Is 8% O2 more normoxic than 21% O2 for long-term in vitro cultures of human primary term cytotrophoblasts?

STUDY QUESTION: Is 8% O2 a better percentage of atmospheric oxygen for long-term cultures of human primary term cytotrophoblasts than the conventional 21% O2 traditionally used in cell culture? SUMMARY ANSWER: Human primary term cytotrophoblasts are able to differentiate into syncytiotrophoblasts under both atmospheric oxygen levels. WHAT IS KNOWN ALREADY: Cell culture is traditionally done under 21% O2, which is equal to a pO2 of ~160 mm Hg. Based on the pO2 measured after instauration of the blood circulation within the placenta, it has been proposed that cytotrophoblasts culture should be under 8% O2, which is equivalent to 60 mm Hg, and that this percentage should be considered as the physiological normoxia for cytotrophoblasts. STUDY, DESIGN, SIZE, DURATION: Cytotrophoblasts were isolated and purified from human term placentas (n > 4). Cells were cultured under 21% O2 and 8% O2 for 12 days. Several cellular parameters were assessed on Days 2, 4, 8 and 12. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placentas were obtained after vaginal or elective cesarean delivery from uncomplicated pregnancies at term (n ≥ 4). Cell viability was measured by a luminescent assay based on quantitation of the ATP content of living cells. Cell fusion was assessed by quantification of syncytin and e-cadherin mRNA expression by real-time PCR and determination of the fusion index by immunofluorescent microscopy. Trophoblast differentiation was assessed by measuring the expression levels of hCGß, inhibin α subunit (InhA) and placental growth factor (PlGF) by real-time PCR and ELISA. Finally, the effect of the two oxygen levels on apoptosis and cellular oxidative stress was also investigated by quantifying caspase 3/7 activation, superoxide dismutase 1 (SOD-1) mRNA expression and H2O2 generation. MAIN RESULTS AND THE ROLE OF CHANCE: There was no difference between 21% O2 and 8% O2 on cell viability. Cell fusion seemed to be enhanced during the first 4 days when the cells were cultured under 21% O2 compared to 8% O2. The expression level of hCGß was equivalent in both oxygen conditions, indicating that there was no difference in trophoblast differentiation. Interestingly, InhA expression was higher under 8% O2, while PlGF expression was inhibited compared to 21% O2. This latter result indicates that 8% O2 may be more hypoxic than normoxic for in vitro culture of primary term cytotrophoblast. This is further corroborated by the fact that 21% O2 did not significantly increase caspase 3/7 activities and the oxidative stress (SOD-1 mRNA expression and H2O2 generation) in our cell cultures. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The in vitro culture of cytotrophoblasts is artificial and does not reflect the in vivo situation. The cell population is nearly 100% pure, cultured as a monolayer, and the cells bath in a chemically defined culture medium deprived of any oxygen carrier. The oxygen molecules available to the cells are passively dissolved in the medium. The gas dissolution properties of the medium and the cellular consumption rate of oxygen may allow the cells to sustain a wide range of oxygen percentages from 8% to 21%. WIDER IMPLICATIONS OF THE FINDINGS: It is possible to culture human primary term cytotrophoblasts for at least 12 days. The O2 percentage of the air does not negatively affect in vitro cytotrophoblast differentiation. For in vitro culture of cytotrophoblasts, it is not necessary to lower the percentage of atmospheric oxygen to 8%. STUDY FUNDING/COMPETING INTEREST(S): This work was fully supported by 'Fetus for Life' charity. The authors state that there is no conflict of interest to declare regarding the publication of this paper.

HIF1A and EPAS1 potentiate hypoxia-induced upregulation of inhibin alpha chain expression in human term cytotrophoblasts in vitro.

