Prenatal Diagnosis of Triploidy in Fetus with Unexpected Chromosomal Translocation of Maternal Origin.

Triploidy is a lethal chromosomal abnormality. Fetuses with triploid condition have a tendency to die in early conception and very few survive to term. In this study, we report the prenatal diagnosis of fetal triploidy with unexpected chromosomal translocation. A 27 years old women was referred to our clinical cytogenetic department due to history of previous conceptus with intrauterine growth retardation at 21-22 weeks of gestation and in present pregnancy, the quadruple marker screen test had suggested a high risk for Trisomy 18 with the risk >1:50. The study was performed on the amniotic fluid and peripheral blood samples received at the clinical cytogenetics department. The interphase FISH and conventional karyotype methods were followed. The prenatal diagnosis using an amniotic fluid sample found a triploid fetus with unexpected balanced chromosomal translocation: 69, XXX,t(2;9)(q11.2;p22)x2. Later the origin of translocation was confirmed by parental chromosomal study. Cytogenetic analysis showed the presence of translocation involving chromosome 2 and 9 in the mother which confirms the maternal origin of translocation in fetal triploidy. Prenatal diagnosis of fetal triploidy with balanced translocation of maternal origin is a rare finding. In present study, the triploidy arises from the failure to expel the second polar body. It is important to perform prenatal fetal imaging with ultrasound at 18-22 weeks to identify any fetal anomalies or intrauterine growth retardation which is associated with triploidy.

Transcriptomic profile of GLCs of PCOS women highlights metabolic dysregulation as a plausible contributor to PCOS pathophysiology.

Polycystic ovary syndrome (PCOS) is a complex heterogeneous disorder with reproductive and metabolic consequences whose aetiology is still elusive. To understand the cellular mechanisms that potentially govern follicular defect in women with PCOS, we performed transcriptomic profiles of granulosa-lutein cells (GLCs) by RNA-Seq analysis. We found differential expression of 876 genes in GLCs between PCOS and controls that belonged to various processes such as cell cycle, extracellular matrix organization, angiogenesis, oxidative stress, metabolism, etc. that support folliculogenesis, oocyte development, and maturation. The cross-talk between oocyte and GLCs is a fundamental cornerstone in determining oocyte quality and highly interlinked pathways of metabolism and redox homeostasis may influence this. We found several genes involved in the metabolism of carbohydrates, nucleotides, cholesterol, and lipids were dysregulated, which may impair the supply of metabolites to the growing oocyte, affecting oocyte development and competence. Additionally, high metabolic activity during folliculogenesis may augment oxidative damage to cells and macromolecules if not counter-balanced. We observed dysregulation of redox homeostasis and AGE-RAGE signalling in the follicular environment. Among the validated genes, prokineticin-1 and growth differentiation factor-15 were found to be negatively regulated, while, S100, calcium-binding protein A9 and angiomotin-like-2 were positively regulated in GLCs of women with PCOS. Comparing our data with previously published relevant transcriptomic studies showed metabolic, cytokine-cytokine receptor interaction, IL-17, and chemokine signalling pathways were most commonly affected in PCOS. Overall, this data can provide insights into mechanisms contributing to PCOS pathophysiology and can be explored as potential indicators for oocyte/embryo quality in IVF settings.

Ovarian granulosa cells from women with PCOS express low levels of SARS-CoV-2 receptors and co-factors.

PURPOSE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is global pandemic with more than 5 million deaths so far. Female reproductive tract organs express coronavirus-associated receptors and factors (SCARFs), suggesting they may be susceptible to SARS-CoV-2 infection; however, the susceptibility of ovary/follicle/oocyte to the same is still elusive. Co-morbidities like obesity, type-2 diabetes mellitus, cardiovascular disease, etc. increase the risk of SARS-CoV-2 infection. These features are common in women with polycystic ovary syndrome (PCOS), warranting further scope to study SCARFs expression in ovary of these women. MATERIALS AND METHODS: SCARFs expression in ovary and ovarian tissues of women with PCOS and healthy women was explored by analyzing publically available microarray datasets. Transcript expressions of SCARFs were investigated in mural and cumulus granulosa cells (MGCs and CGCs) from control and PCOS women undergoing in vitro fertilization (IVF). RESULTS: Microarray data revealed that ovary expresses all genes necessary for SARS-CoV-2 infection. PCOS women mostly showed down-regulated/unchanged levels of SCARFs. MGCs and CGCs from PCOS women showed lower expression of receptors ACE2, BSG and DPP4 and protease CTSB than in controls. MGCs showed lower expression of protease CTSL in PCOS than in controls. Expression of TMPRSS2 was not detected in both cell types. CONCLUSION: Human ovarian follicle may be susceptible to SARS-CoV-2 infection. Lower expression of SCARFs in PCOS indicates that the risk of SARS-CoV-2 infection to the ovary may be lesser in these women than controls. This knowledge may help in safe practices at IVF settings in the current pandemic.

