Detection of early placental hormone production in embryo transfer cycles lacking a corpus luteum.

PURPOSE: This study sought to identify the initiation of placental hormonal production as defined by the production of endogenous estradiol (E2) and progesterone (P4) in a cohort of patients undergoing programmed endometrial preparation cycles with single embryo transfers resulting in live-born singletons. METHODS: In this retrospective cohort study, patients undergoing either programmed frozen-thawed embryo transfer (FET) with autologous oocytes or donor egg recipient (DER) cycles with fresh embryos were screened for inclusion. Only patients who underwent a single embryo transfer, had a single gestational sac, and a resultant live-born singleton were included. All patients were treated with E2 patches and intramuscular progesterone injections. Main outcome measures were serial E2 and P4, with median values calculated for cycle days 28 (baseline), or 4w0d gestational age (GA), through 60, or 8w4d GA. The baseline cycle day (CD) 28 median value was compared to each daily median cycle day value using the Wilcoxon signed rank test. RESULTS: A total of 696 patients, 569 using autologous oocytes in programmed FET cycles and 127 using fresh donor oocytes, from 4/2013 to 4/2019 met inclusion criteria. Serum E2 and P4 levels stayed consistent initially and then began to increase daily. Compared to baseline CD 28 E2 (415 pg/mL), the serum E2 was significantly elevated at 542 pg/mL (P < 0.001) beginning on CD 36 (5w1d GA). With respect to baseline CD 28 P4 (28.1 ng/mL), beginning on CD 48 (6w6d GA), the serum P4 was significantly elevated at 31.6 ng/mL (P < 0.001). CONCLUSION: These results demonstrate that endogenous placental estradiol and progesterone production may occur by CD 36 and CD 48, respectively, earlier than traditionally thought.

Steroid hormone synthesis in mitochondria.

Mitochondria are essential sites for steroid hormone biosynthesis. Mitochondria in the steroidogenic cells of the adrenal, gonad, placenta and brain contain the cholesterol side-chain cleavage enzyme, P450scc, and its two electron-transfer partners, ferredoxin reductase and ferredoxin. This enzyme system converts cholesterol to pregnenolone and determines net steroidogenic capacity, so that it serves as the chronic regulator of steroidogenesis. Several other steroidogenic enzymes, including 3ß-hydroxysteroid dehydrogenase, 11ß-hydroxylase and aldosterone synthase also reside in mitochondria. Similarly, the mitochondria of renal tubular cells contain two key enzymes participating in the activation and degradation of vitamin D. The access of cholesterol to the mitochondria is regulated by the steroidogenic acute regulatory protein, StAR, serving as the acute regulator of steroidogenesis. StAR action requires a complex multi-component molecular machine on the outer mitochondrial membrane (OMM). Components of this machine include the 18 kDa translocator protein (TSPO), the voltage-dependent anion chanel (VDAC-1), TSPO-associated protein 7 (PAP7, ACBD3), and protein kinase A regulatory subunit 1α (PKAR1A). The precise fashion in which these proteins interact and move cholesterol from the OMM to P450scc, and the means by which cholesterol is loaded into the OMM, remain unclear. Human deficiency diseases have been described for StAR and for all the mitochondrial steroidogenic enzymes, but not for the electron transfer proteins or for the components of the cholesterol import machine.

A novel role of ribonuclease inhibitor in regulation of epithelial-to-mesenchymal transition and ILK signaling pathway in bladder cancer cells.

