Differential proteomics of circulating extracellular vesicles of placental origin isolated from women with early-onset preeclampsia reveal aberrant innate immune and hemostasis processes.

PROBLEM: Early-onset preeclampsia (EOPE) is a severe gestational hypertensive disorder with significant feto-maternal morbidity and mortality due to uteroplacental insufficiency. Circulating extracellular vesicles of placental origin (EV-P) are known to be involved in the pathophysiology of EOPE and might serve as an ideal reservoir for its specific biomarkers. Therefore, we aimed to characterize and perform comparative proteomics of circulating EV-P from healthy pregnant and EOPE women before delivery. METHOD OF STUDY: The EV-P from both groups were isolated using immunoaffinity and were characterized using transmission electron microscopy, dynamic light scattering, nanoparticle tracking analysis, and immunoblotting. Following IgG albumin depletion, the pooled proteins that were isolated from EV-P of both groups were subjected to quantitative TMT proteomics. RESULTS: Circulating term EV-P isolated from both groups revealed ∼150 nm spherical vesicles containing CD9 and CD63 along with placental PLAP and HLA-G proteins. Additionally, the concentration of EOPE-derived EV-P was significantly increased. A total of 208 proteins were identified, with 26 among them being differentially abundant in EV-P of EOPE women. This study linked the pathophysiology of EOPE to 19 known and seven novel proteins associated with innate immune responses such as complement and TLR signaling along with hemostasis and oxygen homeostasis. CONCLUSION: The theory suggesting circulating EVs of placental origin could mimic molecular information from the parent organ-"the placenta"-is strengthened by this study. The findings pave the way for possible discovery of novel prognostic and predictive biomarkers as well as provide insight into the mechanisms driving the pathogenesis of EOPE.

Isolation of serum-derived placental/amnio-chorionic extracellular vesicles across pregnancy by immunoaffinity using PLAP and HLA-G.

In brief: Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. Abstract: Extracellular vesicles (EVs) are membrane-bound nanovesicles secreted from the cells into extracellular space and body fluids. They are considered 'fingerprints of parent cells', which can reflect their physiological and functional states. During pregnancy, EVs are produced by the syncytiotrophoblasts and extravillous trophoblasts and are released into the maternal bloodstream. In the present study, placental alkaline phosphatase (PLAP)-specific extracellular vesicles were isolated from maternal serum-derived EVs (SDE) across pregnancy. Transmission electron microscopy and dynamic light scattering analysis showed that the isolated EVs exhibited a spherical morphology with ~30-150 nm size range. Nanoparticle tracking analysis indicated that the concentration of PLAP+ serum-derived EVs (PLAP+-SDE) increased across the gestation. PLAP+-SDE contained DNA with LINE1 promoter methylation pattern. C19 miRNA cluster miRNAs (miR 515-5p, 519e and 520f) were present in PLAP+-SDE along with other miRNAs (miR-133-3p, miR210-3p and miR-223-3p). PLAP+-SDE confirmed the presence of EV markers (CD63 and CD9), along with placental proteins (PLAP and cullin 7). A modified novel strategy to extract an enriched population of circulating placental/amniochorionic EVs was devised employing an additional marker of extravillous trophoblasts, human leukocyte antigen G (HLA-G), along with PLAP. The isolated pooled placental/amniochorionic (PLAP+&HLA-G+) serum-derived EVs (PP-SDE) showed ~two-fold increased protein levels of HLA-G in the third-trimester pregnant women compared to the non-pregnant controls. Future studies will be focused on validation of this novel strategy to isolate an enriched population of placental/amniochorionic EVs to facilitate a better understanding of placental physiology and pathophysiology.

Clinical Utility of Sperm Function Tests in Predicting Male Fertility: A Systematic Review.

