The role of ß-HCG and VEGF-MEK/ERK signaling pathway in villi angiogenesis in patients with missed abortion.

OBJECTIVE: To analyze the effects of the Human Chorionic Gonadotropin beta (ß-hCG) and the VEGF-MEK/ERK signaling pathway on villi angiogenesis in early missed abortion. METHODS: A total of 12 cases of women with missed abortion and 12 cases of women who had induced abortion voluntarily without any disease were included in the present study. The age, pregnancy time and gestation period in the control group corresponded to the missed abortion group. Wes Simple Western system and qRT-PCR were used to detect the expression of VEGF-MEK/ERK signaling pathway related proteins and genes in villous. Radioimmunoassay and Enzyme-linked immunosorbent assay were used to detect ß-hCG and VEGF levels in serum. The microvascular density (MVD) in villous tissue was analyzed by immunohistochemical staining. RESULTS: The levels of ß-hCG and VEGF in serum, the expression of VEGF-MEK/ERK signaling pathway and MVD in villous tissue of the missed abortion group were lower than those of the control group. In addition, compared with the control group, the layers of trophoblasts of the villous tissue in the missed abortion group became thinner significantly, the number of cells reduced, the cell structures were disorganized, and parts of the trophoblast cells were absent. Correlational analysis showed that the protein expression of ERK1/2 was positively correlated with MVD in missed abortion group. CONCLUSIONS: Our results reveal that decreased production of ß-hCG in early pregnant women could down-regulate the expression of VEGF-MEK/ERK signal pathway, then reduce angiogenesis and eventually leading to the abnormal angiogenesis of villous, which may be an important mechanism of missed abortion.

The recombinant equine LHß subunit combines divergent intracellular traits of human LHß and CGß subunits.

The pituitary LHß and placental CGß subunits are products of different genes in primates. The major structural difference between the two subunits is in the carboxy-terminal region, where the short carboxyl sequence of hLHß is replaced by a longer O-glycosylated carboxy-terminal peptide in hCGß. In association with this structural deviation, there are marked differences in the secretion kinetics and polarized routing of the two subunits. In equids, however, the CGß and LHß subunits are products of the same gene expressed in the placenta and pituitary (LHß), and both contain a carboxy-terminal peptide. This unusual expression pattern intrigued us and led to our study of eLHß subunit secretion by transfected Chinese hamster ovary and Madin-Darby canine kidney cells. In continuous labeling and pulse-chase experiments, the secretion of the eLHß subunit from the transfected Chinese hamster ovary cells was inefficient (medium recovery of 16%-25%) and slow (t1/2 > 6.5 hours). This indicated that, the secretion of the eLHß subunit resembles that of hLHß rather than hCGß. In Madin-Darby canine kidney cells grown on Transwell filters, the eLHß subunit was preferentially secreted from the apical side, similar to the hCGß subunit secretory route (∼65% of the total protein secreted). Taken together, these data suggested that secretion of the eLHß subunit integrates features of both hLHß and hCGß subunits. We propose that the evolution of this intracellular behavior may fulfill the physiological demands for biosynthesis of the LH and CG ß-subunits in the pituitary and placenta, respectively.

TGF-ß-induced hCG-ß regulates redox homeostasis in glioma cells.