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein possibly involved in biological functions other than the inhibition of RNase A and angiogenin activities. We have previously shown that RI can inhibit growth and metastasis in some cancer cells. Epithelial-mesenchymal transition (EMT) is regarded as the beginning of invasion and metastasis and has been implicated in the metastasis of bladder cancer. We therefore postulate that RI regulates EMT of bladder cancer cells. We find that the over-expression of RI induces the up-regulation of E-cadherin, accompanied with the decreased expression of proteins associated with EMT, such as N-cadherin, Snail, Slug, vimentin and Twist and of matrix metalloprotein-2 (MMP-2), MMP-9 and Cyclin-D1, both in vitro and in vivo. The up-regulation of RI inhibits cell proliferation, migration and invasion, alters cell morphology and adhesion and leads to the rearrangement of the cytoskeleton in vitro. We also demonstrate that the up-regulation of RI can decrease the expression of integrin-linked kinase (ILK), a central component of signaling cascades controlling an array of biological processes. The over-expression of RI reduces the phosphorylation of the ILK downstream signaling targets p-Akt and p-GSK3ß in T24 cells. We further find that bladder cancer with a high-metastasis capability shows higher vimentin, Snail, Slug and Twist and lower E-cadherin and RI expression in human clinical specimens. Finally, we provide evidence that the up-regulation of RI inhibits tumorigenesis and metastasis of bladder cancer in vivo. Thus, RI might play a novel role in the development of bladder cancer through regulating EMT and the ILK signaling pathway.

[Secretory expression vector V-pLNCX-s-hri inhibits the growth of mouse B16 melanoma].

BACKGROUND AND OBJECTIVE: Human ribonuclease inhibitor (hRI) extracted and purified from human placenta has been shown to remarkably inhibit some solid tumors in mice. This study was to construct V-pLNCX-s-hri, a secretory expression vector, and explore its inhibition effects on the growth of mouse B16 melanoma cells. METHODS: The hRI gene sequence conjugated with the synthesized signal peptide of mouse IgG was cloned into the retroviral vector V-pLNCX to construct V-pLNCX-s-hri. The PA317 cells were used for viral package and NIH3T3 cells were employed to determine the viral titer. The expression of hRI gene was detected by RT-PCR and Western blot. The content of RI was determined by enzyme-linked immunoabsorption assay (ELISA). The model of B16 melanoma-carrying mouse was established and received different treatments. The tumor weight and microvessle density (MVD) were assessed. Normal saline (NS), V-pLNCX, and V-pLNCX-hri were used as controls. RESULTS: The infection efficiency of V-pLNCX-s-hri on cultured B16 cells reached 38.5%. mRNA and protein levels of hRI were detected in B16 cells infected by V-pLNCX-s-hri. The hRI content in the supernatant of infected B16 cells reached 0.228 microg/mL. The hRI content in the peripheral blood of experimental mice was significantly higher in the V-pLNCX-s-hri group (0.249 microg/mL) than in the NS group (0.035 microg/mL), V-pLNCX group (0.028 microg/mL) and V-pLNCX-hri group (0.169 microg/mL) (P<0.01). The tumor weight and MVD were significantly lower in the V-pLNCX-s-hri group compared with those in the NS, V-pLNCX and V-pLNCX-hri groups (P>0.01). CONCLUSIONS: V-pLNCX-s-hri can effectively infect B16 cells and induce high expression of hRI. V-pLNCX-s-hri is superior to V-pLNCX-hri in inhibiting the growth of B16 cells.

Gene expression of placental hormones regulating energy balance in small for gestational age neonates.

OBJECTIVE: Fetal growth restriction is associated with an increased risk for metabolic and cardiovascular disease in later life. To further elucidate mechanisms that might be involved in the process of prenatal programming, we measured the adipokines leptin, resistin, and adiponectin and the GH-releasing hormone ghrelin in the placenta of small for gestational age (SGA) neonates. STUDY DESIGN: The control group included 24 placentas of appropriate for gestational age (AGA) newborns, in the study group were 16 placentas of SGA neonates. Gene expression of leptin, resistin, adiponectin, and ghrelin was examined. For hormones showing alterations in gene regulation placental protein expression was measured by Western blot. RESULTS: Placental mRNA expression of leptin was significantly increased in SGA placentas (p=0.0035, related to beta-actin). Protein concentration was increased, as well. There were no differences in placental resistin, adiponectin, or ghrelin gene expressions between SGA neonates and controls. Leptin was the only hormone to demonstrate a significant inverse correlation with birth weight (r=-0.44, p=0.01). Adiponectin correlated significantly with leptin (r=0.53, p=0.0023) and ghrelin (r=0.50, p=0.0045). CONCLUSIONS: Placental leptin gene expression and protein concentration showed the expected increase in the SGA group. Leptin was inversely correlated with birth weight. Positive correlation of adiponectin with leptin and ghrelin expression suggests an interaction between these hormones in the placenta. However, the unchanged expression of resistin, adiponectin, and ghrelin in SGA placentas and the absence of correlation with birth weight cast doubt whether these hormones produced in the placenta play a key role in fetal programming.

