Biomarker Identification for Preterm Birth Susceptibility: Vaginal Microbiome Meta-Analysis Using Systems Biology and Machine Learning Approaches.

PROBLEM: The vaginal microbiome has a substantial role in the occurrence of preterm birth (PTB), which contributes substantially to neonatal mortality worldwide. However, current bioinformatics approaches mostly concentrate on the taxonomic classification and functional profiling of the microbiome, limiting their abilities to elucidate the complex factors that contribute to PTB. METHOD OF STUDY: A total of 3757 vaginal microbiome 16S rRNA samples were obtained from five publicly available datasets. The samples were divided into two categories based on pregnancy outcome: preterm birth (PTB) (N = 966) and term birth (N = 2791). Additionally, the samples were further categorized based on the participants' race and trimester. The 16S rRNA reads were subjected to taxonomic classification and functional profiling using the Parallel-META 3 software in Ubuntu environment. The obtained abundances were analyzed using an integrated systems biology and machine learning approach to determine the key microbes, pathways, and genes that contribute to PTB. The resulting features were further subjected to statistical analysis to identify the top nine features with the greatest effect sizes. RESULTS: We identified nine significant features, namely Shuttleworthia, Megasphaera, Sneathia, proximal tubule bicarbonate reclamation pathway, systemic lupus erythematosus pathway, transcription machinery pathway, lepA gene, pepX gene, and rpoD gene. Their abundance variations were observed through the trimesters. CONCLUSIONS: Vaginal infections caused by Shuttleworthia, Megasphaera, and Sneathia and altered small metabolite biosynthesis pathways such as lipopolysaccharide folate and retinal may increase the susceptibility to PTB. The identified organisms, genes, pathways, and their networks may be specifically targeted for the treatment of bacterial infections that increase PTB risk.

Placental exosomes in pregnancy and preeclampsia.

Preeclampsia, poses significant risks to both maternal and fetal well-being. Exosomes released by the placenta play a crucial role in intercellular communication and are recognized as potential carriers of essential information for placental development. These exosomes transport a payload of proteins, nucleic acids, and lipids that mirror the placental microenvironment. This review delves into the functional roles of placental exosomes and its contents shedding light on their involvement in vascular regulation and immune modulation in normal pregnancy. Discernible changes are reported in the composition and quantity of placental exosome contents in pregnancies affected by preeclampsia. The exosomes from preeclamptic mothers affect vascularization and fetal kidney development. The discussion also explores the implications of utilizing placental exosomes as biomarkers and the prospects of translating these findings into clinical applications. In conclusion, placental exosomes hold promise as a valuable avenue for deciphering the complexities of preeclampsia, providing crucial diagnostic and prognostic insights. As the field progresses, a more profound comprehension of the distinct molecular signatures carried by placental exosomes may open doors to innovative strategies for managing and offering personalized care to pregnancies affected by preeclampsia.

Azoospermia factor c microdeletions and outcomes of assisted reproductive technology: a systematic review and meta-analysis.

