Expression and localization of alpha-fetoprotein mRNA and protein in human early villous trophoblasts.

Alpha-fetoprotein (AFP) is a major plasma protein produced during human fetal life. It is a good marker for several possible disorders affecting gestation. We previously reported that afp gene expression, which takes place mainly in yolk sac and fetal liver, also occurs in normal human placenta, specifically in early pregnancy. The aim of the present study was to determine the precise location of AFP synthesis sites within the placental villi. In situ hybridization and immunohistochemical experiments were performed on sections obtained from placentas of first-trimester and full-term pregnancies. We found that the pattern of afp gene expression was restricted to specific villous trophoblastic areas in early placentas. Both afp transcripts and AFP protein were mainly located in discontinuous regions, at junctions between two villi and at budding sites. In contrast, no AFP expression was detected in the cytotrophoblastic extravillous proliferative zone or in other placental cell types. According to the earlier studies, no AFP synthesis was detected in placental villous tissue from full-term pregnancies, using in situ hybridization and immunohistochemistry.

Effect of pregnancy on PDE4 cAMP-specific phosphodiesterase messenger ribonucleic acid expression in human myometrium.

In light of the important role of the second messengers cAMP and cGMP in the mechanism of relaxation in the human myometrium, specific regulation of the phosphodiesterase (PDE) enzymatic system responsible for cyclic nucleotide inactivation is essential. We previously identified in the human myometrium PDE4 cAMP-specific PDE as by far the most abundant isoform. Here we have studied the expression patterns of mRNAs for the four cloned human PDE4 genes in the myometria of pregnant and non-pregnant women. Concurrent expression of the PDE4A, 4B, 4C and 4D genes is demonstrated. We found that the PDE4D transcripts are the most prominently expressed. PDE4A and PDE4B mRNAs also are markedly abundant, whereas lower expression is observed for PDE4C mRNAs. Interestingly, we showed that transcripts of PDE4B2 are more abundant in the myometria of pregnant women than in non-pregnant women, whereas no difference between the two tissues was detected for PDE4A, 4C and 4D mRNAs. Cultured human myometrial cells, which present a high level of PDE4 activity and express the four PDE4 mRNA subtypes, provide us with an appropriate model to further evaluate whether the level of expression of the PDE 4B2 mRNA subtype is under hormonal regulation.

Endothelin-1 and ETA receptor expression in vascular smooth muscle cells from human placenta: a new ETA receptor messenger ribonucleic acid is generated by alternative splicing of exon 3.

Endothelin-1 (ET-1) is a potent vasoactive peptide in stem villi vessels, which are considered to be the major sites of placental vascular resistance. To investigate the influence of pregnancy-specific hormonal environment on ET and ET receptor (ET-R) expression, we first developed and characterized a culture of vascular smooth muscle cells from stem villi vessels. Secondly, we investigated whether the muscular layer of stem villi vessels could be a site of the ET expression described in the placenta, and we examined this expression in placental vascular smooth muscle cells (PVSMCs). Prepro-ET-1 and prepro-ET-3 messenger ribonucleic acid (mRNA) were identified in stem villi vessels, whereas only prepro-ET-1 mRNA was observed in PVSMCs. Third, with the goal of using PVSMCs as ET target cells, we characterized the ET-R expressed by these cells in comparison with the muscular layer of stem villi vessels. Whereas both ETA-R and ETB-R are present in stem villi vessels, we found that PVSMCs express exclusively ETA-R. In addition to the previously reported ETA-R spliced transcripts, we described a new ETA-R transcript, ETA-R delta 3, generated by exclusion of exon 3 in stem villi vessels and PVSMCs. Alternative splicing mechanisms of ETA-R mRNA could constitute a control of the abundance of active ETA-R in terms of contractility. PVSMCs will be a useful model to study the environmental stimuli involved in the regulation of ET and ET-R expression in the muscular layer of feto-placental vasculature.

Alpha-fetoprotein gene expression in early and full-term human trophoblast.