Study Question: Are hypoxia-inducible factors (HIF) responsible for the potentiation of inhibin alpha subunit (INHA) gene expression in primary cultures of human term cytotrophoblasts under low-oxygen tension? Summary Answer: Both HIF1A and endothelial PAS domain protein 1 (EPAS1) are involved in the potentiation of INHA gene upregulation in cytotrophoblasts cultured under hypoxia. What Is Known Already: During the in vitro differentiation of cytotrophoblasts into syncytiotrophoblasts under 21% O2, INHA expression increases. This expression is further increased when cells are cultured under low-oxygen tension (e.g. 2.5% O2). Moreover, in pregnancy-related diseases, such as pre-eclampsia or intrauterine growth restriction (IUGR), in which hypoxia is suspected to be responsible for the abnormal placental development, maternal serum concentration of inhibin A is elevated. Study Design, Size, Duration: Cytotrophoblasts were isolated and purified from human term placentas (n = 6). Cells were cultured under 21% O2, and allowed to differentiate for 48 h. A first group of cells was treated for 16 h under 21% O2 with dimethyloxalylglycine (DMOG) or deferoxamine (DFX), molecules that mimic hypoxia by inhibiting HIF1 proteasomal degradation. Involvement of HIF1A and EPAS1 (also known as HIF2A), two HIF isoforms expressed in trophoblasts, was shown by treating another group of cells cultured under 2.5% O2 with specific inhibitors of HIF1A and EPAS1 for 16 h. INHA mRNA expression was assessed by real-time PCR and secreted inhibin A was quantified by ELISA. The role of HIF1A and EPAS1 in INHA transcriptional regulation was further confirmed by cotransfecting primary cytotrophoblasts with a luciferase reporter plasmid containing a 3.9 kb INHA promoter and plasmids allowing overexpression of HIF1A and EPAS1. Participants/Materials, Setting, Methods: Placentas were obtained after vaginal or elective cesarean delivery from uncomplicated pregnancies at term (n≥ 4). The methods used were hormone measurements in the cell supernatants by enzyme-linked immunosorbent assay, real-time quantitative PCR, western blotting, immunofluorescence microscopy and transient transfection. Main Results and the Role of Chance: HIF1 protein stabilization with DMOG and DFX increased 21% O2-induced INHA mRNA and protein upregulation (P < 0.05 versus control), while hypoxia-induced INHA upregulation was repressed by HIF1A and EPAS1 inhibitors (P < 0.05 versus control). In transfection experiments of primary term cytotrophoblasts, cloned INHA promoter transcriptional activity was increased by 2.5% O2 compared to 21% O2 (P < 0.05). Overexpression of both HIF1A and EPAS1 under 21% O2 increased cloned INHA transcriptional activity (P < 0.001 versus control). Large Scale Data: Not applicable. Limitations, Reasons for Caution: HIF1A and EPAS1 may regulate INHA expression by binding to an hypoxia-responsive element within the promoter, but we were unable to identify such an element. Inhibition of HIF1A and EPAS1 did not completely suppress upregulation of INHA expression suggesting that other transcription factors, not identified or studied here, are involved. Wider Implications of the Findings: Our data suggest that the effect of HIF1 proteins on INHA gene promoter activity may be indirect. By demonstrating the role of HIF1A and especially EPAS1 in INHA gene upregulation under hypoxia, the results suggest that HIF1 proteins may become new therapeutic targets in the treatment of pregnancy-related diseases such as pre-eclampsia or IUGR. Study Funding/Competing Interest(s): This work was fully supported by 'Fetus for Life' charity. C. Depoix was supported by a fellowship 'Fonds de Recherche Clinique' from 'Fondation Saint-Luc', Belgium. The authors declare that there is no conflict of interest regarding the publication of this paper.

[Cancer diagnosis during pregnancy: importance of a national and European registration]. / Cancer pendant la grossesse: importance d'un registre national et Européen.

Cancer during pregnancy is relatively rare, but its incidence has been increasing over recent years. A European study has been launched in 2005 by F. Amant (KUL) to register all pregnant patients with a cancer diagnosis with or without treatment during pregnancy (surgery, chemotherapy and/or radiotherapy). All infants exposed to chemotherapy and/or radiotherapy are also followed up by pediatricians, neurologists, cardiologists and psychologists. In Belgium, French- and Dutch- language hospitals are working in close collaboration to follow these pregnant patients. The national results are summarized in this paper.