OBJECTIVE: To investigate whether Azoospermia Factor c (AZFc) microdeletions affect Assisted Reproductive Technology (ART) outcomes. DESIGN: Systematic review and meta-analysis. SETTING: Not applicable. PATIENTS: Infertile men with and without AZFc microdeletions. INTERVENTION(S): Electronic databases were searched for case-control studies reporting sperm retrieval rates and outcomes of ART in infertile men with and without AZFc microdeletions from inception to April 2023. Study quality was assessed using the Newcastle-Ottawa Scale. Summary effect sizes (odds ratio [OR] with 95% confidence interval [CI]) were calculated for both categories of infertile men. MAIN OUTCOME MEASURES: The primary outcome was successful sperm retrieval and the secondary outcomes were outcomes of ART. RESULTS: Case-control studies reporting sperm retrieval rates and ART outcomes in men with AZFa and AZFb deletions were unavailable. On the basis of the data from 3,807 men, sperm retrieval rates were found to be higher in men with AZFc microdeletions compared to their non-deleted counterparts [OR = 1.82, 95% CI 0.97, 3.41], but the difference was not statistically significant. A significantly lower fertilization rate (OR = 0.61, 95% CI [0.50, 0.74]), clinical pregnancy rate (OR = 0.61, 95% CI [0.42, 0.89]), and live birth rate (OR = 0.54, 95% CI [0.40, 0.72]) were observed in men with AZFc deletions compared with men without deletions. There was no statistically significant difference in rates of embryo cleavage, blastocyst formation, good-quality embryos, implantation, and miscarriage between the two groups. On correcting for female factors, the fertilization rate (OR = 0.76, 95% CI [0.71, 0.82]), cleavage rate (OR = 0.54, 95% CI [0.41, 0.72]), clinical pregnancy rate (OR = 0.39, 95% CI [0.30, 0.52]), and live birth rate (OR = 0.48, 95% CI [0.35, 0.65]) were significantly lower in men with AZFc deletions compared with controls. CONCLUSIONS: Presence of AZFc microdeletions adversely affects outcomes of ART in infertile men. Further in-depth studies delineating the role of the AZF genes in embryonic development are necessary to understand the full-impact of this finding. CLINICAL TRIAL REGISTRATION NUMBER: CRD42022311738.

Relevance of augmented kisspeptin signaling through H364 KISS1R in central precocious puberty.

Understanding the pathophysiology of idiopathic central precocious puberty (ICPP) is essential, in view of its consequences on reproductive health and metabolic disorders in later life. Towards this, estimation of circulating levels of the neuropeptides, viz; Kisspeptin (Kp-10), Neurokinin B (NKB) and Neuropeptide Y (NPY), acting upstream to Gonadotropin-Releasing Hormone (GnRH), has shown promise. Insights can also be gained from functional studies on genetic variations implicated in ICPP. This study investigated the pathophysiology of ICPP in a girl by exploring the therapeutic relevance of the circulating levels of Kp-10, NKB, NPY and characterizing the nonsynonymous KISS1R variant, L364H, that she harbours, in a homozygous condition. Plasma levels of Kp-10, NKB and NPY before and after GnRH analog (GnRHa) treatment, were determined by ELISA. It was observed that GnRHa treatment resulted in suppression of circulating levels of Kp-10, NKB and NPY. Further, the H364 variant in KISS1R was generated by site directed mutagenesis. Post transient transfection of either L364 or H364 KISS1R variant in CHO cells, receptor expression was ascertained by western blotting, indirect immunofluorescence and flow cytometry. Kp-10 stimulated signalling response was also determined by phospho-ERK and inositol phosphate production. Structure-function studies revealed that, although the receptor expression in H364 KISS1R was comparable to L364 KISS1R, there was an enhanced signalling response through this variant at high doses of Kp-10. Thus, elevated levels of Kp-10, acting through H364 KISS1R, contributed to the manifestation of ICPP, providing further evidence that dysregulation of Kp-10/KISS1R axis impacts the onset of puberty.

Endometrial and placental stem cells in successful and pathological pregnancies.

The endometrium is a dynamic tissue that undergoes extensive remodeling during the menstrual cycle and further gets modified during pregnancy. Different kinds of stem cells are reported in the endometrium. These include epithelial stem cells, endometrial mesenchymal stem cells, side population stem cells, and very small embryonic-like stem cells. Stem cells are also reported in the placenta which includes trophoblast stem cells, side population trophoblast stem cells, and placental mesenchymal stem cells. The endometrial and placental stem cells play a pivotal role in endometrial remodeling and placental vasculogenesis during pregnancy. The dysregulation of stem cell function is reported in various pregnancy complications like preeclampsia, fetal growth restriction, and preterm birth. However, the mechanisms by which it does so are yet elusive. Herein, we review the current knowledge of the different type of stem cells involved in pregnancy initiation and also highlight how their improper functionality leads to pathological pregnancy.

The G-protein-coupled estrogen receptor, a gene co-expressed with ERα in breast tumors, is regulated by estrogen-ERα signalling in ERα positive breast cancer cells.

