Forms of Circulating Luteinizing Hormone Human Chorionic Gonadotropin Receptor for the Prediction of Early and Late Preeclampsia in the First Trimester of Pregnancy.

OBJECTIVE: To explore the value of circulating luteinizing human chorionic gonadotropin receptor (LHCGR) forms for the prediction of preeclampsia (PE) in the first trimester of pregnancy. METHODS: Case-control study, based on a cohort of 5,759 pregnancies, including 20 early PE, 20 late PE, and 300 controls. We recorded/measured maternal characteristics, mean arterial pressure (MAP), uterine artery (UtA) Doppler, placental growth factor (PlGF), soluble Fms-like tyrosine kinase-1 (sFtl-1), and LHCGR forms (hCG-LHCGR and soluble LHCGR), and their independent predictive values were analyzed by logistic regression. RESULTS: For early PE, the model included black ethnicity, chronic hypertension, previous PE, MAP, UtA Doppler, PlGF, sFlt-1, and LHCGR forms, achieving detection rates (DR) of 83% at 10% of false-positive rates (FPR) [AUC: 0.961 (95% CI: 0.921-1)]. For late PE, the model included body mass index, previous PE, UtA Doppler, PlGF, sFlt-1, and LHCGR forms, with DR of 75% at 10% of FPR [AUC: 0.923 (95% CI: 0.871-0.976)]. In both early and late PE, LHCGR forms improved DR by 6-15%. CONCLUSIONS: LHCGR forms improved the prediction for early and late PE. These results should be confirmed in larger prospective studies.

The utility of circulating LHCGR as a predictor of Down's syndrome in early pregnancy.

BACKGROUND: Previous studies showed that soluble LHCGR/hCG-sLHCGR concentrations in serum or plasma combined with PAPP-A and free ßhCG significantly increased the sensitivity of Down's syndrome screen at early pregnancy without altering the false positive rate. The goal of the present study was to further examine the role of sLHCGR forms as combinatorial markers and to investigate whether sLHCGR could serve as an independent biomarker for Down's syndrome in first trimester pregnancy screens. METHODS: The PAPP-A, free ßhCG, and hCG-sLHCGR concentrations together with nuchal translucency (NT) were measured in 40 Down's and 300 control pregnancies. The sLHCGR concentration was analysed in 40 Down's and 206 control pregnancies. RESULTS: The hCG-LHCGR in combination with PAPP-A and free ßhCG increased the detection rate (DR) by 35% without altering the false positive rate (FPR). The sLHCGR: hCG-sLHCGR ratio alone detected 80% of Down's pregnancies in first trimester screening, with a false positive rate of 0.5%. CONCLUSIONS: While measurement of sLHCGR forms in combination with PAPP-A and free ßhCG significantly increases the detection rate of Down's syndrome at first trimester, the ratio of sLHCGR: hCG-sLHCGR acts as an independent marker with a detection rate that is significantly higher than the existing biochemical markers individually for prenatal first trimester screening of Down's syndrome.

Quantitative ELISAs for serum soluble LHCGR and hCG-LHCGR complex: potential diagnostics in first trimester pregnancy screening for stillbirth, Down's syndrome, preterm delivery and preeclampsia.

BACKGROUND: Soluble LH/hCG receptor (sLHCGR) released from placental explants and transfected cells can be detected in sera from pregnant women. To determine whether sLHCGR has diagnostic potential, quantitative ELISAs were developed and tested to examine the correlation between pregnancy outcome and levels of serum sLHCGR and hCG-sLHCGR complex. METHODS: Anti-LHCGR poly- and monoclonal antibodies recognizing defined LHCGR epitopes, commerical anti-hCGbeta antibody, together with recombinant LHCGR and yoked hCGbeta-LHCGR standard calibrators were used to develop two ELISAs. These assays were employed to quantify serum sLHCGR and hCG-sLHCGR at first trimester human pregnancy. RESULTS: Two ELISAs were developed and validated. Unlike any known biomarker, sLHCGR and hCG-sLHCGR are unique because Down's syndrome (DS), preeclampsia and preterm delivery are linked to both low (less than or equal to 5 pmol/mL), and high (equal to or greater than 170 pmol/mL) concentrations. At these cut-off values, serum hCG-sLHCGR together with PAPP-A detected additional DS pregnancies (21%) which were negative by free hCGbeta plus PAPP-A screening procedure. Therefore, sLHCGR/hCG-sLHCGR has an additive effect on the current primary biochemical screening of aneuploid pregnancies. More than 88% of pregnancies destined to end in fetal demise (stillbirth) exhibited very low serum hCG-sLHCGR(less than or equal to 5 pmol/mL) compared to controls (median 16.15 pmol/mL, n = 390). The frequency of high hCG-sLHCGR concentrations (equal to or greater than 170 pmol/mL) in pathological pregnancies was at least 3-6-fold higher than that of the control, suggesting possible modulation of the thyrotropic effect of hCG by sLHCGR. CONCLUSIONS: Serum sLHCGR/hCG-sLHCGR together with PAPP-A, have significant potential as first trimester screening markers for predicting pathological outcomes in pregnancy.