Routine semen analysis provides considerable information regarding sperm parameters; however, it is not solely adequate to predict male fertility potential. In the past two decades, several advance sperm function tests have been developed. The present systematic review intends to assess the clinical utility of available advance sperm function tests in predicting the male fertility potential. A systematic literature search was conducted as per PRISMA guidelines using PubMed, MEDLINE, Google Scholar, and Cochrane Library. Different keywords either singly or in combination were used to retrieve the relevant articles related to sperm function tests, male fertility, and pregnancy outcomes. A total of 5169 articles were obtained, out of which 110 meeting the selection criteria were included in this review. The majorly investigated sperm function tests are hypo-osmotic swelling test, acrosome reaction test, sperm capacitation test, hemizona binding assay, sperm DNA fragmentation test, seminal reactive oxygen species test, mitochondrial dysfunction tests, antisperm antibody test, nuclear chromatin de-condensation (NCD) test, etc. The different advance sperm function tests analyse different aspects of sperm function. Hence, any one test may not be helpful to appropriately predict the male fertility potential. Currently, the unavailability of high-quality clinical data, robust thresholds, complex protocols, high cost, etc., are the limiting factors and prohibiting current sperm function tests to reach the clinics. Further multi-centric research efforts are required to fulfil the existing lacunas and pave the way for these tests to be introduced into the clinics.

Dopamine receptor D2 regulates genes involved in germ cell movement and sperm motility in rat testes†.

The function of dopamine receptor D2 (D2R) is well associated with sperm motility; however, the physiological role of D2R present on testicular cells remains elusive. The aim of the present study is to delineate the function of testicular D2R. Serum dopamine levels were found to decrease with age, whereas testicular D2R expression increased. In rat testicular sections, D2R immunolabeling was observed in interstitial cells, spermatogonia, spermatocytes and mature elongated spermatids, whereas tyrosine hydroxylase immunolabeling was selectively detected in Leydig cells. In vitro seminiferous tubule culture following bromocriptine (D2R agonist) treatment resulted in decreased cAMP levels. Microarray identified 1077 differentially expressed genes (511 up-regulated, 566 down-regulated). The majority of differentially expressed genes were present in post-meiotic cells including early and late spermatids, and sperm. Gene ontology elucidated processes related to extra-cellular matrix to be enriched and was supported by differential expression of various collagens and laminins, thereby indicating a role of dopamine in extra-cellular matrix integrity and transport of spermatids across the seminiferous epithelium. Gene ontology and enrichment map also highlighted cell/sperm motility to be significantly enriched. Therefore, genes involved in sperm motility functions were further validated by RT-qPCR. Seven genes (Akap4, Ccnyl1, Iqcf1, Klc3, Prss55, Tbc1d21, Tl18) were significantly up-regulated, whereas four genes (Dnah1, Dnah5, Clxn, Fsip2) were significantly down-regulated by bromocriptine treatment. The bromocriptine-stimulated reduction in seminiferous tubule cyclic AMP and associated changes in spermatid gene expression suggests that dopamine regulates both spermatogenesis and spermiogenesis within the seminiferous epithelium, and spermatozoa motility following spermiation, as essential processes for fertility.

Early prediction of pre-eclampsia using circulating placental exosomes: Newer insights.

Pre-eclampsia (PE), a multifactorial de novo hypertensive pregnancy disorder, is one of the leading causes of foeto-maternal morbidity and mortality. Currently, antihypertensive drugs are the first-line therapy for PE and evidence suggests that low-dose aspirin initiated early in high risk pregnancies may reduce the risk of development or severity of PE. However, an early prediction of this disorder remains an unmet clinical challenge. Several potential serum biomarkers associated with maternal immunoregulation and placental angiogenesis have been evaluated but are ineffective and inconsistent for early prediction. Although placental biomarkers would be more specific and sensitive in predicting the risk of PE, accessing the placenta during pregnancy is not feasible. Circulating placental exosomes (pEXO), originating from foeto-maternal interface, are being evaluated as the placenta's surrogate and the best source of non-invasive placental biomarkers. pEXO appear in the maternal circulation starting from six weeks of gestation and its dynamic biological cargo across pregnancy is associated with successful pregnancy outcomes. Therefore, monitoring changes in pEXO expression profiles could provide new insights into the prediction, diagnosis and treatment of PE. This narrative review comprehensively summarizes the available literature on the candidate predictive circulating biomarkers evaluated for PE to date. In particular, the review elucidates the current knowledge of distinct molecular signatures emanating from pEXO in pre-eclamptic women to support the discovery of novel early predictive biomarkers for effective intervention and management of the disease.

DNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss.

Previously, we showed that DNA methylation defects in spermatozoa from male partners of couples undergoing recurrent pregnancy loss (RPL) could be a contributing paternal factor. In the present study, we aimed to determine whether the methylation levels of selected imprinted genes can be used as diagnostic markers to identify epigenetically abnormal spermatozoa sample in these cases. The methylation levels of selected imprinted genes in spermatozoa, which were previously found to be differentially methylated, were combined into a probability score (between 0-1) using multiple logistic regression. Different combinations of these genes were investigated using Receiver Operating Characteristic analysis, and the threshold values were experimentally validated in an independent cohort of 38 control and 45 RPL spermatozoa samples. Among the different combinations investigated, a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 (AUC = 0.88) with a threshold value of 0.61 was selected with a specificity of 90.41% and sensitivity of 70%. The results from the validation study indicated that 97% of the control samples had probability scores below this threshold, whereas 40% of the RPL samples were above this threshold with a post-hoc power of 97.8%. Thus, this combination can correctly classify control samples and potentially identify epigenetically abnormal spermatozoa samples in the male partners of couples undergoing RPL. We propose that the combined DNA methylation levels of these imprinted genes can be used as a diagnostic tool to identify spermatozoa samples with epigenetic defects which could contribute to the pathophysiology of RPL and the couple could be counselled appropriately.

Prolactin Regulates Testicular Gene Expression and Cell Cycle Processes Predominantly via JAK2/STAT5 Pathway in the Male Rat.

Hyperprolactinemia is prevalent in up to 16% of infertile males. Although the prolactin receptor (PRLR) is present on various testicular cells, the physiological role of this receptor in spermatogenesis remains elusive. The aim of this study is to delineate prolactin actions in rat testicular tissue. Serum prolactin, developmental expression of PRLR, signaling pathways associated, and gene transcription regulation in the testes were investigated. Serum prolactin and testicular PRLR expression was found to be significantly increased at pubertal and adult ages as compared to prepubertal. Further, PRLR activated the JAK2/STAT5 pathway, but not the MAPK/ERK and PI3K/AKT pathway in the testicular cells. Gene expression profiling following prolactin treatment in seminiferous tubule culture resulted in a total of 692 differentially expressed genes, of which 405 were upregulated and 287 were downregulated. Enrichment map analysis showed that prolactin target genes are involved in processes such as cell cycle, male reproduction, chromatin remodeling, and cytoskeletal organization. Novel gene targets of prolactin whose role in testes is unexplored were obtained and validated by qPCR. Additionally, 10 genes involved in cell cycle process were also validated; 6 genes (Ccna1, Ccnb1, Ccnb2, Cdc25a, Cdc27, Plk1) were found to be significantly upregulated, whereas 4 genes (Ccar2, Nudc, Tuba1c, Tubb2a) were found to be significantly downregulated in testes after treatment with prolactin. Taken together, the findings from this study suggest a crucial role of prolactin in male reproduction and identified target genes regulated by prolactin in the testes.

Synthesis and biological evaluation of benzamide-chalcone hybrids as potential c-Met kinase and COX-2 inhibitors.