GPER is a seven transmembrane G-protein-coupled estrogen receptor that mediates rapid estrogen actions. Large volumes of data have revealed its association with clinicopathological variables in breast tumors, role in epidermal growth factor (EGF)-like effects of estrogen, potential as a therapeutic target or a prognostic marker, and involvement in endocrine resistance in the face of tamoxifen agonism. GPER cross-talks with estrogen receptor alpha (ERα) in cell culture models implicating its role in the physiology of normal or transformed mammary epithelial cells. However, discrepancies in the literature have obfuscated the nature of their relationship, its significance, and the underlying mechanism. The purpose of this study was to assess the relationship between GPER, and ERα in breast tumors, to understand the mechanistic basis, and to gauge its clinical significance. We mined The Cancer Genome Atlas (TCGA)-BRCA data to examine the relationship between GPER and ERα expression. GPER mRNA, and protein expression were analyzed in ERα-positive or -negative breast tumors from two independent cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan-Meier Plotter (KM) was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 17ß-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ERα agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ERα knockdown. ERα-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation (ChIP) assay. Clinical data revealed significant positive association between GPER and ERα expression in breast tumors. The median GPER expression in ERα-positive tumors was significantly higher than ERα-negative tumors. High GPER expression was significantly associated with longer overall survival (OS) of patients with ERα-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ERα-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ERα occupancy in the upstream region of GPER. Furthermore, treatment with 17ß-estradiol or PPT significantly reduced the IC50 of the GPER agonist (G1)-mediated loss of MCF-7 or T47D cell viability. In conclusion, GPER is positively associated with ERα in breast tumors, and induced by estrogen-ERα signalling axis. Estrogen-mediated induction of GPER makes the cells more responsive to GPER ligands. More in-depth studies are warranted to establish the significance of GPER-ERα co-expression, and their interplay in breast tumor development, progression, and treatment.

Targeting molecular cross-talk between tumor cells and tumor associated macrophage as therapeutic strategy in triple negative breast cancer.

Triple-negative Breast cancer (TNBC) is a subtype of breast cancer (BC) that lacks expression for ER/PR/Her2 receptors and is associated with aggressive disease pathogenesis and the worst prognosis among other subtypes of BC. Accumulating evidence-based studies indicate the high immunogenic ability of TNBC tumors and the applicability of immunotherapeutic strategies to overcome therapy resistance and tumor recurrence in TNBC patients. However, not all TNBC patients respond equally well to current immunotherapies that mainly target the adaptive immune system for tumor rejection. Recent studies are contemplating the efficacy of tumor-associated macrophage (TAM) targeted therapies since these subpopulations of cells comprise one of the major components of tumor-infiltrating immune cells (TIIs) in the TNBC tumor microenvironment (TME) and play an essential role in priming the adaptive immune response mediators towards both antitumorigenic and pro-tumorigenic response facilitated by intercellular cross-talk between tumor cells and TAM populations present within TNBC-TME. The present review discusses these molecular mechanisms and their consequence on the progression of TNBC tumors. Also, the therapeutic strategies targeting candidate genes/pathways involved in molecular cross-talk between TAM-TNBC cells and their impact on the development and progression of TNBC tumors are also discussed.

LHX2 in germ cells control tubular organization in the developing mouse testis.

In the gonads of mammalian XY embryos, the organization of cords is the hallmark of testis development. This organization is thought to be controlled by interactions of the Sertoli cells, endothelial and interstitial cells with little or no role of germ cells. Challenging this notion, herein we show that the germ cells play an active role in the organization of the testicular tubules. We observed that the LIM-homeobox gene, Lhx2 is expressed in the germ cells of the developing testis between E12.5-E15.5. In Lhx2 knockout-fetal testis there was altered expression of several genes not just in germ cells but also in the supporting (Sertoli) cells, endothelial cells, and interstitial cells. Further, loss of Lhx2 led to disrupted endothelial cell migration and expansion of interstitial cells in the XY gonads. The cords in the developing testis of Lhx2 knockout embryos are disorganized with a disrupted basement membrane. Together, our results show an important role of Lhx2 in testicular development and imply the involvement of germ cells in the tubular organization of the differentiating testis. The preprint version of this manuscript is available at https://doi.org/10.1101/2022.12.29.522214.