Reversible effects of oxygen partial pressure on genes associated with placental angiogenesis and differentiation in primary-term cytotrophoblast cell culture.

Timely regulated changes in oxygen partial pressure are important for placental formation. Disturbances could be responsible for pregnancy-related diseases like preeclampsia and intrauterine growth restriction. We aimed to (i) determine the effect of oxygen partial pressure on cytotrophoblast differentiation; (ii) measure mRNA expression and protein secretion from genes associated with placental angiogenesis; and (iii) determine the reversibility of these effects at different oxygen partial pressures. Term cytotrophoblasts were incubated at 21% and 2.5% O2 for 96 hr, or were switched between the two oxygen concentrations after 48 hr. Real-time PCR and enzyme-linked immunosorbent assays (ELISAs) were used to evaluate cell fusion and differentiation, measuring transcript levels for those genes involved in cell fusion and placental angiogenesis, including VEGF, PlGF, VEGFR1, sVEGFR1, sENG, INHA, and GCM1. Cytotrophoblasts underwent fusion and differentiation in 2.5% O2 . PlGF expression was inhibited while sVEGFR1 expression increased. VEGF and sENG mRNA expressions increased in 2.5% compared to 21% O2 , but no protein was detected in the cell supernatants. Finally, GCM1 mRNA expression increased during trophoblast differentiation at 21% O2 , but was inhibited at 2.5% O2 . These mRNA expression effects were reversed by returning the cells to 21% O2 . Thus, low-oxygen partial pressure does not inhibit term-cytotrophoblast cell fusion and differentiation in vitro. Lowering the oxygen partial pressure from 21% to 2.5% caused normal-term trophoblasts to reversibly modify their expression of genes associated with placental angiogenesis. This suggests that modifications observed in pregnancy diseases such as preeclampsia or growth retardation are probably due to an extrinsic effect on trophoblasts.

Viability of primary term cytotrophoblast cell culture in normoxia and hypoxia.

Cytotrophoblast (CT) cells isolated and purified from term placenta are able to differentiate into syncytiotrophoblast cells. Previous reports suggested that hypoxia is an inhibitor of this differentiation and also increases apoptosis. As visual observations of our CT cell cultures indicated a better development in hypoxia than in normoxia (defined as 2.5 and 21% O(2), respectively), we decided to assess the effect of low oxygen tension on in vitro CT cell differentiation by measuring cell viability, apoptosis and CT cell fusion and differentiation. We observed a 45% decrease in cell viability 24 h after plating both in normoxia and in hypoxia but no difference between the two oxygen conditions. Cell viability remained stable during the 4-day culture. Apoptosis also did not increase in hypoxia. Apoptotic index and caspase activation were even lower in hypoxia than in normoxia at Day 1 and Day 4 of the culture. Finally, we observed a 5-fold and 6-fold increase in Syncytin-1 mRNA expression in normoxia and in hypoxia, respectively, indicating that hypoxia did not inhibit CT cell fusion. CT cells differentiated as well in hypoxia as an increase in inhibin α subunit mRNA was evidenced during the 4-day culture. This increase was even higher in hypoxia than in normoxia. In conclusion, hypoxia defined as 2.5% O(2) based on first trimester placental pO(2) did not decrease term primary CT cell viability and did not increase apoptosis. Moreover, it did not inhibit either CT cell fusion or differentiation.

[Screening strategy and diagnostic criteria for gestational diabetes. Proposals of the GGOLFB]. / Stratégie de dépistage et critères diagnostiques du diabète gestationnel. Propositions du GGOLFB.

