: P = 0.0003; 12-14 weeks: P = 0.0012). In contrast, there was no difference in MMP-2 secreted by either uNK cell or total decidual cultures. uNK cells from early human pregnancy decidua possess innate protease activity, especially MMP-2, providing further evidence for a role for these cells in regulation of trophoblast invasion and spiral artery remodeling in early placentation.

51: Maternal serum proteome profile of early placentation in IVF is distinct from normal placentation

51: Maternal serum proteome profile of early placentation in IVF is distinct from normal placentation

2-methoxy estradiol inhibits angiogenesis in sheep uterine artery endothelial cells

OBJECTIVE: Estrogen plays a key role in priming the endometrium for luteal phase development, embryo implantation, and early placentation. The effects of estrogen metabolites and estrogen metabolizing enzymes in endometrial vasulature are not well studied. Here we investigate the angiogenic and vasodilatory effect of 17b estradiol and 2-methoxy estradiol using the sheep model.

Conceptus Signals for Establishment and Maintenance of Pregnancy in Pigs – Lipid Signaling System

Establishment of pregnancy in pigs requires estrogen secretion by the conceptus. The developmental changes of embryo before implantation and embryo-uterine cross talk are dependent on various biological molecules secreted by the endometrium and conceptus. An integral part of maternal recognition of pregnancy seems to be also the lipid signaling system consisting of prostaglandin (PG) F2 alpha and E2 and/or lysophosphatic acid (LPA). The downstream enzymes in PG synthesis pathway are: microsomal PGE synthase-1 (mPGES-1), PGF synthase (PGFS) and prostaglandin 9-ketoreductase/carbonyl reductase (CBR1) which catalyzes conversion of PGE 2 into PGF2 alpha. In contrast to mPGES-1, endometrial PGFS is highly increased on days 13-15 similarly as CBR1 on days 16-17 of the estrous cycle. Potential mechanism by which a conceptus inhibits luteolysis is changing the PGE2/PGF2 alpha ratio in favor of PGE2. It may be result of high expression of mPGES-1 in trophoblast and endometrium on days 10-13 of pregnancy and simultaneously the down-regulation of PGFS and CBR1 in conceptuses during this period. The conceptus can alter expression of endometrial CBR1 to modulate the PGE2/PGF2 alpha ratio in the uterus during the maternal recognition of pregnancy. High expression of conceptus and endometrial terminal PG synthases and CBR1 after initiation of blastocyst attachment suggest their involvement in early placentation. The higher LPA3 receptor mRNA expression during the early pregnancy compared to corresponding period of estrous cycle could indicate an important role of LPA and its receptor during the peri-implantation stage of pregnancy in pigs. Above results suggest that the lipid signaling system is an integral part of establishment and maintenance of pregnancy in the pig.

Evolution of the placenta and associated reproductive characters in bats

Recent advances in molecular phylogenetics indicate that the order Chiroptera is monophyletic and that one of four lineages of microbats (Rhinolophoidea) shares a common origin with megabats. Against this background we undertook a comprehensive analysis of placental evolution in bats. We defined a range of characters and character states associated with female reproduction, early development, placentation and the neonate. These were then mapped on a pre-existing hypothesis of bat relationships that represents the current view from molecular studies. Our purpose was threefold. First, on the assumption of bat monophyly, we wished to establish the stem species pattern of extant chiropterans. Secondly, we asked whether there are derived character conditions in support of a common origin for Rhinolophoidea and the megabats. Thirdly, we looked for evolutionary character transformations that characterize higher-level clades within Chiroptera, i.e. the megabats and the four lineages of microbats. The character condition occurring in the last common ancestor of Chiroptera was unequivocal for 21 of the 25 characters included in the analysis. The data did not offer support for a megabat-rhinolophoid clade or the implication that microbats are paraphyletic. However, analysis of early development, placentation and other reproductive parameters resulted in derived character conditions for the megabats as well as for each of the four major lineages of microbats.