Cytokines of the placenta and extra-placental membranes: roles and regulation during human pregnancy and parturition.

Summary In an earlier, companion, review, we concluded that cytokines produced by the placenta and associated membranes are likely to be involved in control of the processes of implantation and placental development (Bowen et al., 2002). In this review, we discuss evidence that cytokines continue to be part of a paracrine/autocrine regulatory network in the placenta and membranes throughout the mid and late stages of gestation. Cytokines are involved in regulation of placental growth during these later stages of pregnancy and also function to protect the fetus from pathological organisms. The evidence, while not entirely consistent, suggests that production of certain cytokines within the extraplacental membranes is altered during normal term parturition, whereas in the villous placenta evidence of labour-associated changes is much more equivocal. Roles for cytokines have been postulated in many facets of parturition, including expulsion of the fetus by uterine contractions, membrane rupture, and dilation of the cervix. Imbalances and disruptions to the cytokine milieu have been implicated in a number of diseases of pregnancy involving abnormalities of both placental growth/establishment and initiation of parturition. Cytokine secretion induced by intrauterine infection is associated with increased occurrence or severity of some neonatal diseases. This wealth of data supports the view that cytokines are an integral part of a functional regulatory/communication network operating within the placental-maternal unit during normal gestation.

Effects of glucose on placental hormones in the human term placenta in vitro.

Glucose intake during pregnancy results in a decrease in endogenous insulin-like growth factor binding protein-1 (IGFBP-1). However, the exact role of glucose on placental secretion of IGFBP-1 is unclear. This study was designed to investigate the direct effects of glucose on the production of IGFBP-1 and other placental hormones, using an isolated placental preparation. Using the dual recirculating perfusion system for an isolated human placenta lobule, a total of 43 experiments were performed over a duration of 6 hours. Twenty placentae were perfused with a medium containing 141 +/- 10 mg/dL (7.83 +/- 0.56 mmol/L) glucose (group I) and 23 placentae with 242 +/- 12 mg/dL (13.43 +/- 0.67 mmol/L) glucose (group II). Levels of insulin, glucose, lactate, insulin-like growth factor (IGF-I), IGFBP-1, human placental lactogen (hPL) and beta-human chorionic gonadotropin (beta-hCG) were measured at 30 minute intervals during perfusion. Insulin and IGF-I were barely detectable in the perfusates and their levels were not modulated by glucose. IGFBP-1 was predominantly detected in the maternal rather than the fetal compartment of the placental circulation. Glucose increased the levels of IGFBP-1 in the maternal circulation in groups I and II during the first two hours of perfusion (188 +/- 58% and 193 +/- 31%, respectively). However, during the subsequent 4 hour period, the increase in IGFBP-1 concentration was significantly higher in group II (926 +/- 427%) than in group I (428 +/- 216%) (p < 0.05). There was no difference in the levels of hPL or beta-hCG between the two groups in the maternal circulation. Thus, glucose stimulates the production of IGFBP-1 in the maternal circulation of a placenta in vitro. This increase in IGFBP-1 by glucose in vitro, as opposed to the decrease of IGFBP-1 in vivo, may be due to a lack of circulatory maternal insulin in the isolated placental preparation. These results also suggest that there may be a functional barrier within the placenta that prevents an increase in the level of IGFBP-1 in the fetal circulation.

Ethanol alters hormone production in cultured human placental trophoblasts.