For nearly 50 years, the strategy of screening and the diagnostic criteria for gestational diabetes have been the subject of endless controversies. They differ between countries and from one center to another, mainly because of the lack of hard data allowing to define glycemic thresholds at which a therapeutic management is needed. Recently, a large observational study has demonstrated the existence of a robust relationship between maternal blood sugar and several fetomaternal perinatal complications. This relationship is linear, with no clear threshold that would define gestational diabetes unambiguously. Meanwhile, two randomized intervention trials have shown that the therapeutic management of mild gestational diabetes was associated with improved perinatal outcomes. Based on these data, the " International Association of Diabetes and Pregnancy Study Group "(IADPSG) released new recommendations on screening methods and diagnostic criteria for gestational diabetes. Although already endorsed by several international associations and implemented in some countries, these recommendations still raise questions and criticisms. This is why the "Groupement des Gynécologues Obstétriciens de Langue Française de Belgique " (GGOLFB) organized a meeting between diabetologists and gynecologists which allowed to reach a consensus on the strategy that we intend to implement in our respective centers. The purpose of this paper is to briefly overview the recent advances in gestational diabetes and more particularly to make our key conclusions known to the medical community. This will enable the standardization of the management of gestational diabetes in the French-speaking part of Belgium.

[Screening strategy and diagnostic criteria for gestational diabetes. Proposals of the GGOLFB]. / Stratégie de dépistage et critères diagnostiques du diabète gestationnel. Propositions du GGOLFB.

For nearly 50 years, the strategy of screening and the diagnostic criteria for gestational diabetes have been the subject of endless controversies. They differ between countries and from one center to another, mainly because of the lack of hard data allowing to define glycemic thresholds at which a therapeutic management is needed. Recently, a large observational study has demonstrated the existence of a robust relationship between maternal blood sugar and several fetomaternal perinatal complications. This relationship is linear, with no clear threshold that would define gestational diabetes unambiguously. Meanwhile, two randomized intervention trials have shown that the therapeutic management of mild gestational diabetes was associated with improved perinatal outcomes. Based on these data, the "International Association of Diabetes and Pregnancy Study Group" (IADPSG) released new recommendations on screening methods and diagnostic criteria for gestational diabetes. Although already endorsed by several international associations and implemented in some countries, these recommendations still raise questions and criticisms. This is why the "Groupement des Gynécologues Obstétriciens de Langue Française de Belgique" (GGOLFB) organized a meeting between diabetologists and gynecologists which allowed to reach a consensus on the strategy that we intend to implement in our respective centers. The purpose of this paper is to briefly overview the recent advances in gestational diabetes and more particularly to make our key conclusions known to the medical community. This will enable the standardization of the management of gestational diabetes in the French-speaking part of Belgium.

Prevention of preterm labour: 2011 update on tocolysis.

The aim of this paper is to review available data about drugs for preventing preterm labour. Tocolytic therapy includes ß adrenergic receptor agonists, NO donors, magnesium sulphate, prostaglandin-synthase inhibitors, oxytocin receptor antagonists, calcium-channel blockers, progesterone, 17-α-hydroxyprogesterone caproate, and antibiotics. Their specific effects on myometrial contractility, their safety, their efficiency, and side effects profile for the mother and the fetus are presented. The main question of why and for what reasons tocolysis should be administrated is discussed.

Transcription factor AP2 regulates human inhibin α subunit gene expression during in vitro trophoblast differentiation.

During human trophoblast differentiation, inhibin α subunit mRNA expression and protein secretion are increased. To understand how inhibin α subunit gene was regulated during syncytialization, we firstly cloned the inhibin α promoter and found a region with transcriptional activity related to the differentiation state. In this paper, we identified this protein and defined its DNA-binding site. Protein purification and identification were done by DNA affinity chromatography followed by mass spectrometry and western blotting. In order to confirm the identity of the protein, characterize its DNA-binding properties and to measure its expression during in vitro trophoblast differentiation, gel retardation assays and real-time polymerase chain reaction were done. We found that the cytotrophoblastic protein interacting with the inhibin α promoter was the transcription factor activating protein 2 (AP2). Western blotting using specific antibodies against AP2α and AP2γ confirmed that AP2α was the main subtype present in trophoblast cells, while AP2γ was barely detectable. Supershift experiments indicated that these two factors were able to bind to the sequence 5'-GCCtcaAGC-3'. We also observed an increase in AP2α mRNA and protein during in vitro trophoblast differentiation correlated with an increase in inhibin α subunit gene expression. Furthermore, AP2α and AP2γ overexpression in these cells was responsible for an increase in inhibin α subunit mRNA expression. We conclude that AP2 regulates the inhibin α subunit gene expression during trophoblast differentiation and may be a key regulator of syncytialization.