Effects of corticotropin-releasing hormone and stresscopin on vascular endothelial growth factor mRNA expression in cultured early human extravillous trophoblasts

Corticotropin-releasing hormone (CRH) takes a role in the regulation of the onset of parturition. Stresscopin (SCP) is a high affinity ligand for CRH receptor (CRHR)-2. CRHR-2 inhibits VEGF-induced neovascularization. In the present study, we investigated the effects of CRH and SCP on VEGF expression in early placental extravillous trophoblasts (EVTs). Isolation and culture of trophoblasts differentiating into EVTs were performed by the enzymatic digestion of anchoring early placental villi. The presence of CRH, SCP, CRHR-1, and CRHR-2 in cultured EVTs was examined by RT-PCR and immunocytochemistry. The effects of CRH and SCP on VEGF mRNA levels in cultured EVTs were assessed by real-time RT-PCR. CRH, SCP, CRHR-1, and CRHR-2 were expressed in cultured EVTs at mRNA and protein levels. Treatment with either 100 nM CRH or 100 nM SCP for 24 h decreased VEGF mRNA levels in cultured EVTs. The CRH- and SCP-induced decrease in VEGF mRNA levels was counteracted by the concomitant treatment with CRHR-2 antagonist antisauvagine-30, but not with CRHR-1 antagonist antalarmin. We demonstrated that CRH and SCP inhibited VEGF mRNA expression in cultured EVTs through the interaction with CRHR-2, suggesting that CRH and SCP may inhibit angiogenesis during early placentation.

256: Low-dose aspirin influences early pregnancy placentation in IVF /ICSI patients: Evidenced by a distinct maternal serum proteome profile

PATIENTS: EVIDENCED BY A DISTINCT MATERNAL SERUM PROTEOME PROFILE MERVI HAAPSAMO, JUHA RASANEN, MICHAEL GRAVETT, SRINIVASA NAGALLA, University of Oulu, Obstetrics and Gynecology, Oulu, Finland, Oregon Health Sciences University, Obstetrics and Gynecology, Portland, Oregon, University of Washington, Obstetrics and Gynecology, Seattle, Washington, Proteogenix, Inc., Beaverton, Oregon OBJECTIVE: To investigate whether low-dose aspirin therapy when started prior to pregnancy affects maternal serum proteome profile reflecting placentation in the first trimester of pregnancy. STUDY DESIGN: Thirty pregnant women who had undergone IVF/ICSI and had been randomized to receive 100 mg aspirin (n 15) or placebo (n 15) daily starting on the first day of controlled ovarian hyperstimulation were included in this study. Maternal blood sample was taken at 10-13 weeks gestation for proteome analysis. Sera were processed to remove high-abundance proteins using immunoaffinity columns. Total serum protein from control, aspirin treated groups were labeled with Cy2, Cy3 and Cy5 fluorescent dyes and analyzed using multiplex 2-dimensional gel electrophoresis (2D-DIGE). Image analysis and spot quantification was performed using Phoretix software. Protein identification was performed utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS) and MALDI-TOF-MS. RESULTS: Gestational age at delivery or perinatal outcome parameters did not differ between the groups. 2D-DIGE analysis of the serum proteome identified a distinct differential expression pattern between placebo and aspirin groups. Analysis of 176 spots by MALDI-TOF-MS and LC-MS/MS identified 62 unique proteins. Potential proteins influencing various pathways in the placentation process included extra-cellular matrix proteins, cytoskeletal proteins, apolipoproteins, complement proteins and transport proteins. CONCLUSION: Maternal serum proteome profiling suggests that low-dose aspirin therapy started prior to pregnancy could significantly influence placentation and pregnancy outcome.

Cytokines and Chemokines during Human Embryo Implantation: Roles in Implantation and Early Placentation

The complex processes of embryo implantation and early placentation require a plethora of locally acting molecules, which are themselves tightly regulated. Among these are cytokines (including chemotactic chemokines), which are synthesized by several cell types at the maternal-fetal interface. Those produced by endometrial epithelium may be secreted apically into the uterine lumen, where they affect blastocyst development, migration, and attachment, or basally with effects on the transformation of the underlying stroma. Decidualized stromal cells, which subsequently form a major component of the decidua of pregnancy, also produce cytokines that act to drive the decidualization process and chemokines that are chemoattractants both for leukocytes such as uterine natural killer cells and macrophages, and for trophoblast migration. Activated leukocytes within the developing decidua also contribute regulatory cytokines to the local microenvironment. Disturbances in the production of individual cytokines have been demonstrated in the endometrium of some infertile women and in those with recurrent miscarriage. It is important to establish whether a signature of endometrial cytokines may provide a clinically useful indication of women who will experience difficulty in establishing a viable pregnancy.