Maternal alcohol abuse during pregnancy can lead to abnormalities in fetal development, including the fetal alcohol syndrome (FAS). Although intrauterine growth retardation is a hallmark of FAS, the pathophysiology is not fully understood. A contributing factor may be altered placental function, which could affect fetal growth and development. As a major endocrine organ during pregnancy, changes in the production of placental hormones could affect pregnancy and possibly fetal development. In this study, the effect of continued exposure to ethanol on placental hormone production was examined using cultured human placental trophoblasts. Ethanol exposure involved diffusion of ethanol from the atmosphere into the culture medium. This was refreshed daily, leading to daily peak concentrations of 280 to 300 mg/dl (60-65 mM) at 16 to 24 hr. This ethanol exposure for 2 or 4 days significantly increased the production of human chorionic gonadotropin and progesterone by the cultured trophoblasts. However, ethanol treatment had no effect on human placental lactogen production. Acute stimulation (10 min) of cultured trophoblasts with adenosine (50 microM) normally results in increased production of cyclic adenosine 3',5'-monophosphate (cAMP). With ethanol exposure, adenosine-stimulated cAMP production was significantly elevated relative to that in controls. However, the effect of ethanol on adenosine-stimulated cAMP did not appear to be secondary to chronic alterations in adenosine in the culture medium. Measurement of adenosine in the culture medium revealed no difference in concentration or production between control and ethanol treated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

A cytotoxic ribonuclease. Study of the mechanism of onconase cytotoxicity.

Onconase, or P-30, is a protein initially purified from extracts of Rana pipiens oocytes and early embryos based upon its anticancer activity both in vitro and in vivo. It is a basic single-chain protein with an apparent molecular mass of 12,000 daltons and is homologous to RNase A. In cultured 9L glioma cells, onconase inhibits protein synthesis with an IC50 of about 10(-7) M. The inhibition of protein synthesis correlates with cell death determined by clonogenic assays. 125I-Labeled onconase binds to specific sites on cultured 9L glioma cells. Scatchard analysis of the binding data shows that onconase appears to bind to cells with two different affinities, one with a Kd of 6.2 x 10(-8) and another of 2.5 x 10(-7) M. Each cell could bind about 3 x 10(5) molecules of onconase at each of the two affinity sites. The low affinity Kd is similar to the IC50 for onconase toxicity. Onconase also demonstrates a saturability of cytotoxicity at a concentration that would saturate the low affinity binding site. Incubation at 4 degrees C increased the binding of onconase to cells relative to 37 degrees C binding and also increased the sensitivity of cells to onconase toxicity, indicating that receptor binding may be an initial step in cell toxicity. Onconase cytotoxicity can be blocked by metabolic inhibitors, NaN3 and 2-deoxyglucose, and cytotoxicity is potentiated 10-fold by monensin. Ribonuclease activity appears necessary for onconase toxicity because alkylated onconase, which only retains 2% of the ribonuclease activity, was at least 100-fold less potent in inhibiting protein synthesis in cells. Onconase inhibition of protein synthesis in 9L cells coincides with the degradation of cellular 28 S and 18 S rRNA. In contrast to RNase A, onconase is resistant to two RNase inhibitors, placental ribonuclease inhibitor and Inhibit-Ace. Northern hybridization with placental ribonuclease inhibitor cDNA probe indicates that 9L glioma cells contain endogenous placental ribonuclease inhibitor mRNA. Based on these results, we propose that onconase toxicity results from onconase binding to cell surface receptors, internalization to the cell cytosol where it degrades ribosomal RNA, inhibiting protein synthesis and causing cell death.

Early hypophysectomy of sheep fetuses: effects on growth, placental steroidogenesis and prostaglandin production.