To treat or not to treat euthyroid autoimmune disorder during pregnancy?

BACKGROUND: Subclinical autoimmune hypothyroidism during pregnancy is associated with an increased risk of miscarriage and has a deleterious effect on fetal development. The aim of this study was to evaluate a screening and treatment strategy of subclinical hypothyroidism, and to establish normal ranges of thyroid-stimulating hormone (TSH) and thyroxine (T(4)) during pregnancy. METHODS: A retrospective study was carried out on 784 consecutive files of pregnant women; the files were systematically searched for thyroid function and antithyroid antibodies in order to determine the effect and the prevalence of anti-thyroid peroxidase antibodies (TPO-Ab) during pregnancy, and to evaluate treatment with levothyroxin (LT(4)) in TPO-Ab carriers. RESULTS: Among the 75 TPO-Ab-positive patients, 42 received LT(4) treatment during pregnancy. Although the range of TSH serum levels was wide, the mean TSH level was significantly higher in TPO-Ab-positive women (3 vs. 1 mIU/l, p < 0.01). No significant difference in the obstetrical complications rate was observed between TPO-Ab-positive and TPO-Ab-negative populations. CONCLUSIONS: Our study provides information on normal ranges of serum TSH and free T(4) for Belgian pregnant women receiving iodide supplementation. Based on our results, we suggest supplementation of TPO-Ab-positive pregnant women with 50 microg/day of LT(4), unless their TSH levels are lower than 1 mIU/l, to avoid the risk of hypothyroidism during pregnancy.

Contemporary neonatal outcome following rupture of membranes prior to 25 weeks with prolonged oligohydramnios.

BACKGROUND: Prolonged oligohydramnios following early preterm prelabour rupture of membranes (PPROM) is traditionally associated with high neonatal mortality and significant risk of pulmonary hypoplasia. However, recent evidence points to an apparent improvement in outcome. AIMS: To document current neonatal outcomes following rupture of membranes prior to 25 weeks with severe persistent oligohydramnios and a latency to delivery of at least 14 days. METHODS: A retrospective case note analysis over a 28-month period at Saint Luc University Hospital, Brussels. RESULTS: From 23 pregnancies that were complicated by PPROM prior to 25 weeks, 15 infants were born after 24 weeks with a latency of more than 14 days and persistent oligohydramnios. Nine infants (60%) had severe respiratory failure and clinical signs compatible with pulmonary hypoplasia. Seven of these infants (78%) responded to high frequency ventilation and inhaled nitric oxide therapy with good clinical outcome but two died from severe respiratory failure. Five infants showed no clinical signs of pulmonary hypoplasia and responded to conventional neonatal management. One of these infants died at 77 days of age of necrotising enterocolitis. One infant was not resuscitated and died within minutes of birth, following prior discussion with the perinatal team and the parents. Survivors in this high-risk group (73%) had low morbidity at the time of discharge. SUMMARY: The favourable neonatal survival and morbidity figures are in keeping with recent published evidence. This study confirms improved outcome even amongst the highest risk infants with documented persistent oligohydramnios.

[Dissection of renal artery aneurysm in a pregnant woman with Elhers-Danlos disease type IV]. / Dissection d'un anévrysme de l'artère rénale chez une femme enceinte atteinte de maladie Elhers-Danlos de type IV.