Association study of protease activated receptor 1 gene polymorphisms and adverse pregnancy outcomes: Results of a pilot study in Israel

Protease activated receptor 1 (PAR1), the thrombin receptor, is implicated in hemostasis, tissue remodeling, and is critical in early placentation. PAR1 polymorphisms that influence coagulation and adhesion molecule expression may compromise formation of a normal placenta, thereby resulting in adverse outcomes. This study is a prospective, case-control comparison of Israeli mother-neonate (singletons) pairs of complicated pregnancies: 33 preterm deliveries (PTDs), 20 preeclampsia (PE), and 28 idiopathic small for gestational age neonates (SGA) versus 98 pairs of uncomplicated singleton deliveries. PCR amplification identified polymorphisms in the 5' regulatory region of the PAR1 gene at [-1426(C/T)], insertion at [-506 (I/D)], and IVS at [-14 (A/T)]. We found that 15.2% mothers with PTD were heterozygous for [-1426] versus 3% in uncomplicated deliveries; however, maternal allele frequency was not significantly different between pregnancies complicated by PE or SGA versus uncomplicated pregnancies. Maternal allele frequencies for [-506] and [-14] polymorphisms were not significantly different between any of the study groups compared to the uncomplicated group. Haplotype analysis recapitulated the genotype pattern. Maternal homozygous allele frequency for each of the polymorphic variants was low (<2%) in all pregnancies, and equally distributed. Neonatal genotypes did not differ between groups and were not associated with adverse outcomes. Maternal heterozygous allele state for PAR1 polymorphism [-1426] and the haplotype with [-506 wild-type]/[-1426T]/[IVS-14A] is associated with PTD.

Impact of Periconceptional Undernutrition on the Development of the Hypothalamo-Pituitary-Adrenal Axis: Does the Timing of Parturition Start at Conception?

There are a number of critical windows during prenatal and postnatal life and a range of potential agents including exposure to maternal and fetal stressors, nutrition, and antenatal administration of synthetic glucocorticoids and postnatal maternal care and behaviour that are important in programming the subsequent reactivity of the HPA axis. Recently, it has become clear that the periconceptional period is also an important critical period during which changes in the level of maternal nutrition result in altered development of the fetal HPA axis. These findings have potential implications for the ability of the fetus to respond to acute and chronic stressors, for the timing of parturition and have potential implications for adult cardiovascular and metabolic health outcomes. In this review we focus on the different models which have been used to investigate the impact of maternal undernutrition during the periconceptional period on the prepartum activation of the fetal HPA axis. We propose that the term "periconceptional" should be used to refer to the developmental stages which include some or all of the following early events: oocyte maturation, follicular development, conception, and embryo/blastocyst growth up until implantation. When maternal undernutrition extends beyond implantation, up until early placentation, then it is appropriate to describe maternal undernutrition as occurring during 'early gestation'. Further work is required to define the relative contributions of nutritional factors operating in the periconceptional and early gestational periods on the programming of the subsequent development of the HPA axis and is of importance for fetal, postnatal and subsequent adult cardiovascular and metabolic health.

Angiogenesis in implantation

Implantation failure and early pregnancy loss are common following natural conceptions and they are particularly important clinical hurdles to overcome following assisted reproduction attempts. The importance of adequate vascular development and maintenance during implantation has recently become a major focus of investigation. Review of current published literature was undertaken to summerize the cells and cell products that regulate tissue vascularity during implantation. Vascular development at the maternal fetal interface can be regulated by a number of different cell types; two principal candidates are trophoblast and natural killer cells. A wide range of soluble factors, some with well established angiogenic functions as well as other more novel factors, can contribute to vascular development and maintenance at the maternal-fetal interface. Robust vascular development occurs during implantation and early placentation of normal pregnancies. Studies to define the extent and mechanisms by which defects in vascularity contribute to human implantation failure and early miscarriage need to be undertaken.