Circulating LH/hCG receptor (LHCGR) may identify pre-treatment IVF patients at risk of OHSS and poor implantation.

BACKGROUND: Successful pregnancy via in vitro fertilization (IVF) depends on the recovery of an adequate number of healthy oocytes and on blastocyst implantation following uterine transfer. Two hormones, LH and hCG, utilize a common LH/hCG receptor (LHCGR), variations in which have profound implications in human reproduction. Soluble LHCGR (sLHCGR) is released from experimental cell lines and placental explants and it can be detected in the follicular fluid and serum. METHODS: To evaluate the impact of circulating soluble LHCGR (sLHCGR) in fertility treatment, we measured sLHCGR and LH-sLHCGR complex in serum from women seeking IVF using specifically developed quantitative enzyme-linked immunosorbent assays (ELISA). Following an IVF cycle of treatment, patients were grouped according to oocyte yield into low (lower than or equal to 7 oocytes), intermediate (8-14 oocytes) and high (greater than or equal to 15 oocytes) responders and pregnancy outcome noted. RESULTS: Pre-treatment sLHCGR identified many women at risk of ovarian hyperstimulation. Low levels of sLHCGR were associated with pregnancy in both high and low responders but sLHCGR did not significantly affect the treatment outcome of intermediate responders. Low responders who failed to become pregnant had high levels of circulating sLHCGR bound to LH (LH-sLHCGR). CONCLUSIONS: Pre-treatment measurement of sLHCGR could be used to tailor individual fertility treatment programs and improve outcomes by avoiding ovarian hyperstimulation and poor embryo implantation.

Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR) from transfected cells and placenta explants.

Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR). We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm). Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.

Mouse models for preeclampsia: disruption of redox-regulated signaling.

The concept that oxidative stress contributes to the development of human preeclampsia has never been tested in genetically-defined animal models. Homozygous deletion of catechol-O-methyl transferase (Comt-/-) in pregnant mice leads to human preeclampsia-like symptoms (high blood pressure, albuminurea and preterm birth) resulting from extensive vasculo-endothelial pathology, primarily at the utero-fetal interface where maternal cardiac output is dramatically increased during pregnancy. Comt converts estradiol to 2-methoxyestradiol 2 (2ME2) which counters angiogenesis by depleting hypoxia inducible factor-1 alpha (HIF-1 alpha) at late pregnancy. We propose that in wild type (Comt++) pregnant mice, 2ME2 destabilizes HIF-1 alpha by inhibiting mitochondrial superoxide dismutase (MnSOD). Thus, 2ME2 acts as a pro-oxidant, disrupting redox-regulated signaling which blocks angiogenesis in wild type (WT) animals in physiological pregnancy. Further, we suggest that a lack of this inhibition under normoxic conditions in mutant animals (Comt-/-) stabilises HIF-1 alpha by inactivating prolyl hydroxlases (PHD). We predict that a lack of inhibition of MnSOD, leading to persistent accumulation of HIF-1 alpha, would trigger inflammatory infiltration and endothelial damage in mutant animals. Critical tests of this hypothesis would be to recreate preeclampsia symptoms by inducing oxidative stress in WT animals or to ameliorate by treating mutant mice with Mn-SOD-catalase mimetics or activators of PHD.

Is vitamin E a safe prophylaxis for preeclampsia?

The prophylactic use of vitamins E and C for the prevention of preeclampsia is currently being evaluated in multiple clinical trials in Canada, Mexico, the United Kingdom, the United States, and other developing countries. In addition to its antioxidant capacity, exogenous vitamin E may prevent an immunologic switch (Th1 to Th2) that is vital for early-to late transition in normal pregnancies. Moreover, vitamin E could be a potential interferon-gamma (IFN-gamma) mimic facilitating persistent proinflammatory reactions at the fetal-maternal interface. These untoward effects of dietary intake of vitamin E may be more pronounced in those treated cases that fail to develop preeclampsia. A critical test of this hypothesis would be to establish whether, under variable O2 tension, vitamin E is capable of affecting cytokine signaling in placental trophoblasts and maternal immune effector cells, both in early and late human pregnancies.

Natural antisense LHCGR could make sense of hypogonadism, male-limited precocious puberty and pre-eclampsia.

The pleiotropic effects of human chorionic gonadotrophin (hCG), the key regulator of human pregnancy, are dependent upon cell surface expression of its functional cognate receptor LHCGR in the placental trophoblasts, corpus luteum, uterus, vascular endothelial and smooth muscle cells. Additionally, lutenizing hormone-mediated signalling failure has often been linked to activating/inactivating mutations in LHCGR. One of the intriguing aspects of these studies is that the mutations are most frequently located within C-terminal 200-350 residues of the receptor protein. In an attempt to reconcile the mechanistic basis of LHCGR regulation and mutations, we have carried out bioinformatic analyses to identify the CpG-rich regions and the major potential scaffold/matrix attachment sites (S/MARs) in LHCGR and neighbouring gene (ALF) at human chromosome 2p21. Based on these analyses, we propose a chromatin-loop model, which may explain the temporal regulation and susceptibility to mutation of the human LHCGR. One of the characteristic features of the model, is that the major potential S/MAR sequences of the human LHCGR gene (68 kb) are located at the 3' end of the gene, and unlike mouse, the transmembrane and C-terminal protein coding sequences at exon 11 are embedded in this S/MAR site. Moreover, this region is subject to antisense transcription from the neighbouring gene ALF, which is gonad-specific and is only activated in meiotic spermatocytes and oocytes. Together, these analyses suggest that exon 11 of human LHCGR could be more susceptible to mutation than the other 10 exons together and that activation of LHCGR, contingent to the somatic silencing of neighbouring ALF, could be linked to male-limited precocious puberty and pre-eclampsia.

