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.

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.

Robust Recombinant Expression of Human Placental Ribonuclease Inhibitor in Insect Cells.

Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in E. coli, the robust expression is complicated by maintaining the redox potential and solubility by various expression tags. In the present paper we describe the expression of RI in baculovirus-infected High Five cells in large scale utilizing a modified transfer vector combining the beneficial properties of Profinity Exact Tag and pONE system. The recombinant RI is expressed at a high level in a fusion form, which is readily cleaved during on-column chromatography. A subsequent anion exchange chromatography was used as a polishing step to yield 12 mg native RI per liter of culture. RI expressed in insect cells shows higher thermal stability than the commercially available RI products (mainly produced in E. coli) based on temperature-dependent RNase inhibition studies. The endotoxin-free RI variant may also be applied in future therapeutics as a safe additive to increase mRNA stability in mRNA-based vaccines.

[Morphofunctional characteristics of the fetoplacental complex in pregnant women with an exacerbation of herpes virus infection and pathomorphological changes in fetal organs].

Impaired placental cholesterol synthesis in a pregnant woman with an exacerbation of herpesvirus infection is characterized by disturbances in placental hormone biosynthesis, causing severe morphological changes in fetal organs (liver, adrenals, lungs, and thymus).

Changes in the mRNA expression of structural proteins, hormone synthesis and secretion from bovine placentome sections after DDT and DDE treatment.

Disorders in the barrier function and secretory activity of the placenta can be caused by xenobiotics (XB) present in the environment and their accumulation in tissues of living organisms. Thus, the aim of this study was to investigate the effect of 1,1,1-trichloro-2,2,-bis-4-chlorophenyl-ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis-4-chlorophenyl-ethene (DDE) (for 24 or 48h) at doses of 1, 10 or 100ng/ml on the function of cow placentome sections in the second trimester of pregnancy. DDT and DDE affected neither (P>0.05) the viability nor hypoxia inducible factor 1 (HIF1α) mRNA expression of the sections. XB decreased (P<0.05) connexin (Cx) 26, 32, 43 and placenta-specific 1 (PLAC-1) mRNA expression but did not affect (P>0.05) keratin 8 (KRT8) mRNA expression. DDT and DDE also reduced (P<0.05) prostaglandin F2α (PGF2α) synthase (PGFS) mRNA expression, while DDT increased (P<0.05) prostaglandin E2 (PGE2) synthase (PGES) mRNA expression. Neither cyclooxygenase 2 (COX-2) mRNA expression nor PGF2α and PGE2 secretion were affected. Both DDT and DDE increased (P<0.05) neurophysin I/oxytocin (NP1/OT) mRNA expression and oxytocin (OT), oestradiol (E2) and progesterone (P4) secretion while DDT stimulated only 3ß-hydroxysteroid dehydrogenase (3ßHSD) and cholesterol side-chain cleavage enzyme (CYP11A1) mRNA expression (P<0.05). In summary, DDT and DDE impaired the barrier function and secretory activity of the placenta. Thus, these compounds can disrupt trophoblast invasion, myometrium contractility and gas/nutrient exchange throughout pregnancy in cows.

Nutritional influences on implantation and placental development.

The placenta is critical for nourishing the fetus throughout pregnancy, and also produces hormones that alter the metabolic functions of the mother. While the effects of nutrition on fetal development and long-term outcome have been very well documented, there are only a few reports based on studies in rat, sheep, and guinea pigs on how specific nutrients or general nutritional status affect the development of the blastocyst, its implantation, and the subsequent placenta. The data suggest that placental development is highly adaptable and that many types of compensation are possible for suboptimal nutrition.

Analysis of placental regulation of hematopoiesis.

Placental hormones contribute to changes in maternal physiology, especially to changes in the blood system. Methods are described to express a placental hormone from a cloned cDNA by transfection into a mammalian cell line, to purify the hormone, and to assess the activities of the hormone in primary mouse bone marrow cell cultures. The example used in this chapter is prolactin-like protein F (PLP-F), a recently discovered mouse placental hormone that acts on the myeloid lineage. This hormone has been expressed at high levels in stably transfected Chinese hamster ovary cells. The protein is secreted from these cells after cleavage of the signal sequence and the addition of N-linked carbohydrate. A series of chromatographic steps are used to purify the protein to homogeneity, which is verified by gel electrophoresis and silver staining; the identity of the purified protein is confirmed by immunoblot analysis. Purified protein is then assayed by addition to primary bone marrow cells and scoring the growth and the differentiation of the megakaryocyte progenitor, colony forming unit-megakaryocyte.