Alpha-fetoprotein (AFP) is a major serum glycoprotein synthesized during fetal life mainly by the yolk sac and the fetal liver. At term, it reaches high concentrations in the maternal intervillous blood, which is in direct contact with the placental trophoblastic microvillous membrane, and this suggests the placental origin of the AFP at the fetal-maternal interface. We used several experimental approaches to investigate the expression of AFP gene and fetal protein production in early gestation and term placentas. RT-PCR and immunological studies clearly identified AFP messenger RNA and AFP protein in the placental villi from first trimester of pregnancy. The AFP gene was also expressed in highly purified cytotrophoblasts from early placentas, and enzymo-immunoassay showed that AFP protein was synthesized and secreted by early cytotrophoblasts. AFP was also detected in the cytoplasm of these cells by immuno-cytochemistry. However, none of these methods detected any expression of the AFP gene in full-term placental villi or in cultured trophoblasts. These findings demonstrate that both AFP mRNA and protein are present in trophoblastic cells early in pregnancy. The absence of AFP gene expression in term placental villi also suggests, that the AFP at the fetal-maternal interface is attributable to a notable transplacental passage of AFP from fetal blood in late pregnancy.

Expression of endothelin precursor genes in human trophoblast in culture.

We have shown previously the presence of immunoreactive endothelin in cultured trophoblastic cells from human term placenta as well as in the trophoblast-conditioned medium. To confirm whether or not the differentiated syncytiotrophoblast is a site for endothelin synthesis, we investigated, by reverse transcription and polymerase chain reaction, the expression of the three preproendothelin genes in 3-day cultured trophoblast. While no endothelin-2 precursor mRNA was detected, preproendothelin-1 mRNA was found to be expressed by the trophoblast. The endothelin-3 precursor gene was also expressed, but at low level and it was detected only after Southern blotting and oligonucleotide hybridization. The ability of trophoblast in culture to express the endothelin precursor genes supports the idea that, in human term placenta, villous syncytiotrophoblast that lines the intervillous space containing maternal blood acts as an endothelial layer.

Does high polyunsaturated free fatty acid level at the feto-maternal interface alter steroid hormone message during pregnancy?

Polyunsaturated fatty acids (PUFA) are important in pregnancy, fetal development and parturition. We measured free fatty acids (FFA), albumin and alpha-fetoprotein (AFP) in the maternal and fetal circulations of women undergoing elective Caesarean section at term. We also studied the impact of PUFAs on estrogen (ER) and progesterone receptors (PR) binding properties in vitro in the myometria of pregnant women and ex vivo in human myometrial cells in culture. FFA in intervillous blood (I) (feto-maternal interface) and maternal peripheral blood (M) were similar, while those in the umbilical vein (V) and arteries (A) were 2-4 fold lower (P<0.001). PUFA levels were low in M and 3 fold higher in I, A and V (P< 0.001); consequently C20:4 and C22:6 were most abundant in intervillous space. Albumin was uniformly distributed throughout the maternal-fetal unit, but there was a transplacental gradient in AFP. The AFP in the intervillous space had a special conformation (less immuno-reactive, more anionic), suggesting loading with PUFA. Physiological concentrations of C20:4 stimulated estradiol binding, but inhibited progestin binding. C20:4 inhibited progesterone binding by decreasing the number of binding sites, with no change in apparent affinity, in vitro in myometrial tissue and ex vivo in myometrial cells. Thus PUFA may modulate the steroid hormone message, so that the high C20:4 concentration at the maternal-fetal interface at term may help amplify the estrogen signal and inhibit the progesterone signal.

Predictive value of the active renin assay for the diagnosis of ectopic pregnancy.

The increasing frequency of EP and the need for its early diagnosis have focused our interest on the research of biochemical markers. We have established hormonal values in the plasma of 99 spontaneous ongoing pregnancies between the 4th and 10th weeks of amenorrhea, in 21 EPs, and 20 cases of early abortion. We have examined the predictive values of trophoblastic and CL production in pathological pregnancies. The association of low hCG and low active renin appears to be able to discriminate between ectopic and abortive spontaneous gestations.