Abortion is associated with increased expression of FasL in decidual leukocytes and apoptosis of extravillous trophoblasts: a role for CRH and urocortin

Human reproduction is remarkably inefficient, with more than half of spontaneous conceptions failing to complete the first trimester. However, little is known on the molecular events that take place at the implantation site during abortion. Here, we examined the hypothesis that the expression of the proapoptotic Fas/FasL system at the implantation site is impaired in abortions. We found that, in contrast to normal pregnancy, abortive deciduas contain leukocytes that are positive for FasL and extravillous trophoblasts (EVTs), which show increased expression of Fas and increased rates of apoptosis. In addition, the neuropeptides, corticotropin-releasing hormone and urocortin, were elevated in placental material obtained from abortions. In vitro, these peptides induced the expression of FasL in decidual lymphocytes (DL) obtained from elective termination of pregnancy placentas and thus potentiated the cells' ability to induce Fas-mediated apoptosis in an EVT-based hybridoma cell line. Finally, DL from abortion sites effectively induced apoptosis of EVT without prior treatment. It is possible that these events may impede successful early placentation and thus contribute to the pathophysiology of human abortion.

Membrane-Bound Peptidases Regulate Human Extravillous Trophoblast Invasion

During human placentation, the extravillous trophoblast (EVT) invades maternal decidua and spiral arteries. However, the precise regulatory mechanisms by which EVT invasion is induced toward maternal arteries or limited within the uterus have not been well characterized. Recently, we found that dipeptidyl peptidase IV, a membrane-bound cell surface peptidase that can degrade chemokines, including RANTES, was expressed on EVT that had already ceased invasion. Another cell surface peptidase, carboxypeptidase-M, was also detected on EVT including the endovascular trophoblast in the maternal arteries. The inhibition of these peptidases increased cell invasion of choriocarcinoma-derived JEG-3 cells. On the other hand, CCR-1, a chemokine receptor for RANTES, was specifically expressed on EVT that migrated toward maternal arteries, while RANTES enhanced invasion of EVT that were isolated from primary villous explant culture. Platelets, which secrete RANTES and other chemokines, were detected among the endovascular trophoblast, and platelets were shown to enhance invasion of cultured EVT. Furthermore, a novel membrane-bound cell surface peptidase, laeverin, was found to be specifically expressed on EVT at deep sites in the maternal decidua. These findings suggest that membrane-bound peptidases regulate EVT invasion in cooperation with a chemokine system during early human placentation.

Hypoxia Represses the Differentiation of Rcho-1 Rat Trophoblast Giant Cells

Background: A hypoxic environment is known to be essential for early placentation. A low oxygen tension induces hypoxia-inducible factor-1 (HIF-1α) which may play an important role as a transcription factor in maintaining the proliferative and undifferentiated phenotype in human trophoblasts. Methods: We analyzed the effect of a low oxygen tension on the rat trophoblast giant cell differentiation pathway using Rcho-1 cells which were derived from rat choriocarcinomas and consist of trophoblast stem cells. Results: We found that a low oxygen tension suppressed the morphological changes and steroidogenesis during differentiation. The anticipated downregulation of the Id-1 transcription factor, a negative regulator of trophoblast giant cell differentiation, was not observed in the hypoxic environment. On the other hand, deferoxamine, which mimics hypoxia and induces HIF-1α, caused downregulation of Id-1 transcription factor and trophoblast giant cell differentiation. Conclusion: These results indicate that hypoxia represses rat trophoblast giant cell differentiation via an HIF-1α-independent pathway.

P-80: Does a vanishing twin identified at 6-7 week ultrasound impact perinatal outcome?

The It has been reported that 10-15% of singleton IVF/ICSI births start as a higher order gestation. Research has shown that IVF/ICSI singletons have a greater incidence of preterm delivery and low birth weight than spontaneously conceived singletons. Multiple explanations have been suggested including abnormalities established during early placentation when multiple implantation sites occur or underlying causes of infertility itself rather than the IVF treatment leads to the adverse perinatal outcomes.

In vivo and in vitro investigations of the effects of the antimalarial drug dihydroartemisinin (DHA) on rat embryos