Transforming growth factor (TGF-ß) is associated with the progression of glioblastoma multiforme (GBM)-the most malignant of brain tumors. Since there is a structural homology between TGF-ß and human chorionic gonadotropin (hCG) and as both TGF-ß and hCG-ß are known regulators of oxidative stress and survival responses in a variety of tumors, the role of TGF-ß in the regulation of hCG-ß and its consequences on redox modulation of glioblastoma cells was investigated. A heightened hCG-ß level was observed in GBM tumors. TGF-ß treatment increased hCG-ß expression in glioma cell lines, and this heightened hCG-ß was found to regulate redox homeostasis in TGF-ß-treated glioma cells, as siRNA-mediated knockdown of hCG-ß (i) elevated reactive oxygen species (ROS) generation, (ii) decreased thioredoxin Trx1 expression and thioredoxin reductase (TrxR) activity, and (iii) abrogated expression of TP53-induced glycolysis and apoptosis regulator (TIGAR). Silencing of hCG-ß abrogated Smad2/3 levels, suggesting the existence of TGF-ß-hCG-ß cross-talk in glioma cells. siRNA-mediated inhibition of elevated TIGAR levels in TGF-ß-treated glioma cells was accompanied by an increase in ROS levels. As a farnesyltransferase inhibitor, Manumycin is known to induce glioma cell apoptosis in a ROS-dependent manner, and we investigated whether Manumycin could induce apoptosis in TGF-ß-treated cells with elevated hCG-ß exhibiting ROS-scavenging property. Manumycin-induced apoptosis in TGF-ß-treated cells was accompanied by elevated ROS levels and decreased expression of hCG-ß, Trx1, Smad2/3, and TIGAR. These findings indicate the existence of a previously unknown TGF-ß-hCG-ß link that regulates redox homeostasis in glioma cells.

CGB activates ERK and AKT kinases in cancer cells via LHCGR-independent mechanism.

Expression of human chorionic gonadotropin free beta subunit (hCGß) and its hyperglycosylated variant (hCGß-H) is a phenomenon confirmed for tumors of different origin. Despite numerous studies, the mechanism of hCGß action in cancer remains unknown especially that not all tumors secreting hCGß express the receptor for human chorionic gonadotropin (LHCGR). In the presented study, we verified the hypothesis of hCGß potential to activate signaling pathways involving extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) kinases with and without the contribution of LHCGR. To achieve this goal, human ovarian carcinoma cells OVCAR-3 expressing LHCGR and SKOV-3 not expressing LHCGR were either transfected with a vector coding for hCGß or stimulated with recombinant hCGß and the level of pERK and pAKT was measured. The results of the experiments showed that hCGß action leads to the increase in ERK and AKT kinases phosphorylation in cancer cells and indicate that these biological effects can be achieved independently of LHCGR presence. The study also demonstrated that the presence of the receptor is a key factor influencing the magnitude of cells' response.

Human chorionic gonadotropin and its free ß-subunit stimulate trophoblast invasion independent of LH/hCG receptor.

Both paracrine and autocrine factors are involved in the regulation of trophoblast invasion. One of these factors is human chorionic gonadotropin (hCG), which stimulates trophoblast invasion. The stimulatory activity has especially been ascribed to a hyperglycosylated form of hCG (hCG-h) that is expressed in early pregnancy. We compared the stimulatory activities of different forms of hCG and its free ß-subunit (hCGß) on trophoblast invasion. hCG, hCG-h, hCGß, and its hyperglycosylated form (hCGß-h) stimulated the invasion of JEG-3 choriocarcinoma cells. The stimulatory effect of hCGß was also confirmed with primary human trophoblasts. Down-regulation of the LH/hCG receptor by RNA-interference did not significantly reduce the effect of hCGß and hCG on cell invasion. Increased invasion was associated with increased levels of MMP-2, MMP-9 and activity of uPA. Our findings suggest that hCG, hCGß and their hyperglycosylated forms stimulate the invasion of trophoblast cells independent of the classical LH/hCG-receptor.

Does hCG or hCGß play a role in cancer cell biology?

The role that hCG might play in the oncogenic process in cancer is certainly complex. We know that the expression of hCG and its beta subunit is a widespread phenomenon which has been described in many cancer subtypes. However, hCG's involvement in breast cancer has been antithetical: the detection of ectopically expressed hCG(ß) by breast tumors has been employed as a biomarker of malignancy, and hCG has been proposed as a ligand vehicle for toxic drugs, with the aim of targeting the LH/hCG receptor which is reported to be expressed by malignant breast tissue. However, it has also been proposed that hCG is a protective agent against the development of breast cancer, leading some to advocate hCG administration to non-pregnant women as a prophylactic measure against cancer. Nevertheless, suggestions that hCG is involved in the angiogenesis, metastasis and immune escape that are central to cancer progression - are phenomena which clearly apply to breast cancer. Indeed, a tumor vaccine based upon hCG has very recently been shown to protect against mammary tumors in mice. We propose that this apparent paradox is resolved if the free beta subunit of hCG produced by tumors acts as an autocrine anti-apoptotic and angiogenic growth factor, whilst intact heterodimeric hCG, as in pregnancy, is part of developmental signaling that initiates tissue differentiation (including breast ductal tissue development), and hence reduces the population of stem-like cells which are susceptible to oncogenic factors.