Inhibin concentration in the culture media of human oocyte-cumulus-corona cell complexes is not related to subsequent embryo cleavage.

We have demonstrated an in vitro secretion of inhibin by human OCCC, not related to subsequent egg cleavage. A putative signal, released by OCCC, that could modulate inhibin secretion by granulosa cells has not been discovered. Inhibin secretion by OCCC has no predictive value concerning the further fertilization and cleavage of the oocytes.

Embryo-maternal interactions at the implantation site: a delicate equilibrium.

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal environment. During preimplantation, maternal/embryo communication is mediated by the trophectoderm. In the late luteal phase, physiological changes occur in the endometrium to allow blastocyst implantation. The "window of implantation" represents the period of maximum uterine receptivity for implantation. In response to signals from the embryo, pregnancy-specific proteins are released in maternal serum and a series of morphological, biochemical and immunological changes occur in the uterine environment. These systemic and local modifications can be considered to constitute "the maternal recognition of pregnancy". The human hemochorial placenta arises primarily through proliferation, migration and invasion of the endometrium and its vasculature by the embryonic trophoblast. The complex invasive processes accompanying implantation of the embryo are controlled at the embryo-maternal interface by factors from decidualized endometrium and the trophoblast itself. An inflammatory reaction and a proper maternal immune response allow survival and development of the feto-placental unit. In this review, we focus on interactions between trophoblast and uterine tissues and on cellular mechanisms and molecular signals involved in the closely regulated process of implantation.

Changes in plasma and amniotic fluid endothelin levels during pregnancy: facts or artefacts?

OBJECTIVE: To describe the evolution of immunoreactive endothelins (irETs) in maternal peripheral plasma and in amniotic fluid at different stages of pregnancy using two different radioimmunoassays. STUDY DESIGN: Peripheral blood samples were obtained from ten non-pregnant and eighty-four pregnant patients at different stages of pregnancy, but not in labor. Amniotic fluid samples were obtained in ten patients during the second trimester of pregnancy and in twenty-two patients at term. Endothelin concentrations were assayed using two different kits: by antigenic cross-reactions, the RPA 545 assay allowed the detection of ET-1, ET-2 and big ET-1; the RPA 555 assay allowed the detection of ET-1, ET-2 and ET-3. RESULTS: Using the RPA 555 kit, no differences were observed in irET plasma levels between non-pregnant and pregnant patients whatever the gestational age. With the RPA 545 kit, the irET levels were significantly lower in pregnant patients during the first and second trimesters of pregnancy when compared to non-pregnant patients. Immunoreactive ET levels then increased significantly in the last month of pregnancy when compared to mid-pregnancy levels. In amniotic fluid, irET levels were significantly higher at term than during the early second trimester, without any difference between the two RIA kits. CONCLUSION: Our results are indicative of a differential evolution in ET isoforms during pregnancy in maternal peripheral blood. The increase in irET observed towards the end of pregnancy in maternal plasma and in amniotic fluid suggests that ET could play a role in the onset of parturition.

Cullins in human intra-uterine growth restriction: expressional and epigenetic alterations.

Intra-uterine growth restriction (IUGR) is defined by a restriction of fetal growth during gestation. It is a prevalent significant public health problem that jeopardizes neonatal health but also that can have deleterious consequences later in adult life. Cullins constitute a family of seven proteins involved in cell scaffold and in selective proteolysis via the ubiquitin-proteasome system. Most Cullins are critical for early embryonic development and mutations in some Cullin genes have been identified in human syndromes including growth retardation. Our work hypothesis is that Cullins, particularly CUL4B and CUL7, are involved in placental diseases and especially in IUGR. Thus, expression of Cullins and their cofactors was analyzed in normal and pathological placentas. We show that they present a constant significant over-expression in IUGR placentas, whose extent is dependent on the position of the interrogated fragment along the cDNAs, suggesting the existence of different isoforms of the genes. Particularly, the CUL7 gene is up-regulated up to 10 times in IUGR and 15 times in preeclampsia associated with IUGR. The expression of cofactors of Cullins participating to functional complexes has also been evaluated and showed a similar significant increase in IUGR. Promoters of Cullin genes appeared to be under the control of the SP1 transcription factor. Finally, methylation levels of the CUL7 promoter in placental tissues are modulated according to the pathological conditions, with a significant hypomethylation in IUGR. These results concur to pinpoint the Cullin family as a new set of markers of IUGR.