A link between high serum levels of human chorionic gonadotrophin and chorionic expression of its mature functional receptor (LHCGR) in Down's syndrome pregnancies.

Human chorionic gonadotrophin (hCG) is released from placental trophoblasts and is involved in establishing pregnancy by maintaining progesterone secretion from the corpus luteum. Serum hCG is detected in the maternal circulation within the first 2-3 wks of gestation and peaks at the end of the first trimester before declining. In Down's syndrome (DS) pregnancies, serum hCG remains significantly high compared to gestation age-matched uncompromised pregnancies. It has been proposed that increased serum hCG levels could be due to transcriptional hyper-activation of the CGB (hCG beta) gene, or an increased half life of glycosylated hCG hormone, or both. Another possibility is that serum hCG levels remain high due to reduced availability of the hormone's cognate receptor, LHCGR, leading to lack of hormone utilization. We have tested this hypothesis by quantifying the expression of the hCG beta (CGB) RNA, LHCGR RNA and LHCGR proteins in chorionic villous samples. We demonstrate that chorionic expression of hCG beta (CGB) mRNA directly correlates with high serum hCG levels. The steady-state synthesis of LHCGR mRNA (exons 1-5) in DS pregnancies was significantly higher than that of controls, but the expression of full-length LHCGR mRNA (exons 1-11) in DS was comparable to that of uncompromised pregnancies. However, the synthesis of high molecular weight mature LHCGR proteins was significantly reduced in DS compared to uncompromised pregnancies, suggesting a lack of utilization of circulating hCG in DS pregnancies.

Placental expression of interferon-gamma (IFN-gamma) and its receptor IFN-gamma R2 fail to switch from early hypoxic to late normotensive development in preeclampsia.

The inability of the mother to switch from T helper cell type 1 (Th1) to Th2 cytokine profiles at the fetal-maternal interface has been proposed as one of the primary causes of miscarriage, intrauterine growth restriction (IUGR), and preeclampsia (PE). The Th1 [interferon-gamma (IFN-gamma), TNF-alpha, and IL-12] and Th2 (IL-4 and IL-10) cytokines have opposite effects on human pregnancy. Leukemia inhibitory factor (LIF) promotes embryo implantation and sustains pregnancy, whereas IFN-gamma and TNF-alpha are detrimental to pregnancy. Both IFN-gamma and LIF are produced by maternal cells and tissues at the fetal-maternal interface, whereas the IFN-gamma receptors (IFN-gamma R1 and IFN-gamma R2) and LIF receptor are abundantly expressed on the surface of placental trophoblasts. The effect of IFN-gamma on T lymphocyte activation is influenced by the relative membrane density of its two receptors, particularly IFN-gamma R2. In this study we report that in PE (25-40 wk gestation) and PE complicated by IUGR, IFN-gamma R2 protein expression is severely down-regulated and is similar to that observed in early placenta (7-10 wk gestation) developing under low O(2) tension. IFN-gamma production was found to be inversely related to the IFN-gamma R2 protein expression, and LIF receptor protein expression in PE mimicked that in early placental development. These results show that in PE, placental trophoblasts fail to establish an early to late switch with respect to IFN-gamma and IFN-gamma R2 expression. This supports the hypothesis that trophoblasts control the polarization of maternal immune effectors and cytokine profiles at the fetal-maternal interface that could be subject to oxidative stress in PE.

Trichinella spiralis secretes a homologue of prosaposin.

Infective larvae and adult stage Trichinella spiralis secrete a protein homologous to prosaposin, the precursor of sphingolipid activator proteins (saposins) A-D originally defined in vertebrates. The protein contains four saposin domains, with the six cysteine residues which form the three intramolecular disulphide bonds in close register in each case. It differs substantially from vertebrate prosaposins in the N-terminal prodomain, the region separating saposins A and B, and completely lacks the C-terminal domain which has been demonstrated to be essential for lysosomal targetting in these organisms. The protein is secreted in unprocessed form with an estimated mass of 56 kDa, and contains a single N-linked glycan which is bound by the monoclonal antibody NIM-M1, characteristic of the TSL-1 antigens which are capped by tyvelose (3,6-dideoxy-D-arabinohexose). Immuno-electron microscopy localised the protein to membrane-bound vesicles and more complex multi-lamellar organelles in diverse tissues including the hypodermis, intestine and stichosomes, although it was absent from the dense-core secretory granules typical of the latter. Possible functions of a secreted prosaposin are discussed.