Evolution of the human brain, chorionic gonadotropin and hemochorial implantation of the placenta: insights into origins of pregnancy failures, preeclampsia and choriocarcinoma.

Hyperglycosylated chorionic gonadotropin (CG-H) signals placental cytotrophoblast cell growth and invasion, and chorionic gonadotropin (CG) promotes uterine vascularization. A hypothesis is presented relating the evolution of these molecules to the evolution of human hemochorial implantation and that of the human brain. Deep placental invasion, vascularization and hemochorial placentation, under the influence of CG and CG-H, are a critical part of the nutrition and energy-generating mechanisms needed for human brain development and thus for the evolution of humans. Insufficient CG-H production and the resulting inappropriate implantation is associated with an unduly high incidence of pregnancy failures in humans. Low levels of CG-H and inappropriate hemochorial placentation also appear to be associated with subsequent preeclampsia. It is also of note that human CG-H drives invasion by gestational trophoblastic neoplasms that have been described only in humans.

Biochemistry in the diagnosis and management of abnormal early pregnancy.

PURPOSE OF REVIEW: To examine the current state of knowledge regarding the biochemical parameters of early pregnancy failure. FINDINGS: Earlier studies have concentrated on the role of human chorionic gonadotropin and progesterone in early pregnancy failure and recent studies have also explored newer markers such as the inhibins and insulin growth factor binding proteins. Multiparameter models have now been created for the prediction of successful expectant management of early pregnancy failure. SUMMARY: Examining the role of newer biochemical markers in the early pregnancy failure has led to an improved understanding of the underlying mechanisms and has clinical benefit in the selection of appropriate management for patients.

[Development of the human adrenal glands]. / Le développement de la surrénale humaine.

The human adrenal is an endocrine gland located at the superior part of the kidney. Composed of the adrenal cortex of mesoblastic origin and the adrenal medulla of neuroectoblastic origin, the human fetal adrenal grows considerably during the first three months of development. From 12 to 18 weeks of development (WD), the weight of the adrenals increases seven-fold. The gland's weight doubles from 18 to 28 WD and from 28 to 36 WD. At birth, the two adrenals weigh on average 10 g. At the 8th week, two zones are individualized in the adrenal cortex: the definitive zone and the fetal inner zone. At the second trimester, according to ultrastructural and biochemical studies, a third zone, called the transition zone, is individualized between the definitive zone and the fetal inner zone. The definitive zone persists, but the origin of the three zones (glomerular, fascicular and reticular) of adult adrenal cortex is not known. The fetal inner zone regresses from the 5th month of gestation and disappears totally one year after birth. At the 8th week, the immature neuroblasts migrate to the definitive zone, then to the fetal inner zone to compose the adrenal medulla, which develops essentially after birth and during the first year. Before the 10th week, the human fetal adrenal is able to produce steroid hormones, in particular dehydroepiandrosterone sulfate (DHEA-S); the secretion of cortisol remains discussed. The development of the human fetal adrenal is complex and is under the control of hormones (ACTH, LH and betaHCG), growth factors (ACTH essentially) and transcription factors (essentially SF1 and DAX-1). Knowledge of morphological and molecular phenomena of this development permits to understand the pathophisiology of congenital adrenal deficiencies.

Overexpression of human chorionic gonadotropin causes multiple reproductive defects in transgenic mice.