Artemisinin derivatives are clinically effective and safe antimalarials, but are not recommended during the first trimester of pregnancy because of the resorptions and abnormalities seen in animal reproduction studies. Understanding how, when and what toxicity occurs is crucial to any assessment of clinical relevance. Previously, DHA has been shown in the rat whole embryo culture (WEC) to primarily affect primitive red blood cells (RBCs) causing subsequent tissue damage and dysmorphogenesis. To verify the primary target of DHA in vivo and to detect consequences induced by early damage on embryo development, pregnant female rats were orally treated on gestation days (GD) 9.5 and 10.5 with 7.5 or 15 mg/kg/day DHA and caesarean sectioned on GD11.5, 12.5, 13.5, 15 and 20. A parallel in vitro WEC study evaluated the role of oxidative damage and examined blood islands and primitive RBCs. In accordance with the WEC results, primitive RBCs from yolk sac hematopoiesis were the target of DHA in vivo. The resulting anemia led to cell damage, which depending on its degree, was either diffuse or focal. Embryonic response to acute anemia varied from complete recovery to malformation and death, depending on the extent of cell death. Malformations occurred only in litters with embryonic deaths. DHA induced low glutathione levels in RBCs, indicating that oxidative stress may be involved in artemisinin toxicity; effects were extremely rapid, with altered RBCs seen as early as GD10. In establishing the relevance of these findings to humans, one should consider differences in the development of rodents and humans. While yolk sac hematopoiesis occurs similarly in the two species, early placentation and extent of exposure differ. In particular, early hematopoiesis takes only 7 days in rats (during which RBCs expand in a clonal fashion) compared with 6 weeks in humans; thus the susceptible period in relation to the duration of exposure to an artemisinin-based treatment may be substantially different.

Maternal Obesity, Metabolism, and Pregnancy Outcomes

About one third of all pregnant women in the United States are obese. Maternal obesity at conception alters gestational metabolic adjustments and affects placental, embryonic, and fetal growth and development. Neural tube defects and other developmental anomalies are more common in infants born to obese women; these defects have been linked to poor glycemic control. Preeclampsia, a gestational disorder occurring more frequently in obese women, appears to be due to a subclinical inflammatory state that impairs early placentation and development of its blood supply. Fetal growth and development during the last half of pregnancy depends on maternal metabolic adjustments dictated by placental hormones and the subsequent oxygen and nutrient supply. Maternal obesity affects these metabolic adjustments as well. Basal metabolic rates are significantly higher in obese women, and maternal fat gain is lower, possibly in response to altered leptin function. The usual increase in insulin resistance seen in late pregnancy is enhanced in obese mothers, causing marked postprandial increases in glucose, lipids, and amino acids and excessive fetal exposure to fuel sources, which in turn increases fetal size, fat stores, and risk for disease postnatally. Impaired glucose tolerance, gestational diabetes, and hyperlipidemia are more common among obese mothers. To date, little attention has been given to the role of diet among obese women in preventing these problems. However, studies of women with impaired glucose tolerance show that replacing refined carbohydrates and saturated fat with complex, low-glycemic carbohydrates and polyunsaturated fatty acids improves metabolic homeostasis and pregnancy outcomes. Thus, current dietary guidelines regarding the amount and type of carbohydrates and fat for nonpregnant women seem appropriate for pregnant women as well.

1264: The role of Doppler ultrasound in obstetrics

1264: The role of Doppler ultrasound in obstetrics

Dynamic HIF1A Regulation During Human Placental Development1

The human placenta is a unique organ in terms of oxygenation as it undergoes a transition from a low to a more oxygenated environment. This physiological switch in oxygen tension is a prerequisite for proper placental development and involves the hypoxia inducible factor (HIF). HIF is stable and initiates gene transcription under hypoxia, whereas in normoxia, interaction with the von Hippel-Lindau tumor suppressor protein (VHL) leads to rapid degradation of the HIF1A subunit. The degradation requires formation of a multiprotein complex (VHLCBC) and hydroxylation of HIF1A proline residues via members of the egg-laying-defective nine (EGLN) family. Herein, we have investigated the regulatory mechanisms of HIF1A expression during human placental development. Expression of HIF1A and VHL was high at 7-9 wk of gestation, when oxygen tension is low, and decreased when placental oxygen tension increases (10-12 wk of gestation). During early placentation, HIF1A localized in cytotrophoblasts, while VHL was present in syncytiotrophoblasts. At 10-12 wk, VHL appeared in cytotrophoblast cells, which coincided with the disappearance of HIF1A. At the same time the association of VHL and Cullin 2 as well as ubiquitination of HIF1A was maximal. EGLN1, EGLN2, and EGLN3 were also temporally expressed in an oxygen-dependent fashion, with greatest mRNA expression at 10-12 wk of gestation. Inhibition of EGLN activity increased HIF1A stability in villous explants and stimulated transforming growth factor beta 3 (TGFB3) expression consistent with promoter analyses showing that HIF1A transactivates TGFB3. These data demonstrate that during placental development, HIF1A is regulated by temporal and spatial changes in expression and association of molecules forming the multi-protein VHLCBC complex as well as prolyl hydroxylase activities.