Endothelin-2 down-regulation occurs in parallel with the anti-proliferative effect of dimethylsulfoxide in BeWO human choriocarcinoma cell line.

Endothelins exhibit growth regulating properties in many cell types, and there is now considerable evidence that they play a critical pathophysiological role in human diseases such as carcinogenesis. In the choriocarcinoma cell lines JEG-3, JAR and BeWO, we demonstrate by RT-PCR that prepro endothelin (ET)-1 and prepro ET-2 mRNA were expressed, whereas prepro ET-3 was never expressed. Only ET-1 and ET-2 peptides were identified by HPLC/RIA analyses in culture media from these three choriocarcinoma cell lines. In the BeWO line, the cellular growth measured as the cell count and DNA content decreased with increasing concentrations of dimethyl sulfoxide (DMSO; range 0.5-3%). The expression of prepro ET-2 was also suppressed by DMSO, whereas no change was noticed inprepro ET-1 mRNA. All these effects were reversible when DMSO was replaced by 15% foetal calf serum. These effects of DMSO which are correlated to ET-2 down regulation in dividing BeWO cells suggest a role for this endothelin isoform in trophoblast proliferation.

Are pregnancy-associated plasma protein-A (PAPP-A) and CA 125 measurements after IVF-ET possible predictors of early pregnancy wastage?

Pregnancy-associated plasma protein-A (PAPP-A), a macromolecular glycoprotein of placental origin, was reported to be depressed in established ectopic pregnancies. CA 125 is a known marker for ovarian cancer found to be elevated during the first trimester of pregnancy and in women with pelvic inflammatory disease. The present study investigated the usefulness of these parameters to predict the outcome of pregnancy in asymptomatic patients with a positive pregnancy test after in-vitro fertilization and embryo transfer (IVF-ET). Blood samples (n = 159) were obtained at different periods of time post-ET from 39 women, 21 of whom experienced a normal pregnancy, 12 had an intrauterine abortion and six had an ectopic pregnancy. PAPP-A and CA 125 were measured by radioimmunoassays. From day 30 onwards in normal pregnancies, PAPP-A was significantly increased over non-pregnant controls. In the spontaneous abortion group, the levels of PAPP-A were significantly lower than in normal pregnancy but higher than in non-pregnant controls. In ectopic pregnancy, PAPP-A remained at the level of non-pregnant controls throughout the entire observation period. CA 125 was significantly increased in all types of pregnancy. However, in two cases of hyperstimulation followed by a normal pregnancy and in four cases of ectopic pregnancy with signs of peritoneal irritation (hydrosalpinx, ruptured ectopic or salpingitis) the levels of CA 125 were 15-50 times higher than in normal pregnancies.(ABSTRACT TRUNCATED AT 250 WORDS)

Higher endothelin concentrations in the fetoplacental unit of pregnant women of African ancestry.

Immunoreactive endothelin was assayed in maternal and fetal biologic fluids of women of African and European ancestry with normal singleton pregnancies undergoing cesarean section at term for obstetric reasons. Endothelin concentration was found to be higher in the umbilical vein and artery blood of women of African origin. Higher production of endothelins in the fetoplacental unit may place these women at a greater risk of preeclampsia.

Effects of early tamoxifen treatment on hormonal content of 15-day quail embryo gonads.