An integrated in silico analysis highlighted angiogenesis regulating miRNA-mRNA network in PCOS pathophysiology.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrinopathy and a leading cause of anovulatory infertility. Angiogenesis is vital for ovarian folliculogenesis. The expression of angiogenesis-associated genes/proteins is altered in the ovary of PCOS women. However, information on microRNAs (miRNAs) regulating their expression is limited. This study aims to identify dysregulated angiogenesis-related genes in the ovary of women with PCOS, to identify miRNAs regulating them, and to construct a miRNA-mRNA network associated with angiogenesis. METHODS: A comprehensive literature search and reanalysis of seven ovarian GEO microarray datasets were performed to identify differentially expressed angiogenesis-related genes in PCOS. These target genes were used to predict their regulating miRNAs by querying miRNA databases and their expression in the ovary was verified. Panther and STRING database were used for functional enrichment. Gene expression of shortlisted miRNAs was studied in granulosa cells using digital droplet PCR. RESULTS: The miRNAs expressed in the ovary and potentially targeting dysregulated angiogenesis-related genes in PCOS were identified and those enriched in angiogenesis-related pathways, like VEGF, FGF, PI3K/Akt, Notch signaling, and ECM interaction were shortlisted. Analysis showed PI3K/Akt signaling was the most enriched pathway. MiR-218-5p, miR-214-3p, miR-20a-5p, and miR-140-3p associated with the PI3K/Akt pathway were found to be up-regulated in granulosa cells of women with PCOS. CONCLUSIONS: By in silico analysis, we identified crucial dysregulated angiogenesis-related genes, the miRNA-mRNA interactions, and signaling pathways involved in impaired follicular angiogenesis in PCOS. This work provides a novel insight into the mechanism of aberrant ovarian angiogenesis contributing to PCOS pathophysiology.

Compromised Cumulus-Oocyte Complex Matrix Organization and Expansion in Women with PCOS.

The cumulus-oocyte complex (COC) matrix plays a critical role in the ovulation and fertilization process and a major predictor of oocyte quality. Proteomics studies of follicular fluid showed differential expression of COC matrix proteins in women with polycystic ovary syndrome (PCOS), indicating altered COC matrix in these women. In the present study, we aimed to understand COC matrix gene induction in humans and its probable dysfunction in women with PCOS. Animal studies have shown that amphiregulin (AREG) and growth differentiation factor-9 (GDF-9) are important in the induction of COC matrix genes which are involved in cumulus expansion. The effects of AREG and GDF-9 on expression of tumor necrosis factor alpha induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) on human cumulus granulosa cells (CGCs) and murine COC expansion were evaluated. Further time-dependent effects of growth factor supplementation on these gene expressions in CGCs from PCOS and control women were compared. Follicular fluid from PCOS showed reduced COC matrix expansion capacity, using murine COCs. Expression of COC matrix genes TNFAIP6 and HAS2 were significantly reduced in CGCs of PCOS. Treatment of CGCs with AREG and GDF-9 together induced expression of both these genes in controls and could only restore HAS2 but not TNFAIP6 expression in PCOS. Our results suggest that the reduced potential of follicular fluid to support COC expansion, altered expression of structural constituents, and intrinsic defects in granulosa cells of women with PCOS may contribute to the aberrant COC organization and expansion in PCOS, thus affecting fertilization.

Alteration in angiogenic potential of granulosa-lutein cells and follicular fluid contributes to luteal defects in polycystic ovary syndrome.