Recall the clinical signs and personal and family medical history suggestive of Elhers-Danlos syndrome, mainly type IV. Review of literature and presentation of a clinical case observed in a female patient with Elhers-Danlos disease type IV who developed dissection of a renal artery during pregnancy. The prevalence of the syndrome is increasing, reaching, according to the latest publications 1/5,000 births. It is important to determine the type of syndrome to assess prognosis. In the classic type and in the hyper mobile type, pregnancy is generally well tolerated although certain complications linked to connective tissue dysfunction such as pelvic instability, premature delivery, bleeding postpartum and perineal lacerations, may develop. Ehlers-Danlos syndrome can be associated with serious and even fatal complications for these patients: vascular dissection or uterine rupture. It is important for obstetricians to be aware of the clinical signs and symptoms suggestive of Elhers-Danlos syndrome in a pregnant patient and to know the diagnostic possibilities and potential risks. If a typeIV syndrome is diagnosed, it is very important to inform the patient about the potential implications for pregnancy and maternal health, as well as the risk of transmission to the child and the possibility of antenatal diagnosis.

Activin receptor expression and induction of apoptosis in rat blastocysts in vitro.

BACKGROUND: Apoptosis, a process of normal embryonic development, is enhanced in blastocyst from diabetic rats. Nevertheless, glucose seems not to be the only factor involved. Activin A, a TGF-beta family member, is also increased in maternal serum from diabetic pregnancy. METHODS: Flushing medium, blastocysts and uterine cells were obtained from 5 day old pregnant rats. The presence of activin A in flushing medium was investigated by western blotting. RT-PCR was used to test for the presence of activin betaA subunit mRNA in cultured uterine cells. Blastocysts were stained by immunohistochemistry for activin receptor types IIA and IIB, and chromatin degradation (apoptosis) was investigated by terminal transferase-mediated dUTP nick end labelling in blastocysts exposed in vitro to activin. RESULTS: In this study, we demonstrate the presence of activin A protein in fluid from rat uterine horns at day 5 of pregnancy, as well as the presence of activin A receptors type IIB in the trophectoderm and inner cell mass and activin A receptor type IIA in trophectoderm cells only. Activin A increases the chromatin degradation level in vitro. CONCLUSIONS: Activin A protein was found in fluid from uterine horns, and mRNA expression of betaA activin subunit in cultured uterine cells suggests probable secretion from decidual cells. Moreover, activin A increases specifically the apoptosis level in rat blastocyst in vitro.

Control of the human inhibin alpha chain promoter in cytotrophoblast cells differentiating into syncytium.

Inhibins are dimeric proteins consisting of a common alpha subunit linked to one of the beta subunits, beta A or beta B. During pregnancy, the placenta is the main source of inhibin A production and the in-vitro transformation of cytotrophoblast cells into syncytium is associated with an inhibin alpha subunit mRNA up-regulation. In this study, the 5' region of the human inhibin alpha gene was isolated and sequenced. Three transcription initiation sites were identified. When transiently transfected in trophoblast cells with a luciferase reporter vector, the sequence displayed promoter activity. DNase I footprinting and electrophoretic mobility shift assay (EMSA) analysis showed a specific DNA-protein interaction in the promoter when using cytotrophoblast nuclear proteins. This interaction was weaker with syncytiotrophoblast nuclear proteins. Moreover, the deletion of this DNA-protein interaction region suppressed the promoter activity. In an attempt to identify this factor, the potential binding of known factors delta EF1, AP1 and NFE2 were excluded by competition EMSA experiments. We suggest that it may correspond to an undescribed protein interaction. The identification of the human inhibin alpha promoter could help in understanding the mechanisms modulating inhibin gene transcription. Moreover, the identification of a factor, whose presence is related to the trophoblast cell differentiation state, could help in understanding the transformation of cytotrophoblast cells into syncytium.

Vascular endothelial growth factor and placenta growth factor concentrations in Down's syndrome and control pregnancies.