The factors involved in the control of steroid secretion from the ovine placenta and in fetal growth are as yet unclear. We hypothesized that factors derived from the fetal pituitary may play a role in the production and release of placental steroids and in growth of the fetus, and have investigated the effects of fetal hypophysectomy performed between day 70 and day 79 of gestation (term = 147 days) on systemic concentrations of hormones derived from the placenta, and on fetal growth. Maternal peripheral progesterone, placental lactogen and uterine vein progesterone increased significantly from day 90 in all ewes. Peripheral concentrations of prostaglandin E2 and peripheral and uterine vein oestrone sulfate increased significantly in the control group but not in the fetal hypophysectomy group. Uterine vein prostaglandin E2 increased significantly after day 95 in the control group and after day 105 in the fetal hypophysectomy group. Early fetal hypophysectomy caused marked growth retardation. The weights of the brain, kidneys and liver of hypophysectomized fetuses were the same as those of controls suggesting that their growth is not under pituitary control. In contrast, the weights of heart and lungs were reduced in proportion to body weight, suggesting that heart, lung and carcass growth were under pituitary control. Our data indicate that the fetal pituitary influences the control of placental steroid and prostaglandin E2 biosynthesis after day 90 of gestation in sheep, but that output of other hormones such as placental lactogen is independent of pituitary control, and may determine organ-specific growth parameters that are unaffected by removal of the fetal pituitary.

Chorionic gonadotrophin and c-myc expression in growing and growth-inhibited (intermediate) trophoblasts.

FEG-3 cells are a clonal line of human choriocarcinoma and resemble villous cytotrophoblasts which are the stem cells for the syncytiotrophoblast in the placenta. FEG-3 cells synthesize and secrete the alpha subunit of human chorionic gonadotrophin (hCG). Treatment of FEG-3 cells with the chemotherapeutic drug (1 microM) methotrexate (MTX) results in an increase in nuclear diameter. Cell division is blocked and a decrease in c-myc mRNA levels in observed. The effects on cell growth and c-myc mRNA expression are reversible, and cells treated with MTX for 48 h retain their proliferative potential. Assessment of placental hormone gene expression reveals that a member of the human growth hormone gene family is expressed at extremely low levels and is unaffected by MTX treatment. Alpha and beta chorionic gonadotrophin (hCG) levels are increased by MTX treatment, but levels decrease following removal of MTX. In contrast to hCG in FEG-3 cells, non-trophoblastic or ectopic production of alpha hCG in human cervical carcinoma cells is inhibited by MTX treatment. These data indicate that MTX will induce morphological and biochemical changes in FEG-3 cells. They reveal an inverse relationship between c-myc and hCG RNA expression, and suggest different mechanisms govern trophoblast versus non-trophoblast production of alpha hCG.

Identification and characterization of monolayer cultures of sheep trophoblast cells maintained in bicameral culture chambers.

Enriched epithelial cell and fibroblast fractions were isolated from ovine placentomes by isopycnic centrifugation of collagenase/DNAse-dispersed cells through a density gradient of 45% Percoll. The epithelial cells formed confluent monolayers when plated onto filters impregnated with a 50-microns layer of Matrigel in medium containing 10% fetal bovine serum. These cells were maintained in dual environment culture chambers in serum-free medium for at least 12 days. The epithelium had a polarized appearance similar to that found in vivo only when cells were plated at high density (10(7)/cells/cm2). The epithelial monolayer consisted predominantly of a single population of uninucleate cells with intracellular features similar to those previously described for ovine trophoblast both in vivo and in vitro. These cells stained positively with an antiserum to alpha-keratin, a marker specific to epithelial cells, and no staining was observed with antisera raised against binucleate cells or leucocyte-common antigen. Binucleate cells were detected by microscopy and immunostaining in the pellet of cells obtained from the Percoll gradient but were rarely seen in the epithelium. The epithelial monolayer excluded 3H-inulin, added to the basal chamber, from the apical chamber, thus demonstrating the formation of a permeability barrier similar to that found in vivo. The maintenance of a monolayer of pure ovine trophoblast cells in vitro, which retain the characteristics of the epithelium in vivo, will enable the study of many cellular functions of the trophoblast.

Expression of human placental ribonuclease inhibitor in Escherichia coli.