STUDY QUESTION: Is angiogenic potential of follicular fluid (FF) and granulosa-lutein cells (GLCs) altered in polycystic ovary syndrome (PCOS) and does it play a role in corpus luteum (CL) defect observed in them? SUMMARY ANSWER: FF and GLCs of women with PCOS show reduced expression of pro-angiogenic factors compared to controls and exhibit a diminished capacity to induce angiogenesis. WHAT IS KNOWN ALREADY: In women with PCOS, CL insufficiency and frequent miscarriage are reported, which may be due to defect in CL. The development of new blood vessels is essential to promote ovarian folliculogenesis and functional CL formation. The vasculature formation in CL which is important for its function is still unexplored in these women. STUDY DESIGN, SIZE, DURATION: This case-control study was conducted in 30 healthy control women and 30 women with PCOS undergoing controlled ovarian hyperstimulation for IVF. The FF, GLCs and serum were collected from all participants during ovum pick up. PARTICIPANTS/MATERIALS, SETTING, METHODS: The capacity of FF to induce angiogenesis was assessed by measuring levels of pro-angiogenic factors vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) and its tube formation and wound healing potential using human umbilical vein endothelial cells (HUVECs). We investigated the angiogenic potential and endothelial cell-like nature of GLCs using several approaches such as the expression of angiogenic genes by quantitative PCR, DiI-conjugated acetylated low-density lipoproteins (Dil-Ac-LDL) internalization assay, tube formation assay, expression of endothelial cell markers by immunofluorescence analysis. In addition, correlation of transcript levels of angiogenic genes with oocyte parameters was studied. MAIN RESULTS AND THE ROLE OF CHANCE: FF and serum levels of VEGF and FGF2 were significantly higher and lower, respectively, in PCOS compared to controls. The tube formation and wound healing capacity of HUVECs was found to be reduced when measured after supplementation with FF of women with PCOS compared to controls. This suggests a decreased angiogenic capacity of FF in women with PCOS. Tube formation (P = 0.003) and Dil-Ac-LDL internalization (P = 0.03) ability of GLCs were significantly reduced in women with PCOS compared to controls. Protein expression levels of endothelial markers, vascular endothelial growth factor A (VEGFA) (P = 0.004), vascular endothelial growth factor receptor 2 (VEGFR2) (P = 0.011), TEK Receptor Tyrosine Kinase (Tie-2) (P = 0.026), fibroblast growth factor receptor 1 (FGFR1) (P = 0.026) and CD31 (P = 0.035) and transcript levels of angiogenic genes VEGFA (P = 0.042), hypoxia inducing factor 1A (HIF1A) (P = 0.025), FGF2 (P = 0.038), angiopoietin 1 (ANGPT1) (P = 0.028), heparin sulfate proteoglycan 2 (HSPG2) (P = 0.016), ADAM metallopeptidase with thrombospondin type1 motif, 1 (ADAMTS1) (P = 0.027) and fibronectin 1 (FN1) (P = 0.016) were found to be low in GLCs of PCOS compared to controls. Thus, the findings of this study indicate that endothelial cell-like characteristics of GLCs were significantly decreased in PCOS. Furthermore, transcript levels of VEGFA (r = 0.46, P = 0.009), ADAMTS1 (r = 0.55, P = 0.001), FGF2 (r = 0.42, P = 0.022) and ANGPT2 (r = 0.47, P = 0.008) showed a positive correlation with oocyte fertilization rate. LIMITATIONS, REASONS FOR CAUTION: The vasculature formation in CL is not possible to study in women, but we explored the angiogenic characteristics of FF and GLC obtained from women with PCOS to speculate any vascularization defect of CL in these women. The FF and GLCs were obtained from the stimulated cycle during oocyte retrieval, which may not exactly mimic the in-vivo condition. The small sample size is another limitation of this study. Larger sample size and support by color Doppler studies on CL blood flow would help to strengthen our findings. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that the altered angiogenic potential of FF and GLCs may affect vasculature development required for CL formation and function in PCOS. These findings pave the way to devise therapeutic strategies to support angiogenesis process in follicle of women with PCOS, which may improve CL insufficiency, progesterone levels and prevent frequent miscarriages in these women. Furthermore, our study also hypothesizes that the vascularization around the ovarian follicles is also compromised which may lead to the growth arrest of the follicles in PCOS, however, this needs thorough investigations. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grant BT/PR16524/MED/97/346/2016 from the Department of Biotechnology, Government of India. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Quantitative mass spectrometric analysis to unravel glycoproteomic signature of follicular fluid in women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex endocrinopathy affecting women of reproductive age, and whose etiology is not well understood yet. In these women, the follicular growth is arrested at preantral stage leading to cyst formation, consequently resulting in anovulatory infertility in these women. As the follicular fluid provides the conducive microenvironment for the growth of oocytes, molecular profiling of the fluid may provide unique information about pathophysiology associated with follicular development in PCOS. Post-translational addition of oligosaccharide residues is one of the many modifications of secreted proteins influencing their functions. These glycoproteins play a significant role in disease pathology. Despite glycoproteins having such essential functions, very limited information is available on their profiling in human reproductive system, and glycoproteomic profile of follicular fluid of women with PCOS is yet unexplored. In the present study, we performed a comparative glycoproteomic analysis of follicular fluid between women with PCOS and controls undergoing in vitro fertilization, by enrichment of glycoproteins using three different lectins viz. concanavalin A, wheat germ agglutinin and Jacalin. Peptides generated by trypsin digestion were labeled with isobaric tags for relative and absolute quantification reagents and analyzed by liquid chromatography tandem mass spectrometry. We identified 10 differentially expressed glycoproteins, in the follicular fluid of women with PCOS compared to controls. Two important differentially expressed proteins- SERPINA1 and ITIH4, were consistently upregulated and downregulated respectively, upon validation by immunoblotting in follicular fluid and real-time polymerase chain reaction in granulosa cells. These proteins play a role in angiogenesis and extracellular matrix stabilization, vital for follicle maturation. In conclusion, a comparative glycoproteomic profiling of follicular fluid from women with PCOS and controls revealed an altered expression of proteins which may contribute to the defects in follicle development in PCOS pathophysiology.