Vascular endothelial growth factor (VEGF) and placenta growth factor (PLGF) are considered to play important roles in angiogenesis and vascular permeability during placental development. Since trisomy 21 placentae show trophoblastic hypoplasia and hypovascularity, we investigated PLGF and VEGF synthesis in Down's syndrome pregnancies. Maternal serum was collected from 102 euploid and 24 trisomy 21 pregnancies between 15 and 20 weeks gestation and tested for these two factors by enzyme-linked immunosorbent assays. Protein extracts from 15 normal and six trisomy 21 placentae were also tested. VEGF was not detected in maternal serum, while PLGF increased significantly with gestational age. Serum PLGF, transformed as a multiple of the gestational age median (MoM), in Down's syndrome pregnancies was significantly lower than in euploid controls (mean 0.67 +/- 0.043 MoM versus 1.00 +/- 0.047 MoM, analysis of variance F = 11.605, P < 0.001 ). Both VEGF and PLGF were detected in placental protein extracts without variation according to gestational age. Down's syndrome placentae had significantly less PLGF compared to normal placentae (Mann-Whitney, P < 0.05 ) but no difference was observed in placental VEGF content (Mann-Whitney, P = 0.94 ). Considering the biological properties of PLGF, this decrease may provide new insights into the mechanism(s) leading to the structural and functional anomalies described in trisomy 21 placentae.

Inhibin subunits mRNA expression level in human placenta from normal and Down's syndrome pregnancies.

In order to study the mechanisms leading to increased inhibin A and activin A in maternal serum with advancing gestation and increased inhibin A in Down's syndrome pregnancy, the mRNA expression level of inhibin and activin subunits was quantitatively studied in human placenta using Northern blot and semiquantitative RT-PCR analysis. The corresponding protein level was also determined by specific ELISAs for inhibin A, inhibin B, activin A and inhibin pro alphaC in placental extracts. Normal placenta (n=27) showed a slight significant increase in alpha and betaA subunit mRNA levels in term pregnancy, with no change of the corresponding protein level. Placenta from Down's syndrome pregnancies (n=6) did not differ from controls in either mRNA expression or corresponding protein levels. In conclusion, there is a dissociation between inhibin and activin subunit mRNA levels and the corresponding protein levels in maternal serum, and Down's syndrome inhibin A increase is not explained by mRNA expression upregulation. In an additional study, ovarian cortex tissue from term pregnancies (n=3) were examined. Only the alpha subunit mRNA was expressed, at a higher level than in the placenta, suggesting that ovary could be a source of inhibin pro alphaC during pregnancy.

Inhibin and activin production and subunit expression in human placental cells cultured in vitro.

Inhibins and activins are dimeric proteins, with each subunit being one of three related protein subunits (alpha, betaA or betaB). The mRNA levels of these subunits were studied quantitatively during in-vitro differentiation of human cytotrophoblast cells into syncytium, using Northern blot analysis and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. The corresponding protein concentrations were determined by specific enzyme-linked immunosorbent assays for inhibin A, B, pro alphaC and activin A in cellular protein extracts and culture medium (n = 5). Immunofluorescence studies showed syncytium formation after 48 h. The alpha subunit was present before plating and increased at 48 h (P<0.001) while the betaA subunit was weak before plating and increased at 24 h. The betaB subunit was not detected. With respect to corresponding protein synthesis, inhibin A (alpha + betaA) had risen after 48 h in cellular protein extract and after 72 h in culture medium, while activin A (betaA + betaB) was detected after 24 h, with no significant variations in culture medium. There was a good correlation between inhibin A and alpha subunit expression (r = 0.736, P<0.001), as well as between activin A and betaA subunit expression (r = 0.755, P<0.001). This study showed that mRNA expression parallels protein synthesis of inhibin and activin in trophoblast cells. Inhibin A synthesis appears to be dependent on alpha subunit mRNA expression, rather than on the betaA subunit which controls activin A synthesis. This study has also shown that isolated cytotrophoblast cells do not produce dimeric inhibin. However, during the transformation of cytotrophoblast cells into syncytium, betaA subunit mRNA expression may be an indicator of cell aggregation, while alpha subunit mRNA expression may be an indicator of cell fusion.