Human CG is a pregnancy marker secreted by the placenta, and it utilizes the same receptors as does LH. Human CG is a heterodimer, and its subunits are expressed in tissues other than placenta. Similarly, LH/hCG receptors are also expressed in multiple tissues; however, the physiological significance of this expression is unknown. Free hCGbeta is efficiently secreted in vitro in transfected cells and is highly expressed in many human cancers; however, the biological effects of free hCGbeta in vivo are unknown. To study in vivo consequences of elevated levels of free hCGbeta and hCG dimer in both male and female reproductive physiology, we used mouse metallothionein 1 promoter to generate multiple lines of transgenic mice that overexpressed either one or both subunits of hCG. Although mice expressing the glycoprotein hormone alpha subunit are normal and fertile, both male and female transgenic mice overexpressing only the hormone-specific hCGbeta subunit are infertile. The hCGbeta subunit-expressing transgenic female mice progressively develop cystic ovaries, whereas the male transgenic mice are infertile but otherwise are not phenotypically discernible. In contrast, both the male and female transgenic mice coexpressing high levels of the hCG subunits (i.e., the hCG dimer) demonstrate multiple reproductive defects. The male transgenic mice have Leydig cell hyperplasia, very high levels of serum testosterone, reduced testis size, and dramatically enlarged seminal vesicles and are infertile and display overly aggressive behavior when caged with females. The female transgenic mice are also infertile, have elevated levels of serum estradiol, and progressively develop hemorrhagic and cystic ovaries with thecal layer enlargement and stromal cell proliferation and degenerating kidneys. These results suggest that the in vivo biological effects of ectopically expressed free hCGbeta subunit are distinct from those of the hCG dimer and are gender specific. These transgenic mice are useful models for studying the biology of free hCGbeta subunit, for further analyzing the gain of function effects of hCG during early Leydig cell development, and for studying the roles of hCG in ovarian and kidney pathophysiology and function.

Hyperthyroidism in the setting of gestational trophoblastic disease.

We report the case of a 27-year-old woman who presented with palpitations, hyperemesis, and weight loss. The patient was found to have laboratory values consistent with hyperthyroidism. A serum pregnancy test was positive for an estimated 8-week gestation. After an ultrasound diagnosis of a molar pregnancy, the patient underwent a uterine evacuation with subsequent resolution of her hyperthyroid status. We provide herein the details of this rarely documented presentation of hyperthyroidism in the setting of gestational trophoblastic disease.

Low fatty acid amide hydrolase and high anandamide levels are associated with failure to achieve an ongoing pregnancy after IVF and embryo transfer.

Human reproduction is a rather inefficient process, yet the molecular reasons for this inefficiency remain unknown. IVF and embryo transfer (IVF-embryo transfer) also results in a high frequency of implantation failures and early spontaneous abortions. Here we show that the anandamide (AEA)-degrading enzyme, fatty acid amide hydrolase (FAAH), had significantly lower activity (46 +/- 17 versus 161 +/- 74 pmol/min per mg protein) and protein content (0.10 +/- 0.03 versus 0.23 +/- 0.06 units) in lymphocytes of IVF-embryo transfer patients who failed to achieve an ongoing pregnancy than in those who become pregnant, and this was paralleled by a significant increase in blood AEA (4.0 +/- 2.2 pmol/ml and 0.9 +/- 1.0 pmol/ml respectively). The blood levels of the other endocannabinoid, 2-arachidonoylglycerol, or of the AEA congener, N-palmitoylethanolamine, which are metabolized by enzymes different from FAAH, was not different between the pregnant and nonpregnant women, nor was there any difference in the activity of the AEA membrane transporter or the amounts of cannabinoid receptors in lymphocytes. Taken together with the reported negative effects of AEA on embryo implantation, this study indicates that low FAAH activity and subsequent increased AEA levels in blood might be one of the causes of implantation failure or pregnancy loss, thereby leading to a better understanding of the pathophysiological and therapeutic implications of endocannabinoids in human fertility.

PPAR gamma/RXR alpha heterodimers are involved in human CG beta synthesis and human trophoblast differentiation.