Immune alterations in recurrent implantation failure.

A failure to achieve pregnancy after three or more embryo transfer cycles with high-quality blastocysts is referred to as recurrent implantation failure (RIF). RIF can be due to altered uterine factors or male factors or embryo factors. Disrupted endometrial receptivity, altered expression of genes in several pathways, immunologic disturbances in the peripheral blood and/or the endometrium, and epigenetic alterations are associated with RIF. Amongst the immunologic disturbances, altered Th1/Th2 ratio, altered NK cell and macrophage numbers are observed in women with RIF. However, not all women with RIF have the same kind of immune dysfunction suggesting that RIF is a heterogeneous condition associated with varied immune responses and one size may not fit all. Thus, personalized therapies based on the immune status of the patient are being tested in women with RIF. In general, women with a high Th1/Th2 ratio are offered Tacrolimus, while intravenous IgG is recommended in women with high NK cell numbers/HLA mismatch. Women with hyperactivated immune status in the uterus are offered progesterone support, prednisolone, vitamin E, and intralipid treatment to suppress inflammation and oxidative stress, while endometrial scratching and intrauterine hCG administration are offered to women with hypo-active immune status. There is a need for standardized tests for evaluation of immune status in patients and sufficiently powered randomized controlled trials for personalized therapies to determine which of these will be beneficial in women with RIF. Till then, the ART community should limit the use of such add-on interventions in women with RIF.

Determination of functional similarity of biosimilar H9P2S from an investigational CHO clone with Adalimumab.

Biosimilars, which are replicas of innovator pharmaceuticals, constitute the most significant share of biopharmaceutical products. These products are associated with structural and manufacturing complexities and are hence considered as similar to innovator drugs. Adalimumab is a monoclonal antibody that has been approved by the US FDA for blocking TNF-α. Adalimumab, also known as Humira, is preferred over other anti-TNF-α mAbs because of its lower immunogenicity and enhanced clinical efficacy. As cost-effective mAb development is still a challenging area, we developed an in-house stable CHO-K1 cell line for the production of recombinant monoclonal mAb against TNF-α. This clone yielded H9P2S, as a biosimilar against TNF-α, for which several functional assays were conducted to prove its biosimilarity to Adalimumab. Two batches of H9P2S and their subsequent dilutions were compared with Adalimumab. H9P2S and Adalimumab showed highly similar TNF-α binding and neutralizing activities, confirming the suitability of our clone for yielding biosimilar drugs. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03384-z.

Loss of HOXA10 causes endometrial hyperplasia progressing to endometrial cancer.

Endometrial cancer is the fourth most common malignancy in women and the precursor lesion is endometrial hyperplasia. HOXA10 is a transcription factor that plays key roles in endometrial functions such as the endowment of receptivity, embryo implantation, and trophoblast invasion. Herein, using testicular transgenesis, we developed transgenic mice that expressed a shRNA against HOXA10 and there was a nearly 70% reduction in the expression of HOXA10 in these animals. We observed that downregulation of HOXA10 led to the development of endometrial hyperplasia in the young animals (3 months), and as they aged (>1 year), most animals developed well-differentiated endometrial adenocarcinoma. In the endometrium of animals with reduced HOXA10, there was increased proliferation and elevated levels of ERα and ERß. In parallel, there was increased expression of Wnt4 and ß-Catenin, SOX9, and YAP1. We propose that chronic reduction in HOXA10 expression disrupts multiple pathways in the uterus that aids in the development of endometrial hyperplasia which progresses to endometrial cancer with age.

False-positive detection of Group B Streptococcus (GBS) in chromogenic media (Strep B Carrot Broth) due to presence of Enterococcus faecalis in High Vaginal swabs.