An early treatment of quail eggs with an antiestrogen (Tamoxifen = TAM) modifies the female gonads morphologically. In the left ovary, the proliferation of the germinal epithelium and formation of a cortex is markedly inhibited, while the medulla on both sides becomes more developed and keeps a cordal organization. The effects of TAM on the hormonal potentialities of 15-day embryo gonads have been investigated by radioimmunoassay. The data have been compared with those of normal gonads. The results indicate that the TAM treatment does not mainly modify the hormonal levels and patterns in the gonads of both sexes and does not inhibit the function of the hypothalamo-hypophyseal-gonadal axis. The reduced cortical development is thus not linked to an estrogen decrease but to competitive effects on receptors at the level of this tissue.

Placental endothelin gene expression and endothelin concentration in fetal fluids of the first trimester gestational sac.

We have investigated the distribution of immunoreactive endothelins (irET) in fetal fluids and expression of ET precursor genes in villous tissue during the first trimester. Samples of maternal plasma (n = 6), coelomic fluid (n = 28), amniotic fluid (n = 23) and villous tissue (n = 3) were obtained from 30 pregnancies immediately before surgical termination at 7-12 weeks gestation. irET concentration was measured in plasma and fluids using two different radioimmunoassay kits, i.e. RPA 545 and RPA 555 and high performance liquid chromatography (HPLC). Total RNA was extracted and purified from villous tissue, reverse transcription and polymerase chain reaction (RT-PCR) were performed to evaluate the expression of ET-related genes. The irET concentration as evaluated by both kits was significantly higher (P<0.005) in maternal plasma than in coelomic or amniotic fluid and significantly higher (P<0.005) in coelomic fluid than in amniotic fluid using the RPA 555 kit. The profile of ET obtained by the HPLC- radioimmunoassay (RPA 555 kit) method confirmed significantly (P<0.005) higher ET concentration in coelomic than in amniotic fluid, although a similar distribution pattern for the three ET was observed in both embryonic fliud cavities. ET-3 was the predominant isoform in both fluids, reaching 19.4+/-2.0 pg/ml and 6.3+/-1.6 pg/ml in coelomic and amniotic fluid, respectively. Coelomic or amniotic fluid irET concentration did not change with gestational age irrespective of the kit used. RT-PCR demonstrated that first trimester placenta expresses the genes encoding for prepro-ET-1, -ET-2 and -ET-3. The similar ET distribution pattern in both fluid cavities could reflect their origin from the villous tissue and suggests that ET may play a role in the development of placenta and other fetal organs during organogenesis.

Localization and production of immunoreactive endothelin-1 in the trophoblast of human placenta.

Regarding their endocrine and paracrine activities, endothelins can be considered as peptide hormones and growth factors. The presence of endothelin-1 (ET-1)-binding sites on smooth muscle of placental villous vessels, on villous trophoblast and on purified trophoblast in culture raises the question of the origin of the peptide. In placenta, endothelin could derive from maternal, fetal and/or endogenous sources. Therefore, localization of ET-1 was investigated by use of immunohistochemistry in human term placenta and in cultured trophoblast using the avidin-biotin-peroxidase complex procedure. Specificity of immunostaining was demonstrated by applying ET-1 antibody that has been preabsorbed with excess peptide. In sections of placenta, ET-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the vessels and in the syncytiotrophoblast of the villi. ET-1 IR was also detected in the decidua-like cells and in the extravillous cytotrophoblast of the basal plate, a region where the maternal and fetal cells intermingle closely. The extravillous cytotrophoblast of the chorionic plate and of the placental septa also exhibited a strong ET-1 IR. For trophoblast culture cytotrophoblastic cells were obtained from placental villi by trypsin-DNase dispersion, further purified on Percoll gradient and enriched by employing a monoclonal anti-HLA class-I antibody. The trophoblastic origin of the cells was demonstrated by immunohistochemistry and by studying the secretion of gestational hormones during culture. After different periods of culture of purified cytotrophoblastic cells (1 to 5 days), ET-1 IR was observed in 95% of cells: cytotrophoblastic cells, trophoblast aggregates, and syncytiotrophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)

Evolution of tissue levels of steroids: oestradiol-17 beta, oestrone and progesterone in the macaque myometrium during gestation.