Recent studies performed with null mice suggested a role of either RXR alpha or PPAR gamma in murine placental development. We report here that both PPAR gamma and RXR alpha are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPAR gamma (but not for PPAR alpha or PPAR delta) increase both human CG beta transcript levels and the secretion of human CG and its free beta-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPAR gamma ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPAR gamma/RXR alpha heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CG beta gene (beta 5) were found to bind RXR alpha and PPAR gamma from human cytotrophoblast nuclear extracts, suggesting that PPAR gamma/RXR alpha heterodimers directly regulate human CG beta transcription. Altogether, these data show that PPAR gamma/RXR alpha heterodimers play an important role in human placental development.

Embryo implantation and embryonic stem cell development in primates.

The endocrine dialogue that results in implantation and the successful establishment of pregnancy in primates relies on embryonic secretion of chorionic gonadotrophin (CG). This hormone is a signal of embryo viability and capacity to support the corpus luteum. The expression of CG is apparently restricted to primates. Active or passive immunization of marmoset monkeys against the beta subunit of CG prevented implantation and early pregnancy, without disrupting the ovarian cycle. Studies of individual embryos cultured in vitro showed that CG is secreted at low levels by the blastocyst from before attachment, with secretion increasing exponentially after attachment. Gonadotrophin releasing hormone (GnRH) was also secreted, from mid-blastocyst stages, before the detection of CG. The secretion of GnRH by the embryo continued through the attachment and outgrowth stages of embryonic differentiation in vitro. The hypothetical role of GnRH in regulating CG release during implantation was tested in recently completed experiments. Individual embryos cultured with GnRH, or with agonist or antagonist to GnRH, showed significant variations in their secretion of CG and in their survival in culture, suggesting a causal relationship between these hormones. Embryos cultured with natural GnRH showed enhanced growth and development. Embryonic stem cells, from the inner cell mass of marmoset and rhesus monkeys, were the first primate embryonic stem cells to be isolated and characterized, enabling the subsequent isolation of human embryonic stem cells.

The increase in bladder carcinoma cell population induced by the free beta subunit of human chorionic gonadotrophin is a result of an anti-apoptosis effect and not cell proliferation.

Ectopic production of free beta human chorionic gonadotrophin (hCGbeta) by bladder carcinoma is well described and occurs in approximately 35% of cases. hCGbeta secreting tumours are more aggressive, radioresistant and have a greater propensity to metastasize. We proposed that the ectopic production of hCGbeta was contributing in an autocrine fashion to the radioresistance and metastatic potential of such secreting tumours. Though we demonstrated that the addition of hCGbeta to the culture media of bladder, cervical and endometrial carcinoma cell lines brought about an increase in cell populations this was not accompanied by a significant increase in the rate of replication. Since a cell population size is a balance of mitosis and mortality, we proposed that hCGbeta was inhibiting apoptosis. Here we have demonstrated that following incubation with recombinant hCGbeta, bladder carcinoma cells refrain from undergoing apoptosis. Quantitation of apoptotic bodies was carried out by immunoassay and corrected to cell number as determined by MTT assay. In each cell line, addition of hCGbeta reduced the number of apoptotic bodies dose-dependently, indicating a diminished apoptotic rate. Furthermore, TGFbeta1-induced apoptosis could be dose-dependently inhibited by co-incubation with hCGbeta. We propose, therefore, that such a decline in apoptosis may account for the cell population increase previously reported. It may also explain the radioresistance and aggressive nature of hCGbeta-secreting tumours and the poor prognosis associated therein.

The carboxyl-terminal region is a determinant for the intracellular behavior of the chorionic gonadotropin beta subunit: effects on the processing of the Asn-linked oligosaccharides.

The placental hormone human CG (hCG) consists of two noncovalently linked alpha- and beta-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common alpha subunit but differ in their structurally distinct beta subunits. The CGbeta subunit is distinguished among the beta subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGbeta subunit (CGbeta114) with the alpha-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGbeta and CGbeta114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.