Introduction. Vaginal colonization of Group B Streptococcus (GBS) is associated with preterm births and neonatal sepsis. Thus routine screening of GBS in prenatal care is recommended.Hypothesis. Chromogenic media (carrot broth) aids in specific and rapid detection of GBS.Aim. To investigate the efficiency of Strep B Carrot Broth for detection of GBS in high vaginal swabs from pregnant women.Methods. In this study 201 vaginal swab samples were collected from pregnant women. Swabs were inoculated in chromogenic media (Strep B Carrot Broth). The positive and negative cultures were inoculated on blood agar and crome agar plates. The colonies were subjected to 16S rRNA sequencing and gene-specific PCR for confirmation. The Christie Atkins Munch Peterson (CAMP) and bile esculin agar (BEA) tests were used for biochemical confirmation. PCR was performed on genomic DNA isolated from uncultured vaginal swabs.Results. It was found that 20/201 (9.9 %) vaginal swab samples were positive in the Strep B Carrot Broth and 17/20 (85 %) and 19/20 (95 %) of these samples yielded colonies on blood agar and crome agar, respectively. Of the 181 carrot broth-negative samples, 1 (0.5 %) and 38 (20.9 %) yielded colonies on blood agar and crome agar plates, respectively. However, 16 s rRNA sequencing revealed that none of the 20 carrot broth-positive cultures were GBS and had sequence similarities to Enterococcus faecalis. This was also confirmed by using gene-specific PCR and BEA positivity. Furthermore, E. faecalis was detected by PCR in DNA isolated from 57 uncultured vaginal swabs samples, GBS could only be detected by PCR in four samples.Conclusion. Carrot broth-based culture can lead to false-positive detection due to the presence of E. faecalis. Thus GBS-positive results in carrot broth must be confirmed by the other molecular and biochemical tests before making a final diagnosis.

Clinical Profile of SARS-CoV-2-Infected Neonates.

Background There are conflicting data on the mother-to-child transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and few studies have described the clinical course of neonates infected with SARS-CoV-2. Objectives This study investigates the mother-to-child transmission rate and clinical profile of SARS-CoV-2-infected newborns. Methods Data on 304 newborns of 301 mothers with coronavirus disease 2019 (COVID-19) were prospectively collected and analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) determined the presence of SARS-CoV-2 in the placenta, umbilical cord stump, and nasopharyngeal swabs collected within 24h of birth. Clinical and laboratory data of SARS-CoV-2-infected newborns was entered in a structured proforma. Results A total of 20 neonates (6.5%) were positive for SARS-CoV-2, of which 12 were positive only in the nasopharyngeal swab, four cases had the umbilical stump positive, three were positive in the placenta, and one case was positive in all the three specimens collected. Six of the 20 SARS-CoV-2-positive neonates developed severe symptoms. The SARS-CoV-2-positive symptomatic neonates required a more extended stay in hospital compared to their non-symptomatic infected counterparts. Conclusions A proportion of the babies born to SARS-CoV2-infected mothers tested positive and some of these newborns had severe symptoms.

Outcomes in neonates born to mothers with COVID-19 during the second wave in India.

COVID-19 pandemic has affected all age groups globally including pregnant women and their neonates. The aim of the study was to understand outcomes in neonates of mothers with COVID-19 during the first and second waves of COVID-19 pandemic. A retrospective analysis of 2524 neonates born to SARS-CoV-2-infected mothers was conducted during the first wave (n = 1782) and second wave (n = 742) of the COVID-19 pandemic at five study sites of the PregCovid registry in Maharashtra, India. A significant difference was noted in preterm birth, which was higher in the second wave (15.0%, 111/742) compared to the first wave (7.8%, 139/1782) (P < 0.001). The proportion of neonates requiring NICU admission was significantly higher in the second wave (19.0%, 141/742) as compared to that in the first wave (14.8%, 264/1782) (P < 0.05). On comparing regional differences, significantly higher neonatal complications were reported from Mumbai metropolitan region (P < 0.05). During the second wave of COVID-19, birth asphyxia and prematurity were 3.8- and 2.1-fold higher respectively (P < 0.001). Neonatal resuscitation at birth was significantly higher in second wave (3.4%, 25/742 vs 1.8%, 32/1782) (P < 0.05). The prevalence of SARS-CoV-2 infection in neonates was comparable (4.2% vs 4.6%) with no significant difference between the two waves. CONCLUSION: Higher incidence of adverse outcomes in neonates born to SARS-CoV-2-infected mothers in the second wave of COVID-19 as compared to the first wave. TRIAL REGISTRATION: PregCovid study is registered with the Clinical Trial Registry of India (CTRI/2020/05/025423, Registered on 28/05/2020). WHAT IS KNOWN: • The second wave of COVID-19 was more lethal to pregnant women than the first wave. Newborns are at risk of developing complications. WHAT IS NEW: • Birth asphyxia, prematurity, and neonatal resuscitation at birth were significantly higher in the second wave as compared to those in the first wave of the COVID-19 pandemic in India.