Pathomechanisms of polycystic ovary syndrome: Multidimensional approaches.

Polycystic ovary syndrome is a complex endocrine disorder affecting numerous women of reproductive age across the globe. Characterized mainly by irregular menses, hirsutism, skewed LH: FSH ratios and bulky polycystic ovaries, this multifactorial endocrinopathy results in unfavorable reproductive and metabolic sequelae, including anovulatory infertility, type 2 diabetes, metabolic syndrome and cardiovascular disease in later years. Increasing evidence has shown that the manifestation of polycystic ovary syndrome (PCOS) is attributable to a cumulative impact of altered genetic, epigenetic and protein profiles which bring about a systemic dysfunction. While genetic approaches help ascertain role of causal variants in its etiology, tissue-specific epigenetic patterns help in deciphering the auxiliary role of environmental, nutritional and behavioral factors. Proteomics is advantageous, linking both genotype and phenotype and contributing to biomarker discovery. Investigating molecular mechanism underlying PCOS is imperative in order to gain insight into the pathophysiology of PCOS and formulate novel diagnostic and treatment strategies. In this review we have summarized these three aspects, which have been successfully utilized to delineate the pathomechanisms of PCOS.

An overview of the oxytocin-oxytocin receptor signaling network.

Oxytocin, a nine amino acid long neuropeptide hormone, is synthesized in the hypothalamus and stored and released from the neural lobe of the pituitary gland. Although commonly known for its central role in the regulation of parturition and lactation, oxytocin signaling also plays a key role in modulating social behavior, evoking contentment, initiating maternal behavior, inducing trust, generosity and bonding in humans and animals. Oxytocin signaling can prove to be of great importance in therapeutics and drug targeting because of its diverse range of actions. However, a well annotated map of oxytocin signaling pathway is currently lacking in the publicly available pathway resources. Therefore, we systematically curated the available signaling information of oxytocin from published literature and collated the data to develop a more complete map. We cataloged 66 molecules belonging to oxytocin signaling pathway, which included 9 protein-protein interactions, 39 post-translational modifications, 14 protein translocation events and 22 activation/inhibition events. Further, Oxytocin signaling network data is made freely available to academic fraternity by integrating this into NetPath ( http://www.netpath.org /), a freely available human signaling pathway resource developed previously by our group.