Human placental ribonuclease inhibitor (PRI) has been expressed in and isolated from Escherichia coli. Its apparent molecular weight, immunoreactivity and amino acid composition are virtually identical with those of native PRI. It inhibits the enzymatic activities of either angiogenin, a blood vessel inducing protein homologous to pancreatic RNase (RNase A), or RNase A in a stoichiometry of 1:1. Recombinant PRI binds to angiogenin and RNase A with Ki values of 2.9 x 10(-16) M and 6.8 x 10(-14) M, respectively, comparable to the affinities of native PRI for these enzymes. Thus, these results confirm that PRI inhibits angiogenin more effectively than RNase A.

Endocrinological problems in malformation of the male genitalia.

There are two groups of congenital defects of the male genitalia: defects of organogenesis (e. g., aplasia of the external genitalia) and malformations due to defective hormonal influences on these organs. The pathophysiological mechanisms of the functional malformations can be a defect of androgen biosynthesis in the gonads, defects of conversion of precursor hormones into the biologically active compound, and defects at the receptor site with a faulty hormonal utilisation. All these defects in the production of a biologically active substance can be present and operating very early in life, i. e., in the embryonal or foetal period. Problems can arise from the mother and particularly the placenta, or from the child (pituitary). The production of an antimüllerian hormone in Sertoli cells has been recently reported as an additional important mechanism. These hormonal defects influence the development of the male genitourinary tract system. Testosterone biosynthesis has several enzymatic steps: The synthesis of androgens starts early in the embryonal period, with the final transformation of the indifferent organ into the differentiated external male genitalia occurring under the influence of the foetal testosterone. Targets for these hormonal malfunctions are, besides the urogenital system, an enzyme (5-alpha-hydroxylase) and cytoplasmic and nuclear receptor sites on the skin of the external genital region. One of the clinical manifestations of such a hormonal malfunction is a defective virilisation of the external male genitals, with several types of hypospadias. Complete and partial defects of testosterone biosynthesis are known. Extensive laboratory investigations disclose the specific defect. Besides hypospadias, similar malformations occur in the form of ambiguous external genitalia if testosterone cannot metabolize into dihydrotestosterone in the target cell. In earlier descriptions this congenital genitourinary defect was called pseudovaginal perineoscrotal hypospadias. Today, the specific defect is identified as a 5-alpha-reductase deficiency. The diagnosis is established by a skin biopsy of the genital area. Ambiguous genitalia of variable expression are present if the receptor sites at the target cells are defective. The clinical presentation is known as testicular feminization or hereditary male pseudohermaphroditism. Incomplete partial expression of this disorder is known. The diagnosis is made clinically, and diagnosis is proven by investigation of hormone receptor sites in the target cells in the skin of the genital region.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of hormone production in the human feto-placental unit.

Hormone production in the human feto-placental unit has been studied extensively yet relatively little is known about the regulatory mechanisms involved. A tissue culture approach has been used to examine the effect of potential controlling factors on steroid production by the human mid-term fetal adrenal and mid-term and term placenta. Adrenal. The pituitary peptides corticotropin (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) had the most significant influence on adrenal steroidogenesis in both the fetal and definitive zones. Their effects were not identical: they enhanced dehydroepiandrosterone sulphate (DHA-S) production in a comparable manner but alpha-MSH had much less of a stimulatory effect on cortisol biosynthesis. Medium from homologous fetal pituitary cultures mimicked the effects of alpha-MSH rather than ACTH. Homologous placental culture medium and progesterone enhanced only cortisol production and only in the fetal zone cells. These results demonstrate that specific fetal pituitary and placental factors influence fetal adrenal activity and suggest a functional zonation of the fetal adrenal. Placenta. DHA, DHA-S and 16-hydroxy-DHA stimulated oestrogen biosynthesis while high concentrations of DHA and DHA-S (but not 16-hydroxy-DHA) inhibited progesterone production. Luteinizing hormone-releasing hormone (LRH) inhibited both oestrogen and progesterone biosynthesis. Placental steroidogenesis can therefore be influenced not only by the fetus, through its increasing adrenal output of oestrogen precursors, but also by factors originating within the placenta itself.