Characterization of a Novel POU1F1 Mutation Identified on Screening 160 Growth Hormone Deficiency Patients.

The objective of the study is the functional characterization of a novel POU1F1 c.605delC mutation in combined pituitary hormone deficiency (CPHD) and to report the clinical and genetic details of 160 growth hormone deficiency patients. Screening of GH1, GHRHR, POU1F1, PROP1, and HESX1 genes by Sanger sequencing was carried out in 160 trios and 100 controls followed by characterization of the POU1F1 c.605delC mutation by expression studies including site directed mutagenesis, co-transfection, protein degradation, and luciferase assays to compare the wild type and mutant POU1F1. In vitro studies showed that the POU1F1 c.605delC mutation codes for a truncated protein with reduced transactivation capacity on its downstream effectors, viz., growth hormone (GH) and prolactin (PRL) causing severe CPHD. Experiments using different protease inhibitors reveal rescue of the protein upon blockage of the lysosomal pathway that might be useful in novel drug designing using targeted approach thereby maintaining the milieu and preventing/delaying the disease. The study provides an insight into the disease causing mechanism of POU1F1 c.605delC mutation identified in a CPHD child with severe short stature and failure to thrive. It also shows mutation effect on the expression, function and turnover of protein and highlights mechanistic details by which these potent regulators may operate.

The effects of leptin on human cytotrophoblast invasion are gestational age and dose-dependent.

Introduction: Leptin and its receptors are expressed by the human placenta throughout gestation, yet the role of leptin in early human placental development is not well characterized. Leptin is overexpressed in the placentas from preeclamptic (PE) pregnancies. PE can result from the impaired invasion of fetal placental cells, cytotrophoblasts (CTBs), into the maternal decidua. We hypothesized that elevated leptin levels would impair human CTB invasion. Methods: The effects of leptin on the invasion of human CTBs were evaluated in three cell models, HTR-8/SVneo cells, primary CTBs, and placental villous explants using invasion assays. Further, leptin receptor expression was characterized in all three cell models using RT-PCR. Further phosphokinase assays were performed in HTR-8/SVneo cells to determine signaling pathways involved in CTB invasion in response to differential leptin doses. Results: We found that, prior to 8 weeks gestation, leptin promoted CTB invasion in the explant model. After 11 weeks gestation in explants, primary CTBs and in HTR-8/SVneo cells, leptin promoted invasion at moderate but not at high concentrations. Further, leptin receptor characterization revealed that leptin receptor expression did not vary over gestation, however, STAT, PI3K and MAPK pathways showed different signaling in response to varied leptin doses. Discussion: These data suggest that the excess placental leptin observed in PE may cause impaired CTB invasion as a second-trimester defect. Leptin's differential effect on trophoblast invasion may explain the role of hyperleptinemia in preeclampsia pathogenesis.

Longitudinal profile of sHLA-G during pregnancy and its association with small for gestational age births in North Indian pregnant females: A nested case-control study.

PROBLEM: Small for gestational age (SGA) neonates are vulnerable to various long and short-term adverse health consequences. The expression of HLA-G in the placenta is crucial for establishment and maintenance of pregnancy. Its aberrant expression could lead to perturbed immunological interactions in the placenta which could be associated with SGA births. The objective of this study was to assess the difference in the trajectories of soluble HLA-G in maternal sera during pregnancy between women delivering SGA and appropriate for gestational age (AGA) neonates. METHOD OF STUDY: Soluble HLA-G was estimated in the maternal sera collected at different time points in pregnancy of North-Indian pregnant females delivering SGA (N = 23) or AGA (N = 17) neonates using sandwich ELISA. Linear mixed models were built and compared to study the association between sHLA-G levels during pregnancy and SGA births. RESULTS: No significant difference was observed in the sHLA-G trajectories during pregnancy in mothers delivering SGA as compared to those delivering AGA (P-value = .5677). A trend towards higher sHLA-G levels at the first trimester of pregnancy (< 14 weeks of gestation) was observed in mothers delivering SGA neonates (Median = 41.71, IQR = 21.31-71.38) as compared to those delivering AGA neonates (Median = 37.58, IQR = 19.05-73.57). CONCLUSION: During pregnancy, sHLA-G trajectories do not differ significantly between mothers delivering SGA and those delivering AGA neonates. However, sHLA-G trends towards higher levels during early pregnancy in mothers delivering SGA neonates.

Siglec-6 Signaling Uses Src Kinase Tyrosine Phosphorylation and SHP-2 Recruitment.

Preeclampsia is a pregnancy-specific disorder involving placental abnormalities. Elevated placental Sialic acid immunoglobulin-like lectin (Siglec)-6 expression has been correlated with preeclampsia. Siglec-6 is a transmembrane receptor, expressed predominantly by the trophoblast cells in the human placenta. It interacts with sialyl glycans such as sialyl-TN glycans as well as binds leptin. Siglec-6 overexpression has been shown to influence proliferation, apoptosis, and invasion in the trophoblast (BeWo) cell model. However, there is no direct evidence that Siglec-6 plays a role in preeclampsia pathogenesis and its signaling potential is still largely unexplored. Siglec-6 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif in its cytoplasmic tail suggesting a signaling function. Site-directed mutagenesis and transfection were employed to create a series of Siglec-6 expressing HTR-8/SVneo trophoblastic cell lines with mutations in specific functional residues to explore the signaling potential of Siglec-6. Co-immunoprecipitation and inhibitory assays were utilized to investigate the association of Src-kinases and SH-2 domain-containing phosphatases with Siglec-6. In this study, we show that Siglec-6 is phosphorylated at ITIM and ITIM-like domains by Src family kinases. Phosphorylation of both ITIM and ITIM-like motifs is essential for the recruitment of phosphatases like Src homology region 2 containing protein tyrosine phosphatase 2 (SHP-2), which has downstream signaling capabilities. These findings suggest Siglec-6 as a signaling molecule in human trophoblasts. Further investigation is warranted to determine which signaling pathways are activated downstream to SHP-2 recruitment and how overexpression of Siglec-6 in preeclamptic placentas impacts pathogenesis.

Provocative ideas on human placental biology: A prerequisite for prevention and treatment of neonatal health challenges.

A 2-day invite-only meeting on generating "Provocative Ideas on human placental research" was organized on 1-2 December 2015 at the Translational Health Science and Technology Institute, Faridabad. This meeting was sponsored by Department of Biotechnology, Ministry of Science and Technology, Govt. of India. The objectives of this meeting were the critical evaluation of placental physiology and its development. Special emphasis was placed on understanding the consequences and implications of placental development in sustenance of pregnancy and in pregnancy-associated complications such as preeclampsia, intrauterine growth restriction, and preterm birth. This meeting brought together experienced as well as novice clinicians and biologists who have a keen interest in the field of placental biology, including development of new technologies and methods for evaluating the role of placenta in predicting pregnancy outcomes. The meeting primarily focused on (i) high-throughput "-omics" approaches, (ii) maternal nutrition and placental function, (iii) placental infection and inflammation, (iv) real-time evaluation of placental development: tools for placental research, and (v) epidemiologic relevance of placental-based research. Unanimous consensus emerged among the participants to carry out additional work focused on these areas. In this article, we summarize the talks and review the published literature on the above-mentioned niches. As a direct outcome of this meeting, a request for applications has been announced by the Department of Biotechnology, Government of India, for pursuing research in this vital but understudied domain.