Mechanisms of group B Streptococcus-mediated preterm birth: lessons learnt from animal models.

Group B Streptococcus (GBS) is an opportunistic pathogenic bacterium which upon colonization in the female reproductive tract can cause preterm births, fetal injury, and demise. Several determinants for GBS pathogenesis have been explored so far through the studies using animal models ranging from mice to non-human primates. The results from these experimental data have identified outer membrane vesicles, ß-hemolysin, hyaluronidase, and Cas9 of GBS as major virulence factors leading to preterm births. Most of these factors drive inflammation through activation of NLRP3 and elevated production of IL1-ß. However, the absence of one of the factors from the pathogen reduces but does not completely abolish the pathogenesis of GBS suggesting the involvement of more than one factor in causing preterm birth. This makes further exploration of other virulence factors of GBS pathogenesis important in gaining an insight into the mechanistic basis of GBS-mediated preterm births. Lay summary: Group B Streptococcus (GBS) is a pathogenic bacteria whose infection in the reproductive tract during pregnancy can cause premature delivery. This bacterial infection is one of the major causes of death of mother and baby during pregnancy, and the bacteria is prevalent in all parts of the world. This makes the research on GBS so important and many of the mechanisms behind GBS infection during pregnancy still remain unexplored. In this review, we have outlined how various animal models contributed in finding the mechanism of GBS pathogenesis. The review also focuses on compiling various virulence factors which makes GBS pathogenic in the vulnerable. Understanding the mechanisms of infection by GBS will be crucial in developing drugs and vaccines to protect against the harmful effects of the bacteria.

Association of AMH and AMHR2 gene polymorphisms with ovarian response and pregnancy outcomes in Indian women.

PURPOSE: To evaluate the association of single-nucleotide polymorphisms (SNPs) in the anti-Müllerian hormone (AMH) and AMH type II receptor (AMHR2) genes with ovarian response and clinical pregnancy outcomes in women undergoing controlled ovarian hyperstimulation. METHODS: In this prospective study, we genotyped AMH polymorphisms (c. -649 T > C, c. 146 T > G, c. 252 G > A, and c. 303 G > A) in 365 women and AMHR2 polymorphisms (c. -482 A > G, c. 622-6 C > T, c. 4952 G > A, c. 10 A > G) in 80 women undergoing controlled ovarian hyperstimulation for IVF. RESULTS: Higher doses of exogenous FSH and lower numbers of preovulatory follicles were noted in women having AMH c. -649 T > C and AMH c. -146 T > G polymorphisms, respectively. Overall, we found that the presence of a polymorphic genotype (homozygous or heterozygous) at positions c. -649 T > C, c. 146 T > G, c. 252 G > A, and c. 303 G > A in the AMH gene was associated with higher doses of FSH for ovulation induction (p < 0.001). Interestingly, a higher live birth rate was noted in women with a homozygous polymorphic genotype for all four AMH SNPs investigated while none of the women showing a homozygous polymorphic genotype at all AMHR2 SNPs investigated in this study had a live birth. CONCLUSION: Our results show that presence of AMHR2 SNPs (c. 482 A > G, c. 622-6 C > T, c. 4952 G > A, and c. 10 A > G) negatively correlate with live birth rate. However, these findings need to be validated by using larger sample size.