In vitro induction of trophoblast from human embryonic stem cells.

Human embryonic stem (ES) cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we describe a method by using bone morphogenetic protein (BMP)4, a member of the transforming growth factor (TGF)-beta superfamily, to induce the differentiation of human ES cells to trophoblast. Immunoassays (as well as DNA microarray and reverse-transcription polymerase chain reaction analyses--data not shown) demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). This technique underscores fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.

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.

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.

Syncytiotrophoblastic localization of the human growth hormone variant mRNA in the placenta.

The hGH/hCS genes, clustered on chromosome 17 in the 5' to 3' order GH-N, CS-L, CS-A, GH-V and CS-B, show a high degree of sequence identity. The expression product of the GH-V gene is the placental growth hormone, which replaces pituitary GH in maternal blood throughout pregnancy. By means of mRNA competitive hybridization using 32P-labelled and unlabelled 30 bases long oligonucleotides, we first optimized specific hybridization conditions. In situ hybridization was then performed to locate the GH-V mRNA encoding placental growth hormone. The hGH-V gene appears expressed in the placental syncytiotrophoblast. Unlike the CS-A and CS-B genes (both encoding hPL) which are expressed uniformly in the syncytiotrophoblast, the GH-V mRNA is located in a few syncytiotrophoblast cells only.

Hypothalamic-like releasing hormones of the placenta.

The challenge to define the hypothalamic-like hormones and to determine where they are produced in the placenta and how they control the production of other hormones such as human chorionic gonadotropin, progesterone, and estrogen, is an important current topic of investigation. This article reviews the existing data that have indicated or established the existence of placental hypothalamic-like peptide hormones.

Endocrine and metabolic homeostasis in diabetic pregnancy.

Diabetic pregnancies involve complex alterations in maternal-placental and fetal compartments. This article discusses the unique features of diabetic pregnancies as they pertain to the maternal changes that must occur to accommodate the fetus.

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.

[Steroidogenic function of the intra-arterial trophoblast in the rat. Ultrastructural, histoenzymologic and biochemical data]. / La fonction stéroïdogène du trophoblaste intra-artériel chez la ratte: données ultrastructurales, histoenzymologiques et biochimiques

The ultrastructural study of the intra-arterial trophoblast has revealed in the pregnant Rat a steroidogenic activity which has been confirmed by histoenzymologic observations (presence of delta 5-3 beta-HSDH and 17 beta-HSDH). At the 15th day postcoitum an in vitro investigation upon the metabolism of steroid hormone precursors suggests that the steroids (oestrogens, progestogens and androgens) secreted by the intra-arterial trophoblast have a local action upon the wall of the uterine placental arteries and are actively concerned with an important part upon the utero-placental hemodynamic as a whole.

PIP: The large cells of fetal origin lying in the afferent arterial sinus, myometrial arterioles and segmentary arteries of hemochorial placentas have been demonstrated to have a trophoblastic function, and are investigated here in rats by electron microscope, histochemistry, and biochemistry. At 11 days of gestation these cells begin to migrate into the maternal sinus, and have an undifferentiated appearance with many polysomes. By Day 13 the endoplasmic reticulum occupies most of the cell, with cisternae and Golgi vesicles typical of glycoprotein secretion. A glycoprotein cell coat covered the cell surface. Few lysosomes containing acid phosphatase were present, having no phagocytic activity. Lipid pools with active mitochondria nearby were common. Intraarterial trophoblast tissues were dissected out and incubated for qualitative enzyme tests. Moderate delta-5,3-beta-hydroxysteroid dehydr ogenase and 17beta-hydroxysteroid dehydrogenase, and some 5alpha-3alpha- and 3beta-hydroxysteroid-dehydrogenase were present. No reduced 5beta-c ompounds were detected. The data suggested that both steroids and mucop roteins are formed by these cells, perhaps having immunosuppressive or local vasodilator roles.

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.