In 15 cynomolgus monkeys between days 30-160 of gestation, tissue levels of oestrone (E1), oestradiol 17 beta (E2) and progesterone (P4) were assayed by RIA in the myometrium and the placenta. Myometrial samples were subdivided as follows: inner and outer layers adjacent to the placental area (IMP and OMP) and inner and outer layers from antiplacental areas (IMAP and OMAP). Steroid levels (ng/g wet wt) were in the range of known plasma values (ng/ml) and there was no asymmetric distribution of steroids between the various locations. When the results obtained in all the layers were pooled the gestational profiles indicated a decrease of E1 between days 80-130, whereas at the same time E2 and P4 increased. The ratio P4/E2 was 8 on day 40, 17 on day 80 and 9 on day 160. In the placenta, levels of E2 and P4 were 4 times higher, levels of E1 10 times higher than in the myometrium. Gestational profiles of the three steroids in the placenta increased from day 30 to day 160. Myometrium steroid content therefore does not appear to be a simple reflection of steroid diffusion from the site of production.

[Immunoradiometric assay for active renin and plasma prorenin during cycles stimulated by IVFET or GIFT]. / Dosage immunoradiométrique de la rénine active et de la prorénine plasmatiques au cours des cycles stimulés pour FIVETE ou GIFT.

Prorenin (PR) an inactive high molecular weight form of renin normally circulates in human plasma at a concentration of about 10 times that of active renin (AR) and this proenzyme seems to be linked to the reproductive function. It has been demonstrated that AR and PR are present at high concentrations in follicular fluid when the ovaries are stimulated with gonadotropins and that the PR plasma levels increase steadily after hCG injection with a correlation between blood PR and the number of developing follicles and corpora lutea. From September 1986 we studied the profile of immunoreactive active renin (AR) and prorenin (PR) in plasma during hyperstimulated cycles for IVFET or GIFT. All women were treated with a protocol combining GnRH analog (Decaptyl Ipsen Biotech, Paris France) and human menopausal gonadotropins until injection of 5,000 IU hCG. AR has been assayed in frozen samples by specific immunoradiometry (Renin RIA code 79 795, Pasteur Diagnostic, France) using two complementary monoclonal antibodies. A second assay of total renin was carried out after trypsin activation which revealed the inactive form. Progesterone (P), estradiol (E2) were measured by radioimmuno-assays. During the follicular phase, from day 1 of hMG administration to the day before hCG, no significant difference could be found between two groups, 63 pregnant or 60 nonpregnant cycles matched for age and number of oocytes retrieved, for E2, P, PR and AR. During the periovulatory period (D - 1, Do = day of hCG injection and D 1) no difference could be found for E2, P and PR (tabl. 1). In the 2 groups the mean E2 levels increased after hCG injection, as well as P and PR. But a significant difference appeared for AR which increased in the plasma immediately after hCG administration in the pregnant group whereas it decreased in the non-pregnant group (+2.5 vs -2 pg/ml) the mean variation between Do and D + 1 being significantly different in fertile cycles and in nonfertile cycles.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoreactive renin variations during fertile and infertile hyperstimulated cycles with in-vitro fertilization and embryo transfer or gamete intra-fallopian transfer.

The concentrations of prorenin (PR) and active renin (AR) were determined sequentially during ovarian hyperstimulated cycles for in-vitro fertilization-embryo transfer or gamete intra-Fallopian transfer. AR and PR increased during the early luteal phase and PR was highly correlated with the progesterone concentration. In fertile cycles, AR increased more rapidly after HCG injection and AR and PR levels were significantly higher during the late luteal phase. These results confirm the role of the renin angiotensin system in ovarian physiology.