c-Met kinase and cyclooxygenase 2 (COX-2) enzymes are two significant targets in tumor progression. Chalcone and benzamide moieties were combined using molecular hybridization to assess their potential as c-Met kinase and COX-2 inhibitors. 4-Methylbenzamide and 4-chlorobenzamide chalcone analogs were synthesized, characterized, and evaluated for antiproliferative activity on Michigan Cancer Foundation-7 (MCF-7), HT-29, MDA-MB-231, COLO-205, and A549 cell lines by sulforhodamine-B stain (SRB) assay. Following the SRB assay, compounds were evaluated for their c-Met kinase and COX-2 inhibitory potential. All compounds inhibited COX-2 with half-maximal inhibitory concentration (IC50 ) <10 µM. Compounds 7h, 7i, 7j, 8f, and 8j inhibited c-Met with IC50 <10 µM. Compound 7h was evaluated for its long-term antiproliferative and anti-migratory effects by colony formation and wound healing assay. It exerted these effects in a concentration-dependent manner. Compounds 7j and 8j were further evaluated for in vitro antiangiogenic effects. Compound 7j exhibited moderate antiangiogenic effect while compound 8j exhibited strong effect. Compounds 7h, 7i, 7j, 8f, and 8j were evaluated for the serum protein binding, using the in vitro bovine serum albumin binding assay. The results indicated that the tested compounds bind to bovine serum albumin (BSA) and can be further explored by other studies.

Whole genome bisulfite sequencing of sperm reveals differentially methylated regions in male partners of idiopathic recurrent pregnancy loss cases.

OBJECTIVE: To study the genome wide alterations in sperm DNA methylation in male partners of idiopathic recurrent pregnancy loss (iRPL) cases and note regions as potential diagnostic markers. DESIGN: Case-control study and methylome analysis of human sperm. SETTING: Obstetrics and Gynaecology clinics. PATIENT(S): Control group consists of apparently healthy fertile men having fathered a child within the last 2 years (n = 39); and case group consists of male partners of iRPL cases having ≥2 consecutive 1st trimester pregnancy losses (n = 47). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm DNA samples of controls and cases were selected for whole genome bisulfite sequencing analysis based on the previously set thresholds of global methylation levels and methylation levels of imprinted genes (KvDMR and ZAC). Whole genome bisulfite sequencing of selected sperm genomic DNA was performed to identify differentially methylated CpG sites of iRPL cases compared with fertile controls. Pathway analysis of all the differentially methylated genes was done by Database for Annotation, Visualization, and Integrated Discovery annotation tool and Kyoto Encyclopedia of Genes and Genomes tool. Differentially methylated CpGs within genes relevant to embryo and placenta development were selected to further validate their methylation levels in study population by pyrosequencing. RESULT(S): A total of 9497 differentially methylated CpGs with highest enrichment in intronic regions were obtained. In addition, 5352 differentially methylated regions and 2087 differentially methylated genes were noted. Signaling pathways involved in development were enriched on pathway analysis. Select CpGs within genes PPARG, KCNQ1, SETD2, and MAP3K4 showed distinct hypomethylated subpopulations within iRPL study population. CONCLUSION(S): Our study highlights the altered methylation landscape of iRPL sperm, and their possible implications in pathways of embryo and placental development. The CpG sites that are hypomethylated specifically in sperm of iRPL subpopulation can be further assessed as predictive biomarkers.

Paternal obesity induces epigenetic aberrations and gene expression changes in placenta and fetus.

Paternal epigenome regulates placental and fetal growth. However, the effect of paternal obesity on placenta and its subsequent effect on the fetus via sperm remains unknown. We previously discovered abnormal methylation of imprinted genes involved in placental and fetal development in the spermatozoa of obese rats. In the present study, elaborate epigenetic characterization of sperm, placenta, and fetus was performed. For 16 weeks, male rats were fed either control or a high-fat diet. Following mating studies, sperm, placenta, and fetal tissue were collected. Significant changes were observed in placental weights, morphology, and cell populations. Methylation status of imprinted genes-Igf2, Peg3, Cdkn1c, and Gnas in spermatozoa, correlated with their expression in the placenta and fetus. Placental DNA methylating enzymes and 5-methylCytosine levels increased. Furthermore, in spermatozoa, DNA methylation of a few genes involved in pathways associated with placental endocrine function-gonadotropin-releasing hormone, prolactin, estrogen, and vascular endothelial growth factor, correlated with their expression in placenta and fetus. Changes in histone-modifying enzymes were also observed in the placenta. Histone marks H3K4me3, H3K9me3, and H4ac were downregulated, while H3K27me3 and H3ac were upregulated in placentas derived from obese male rats. This study shows that obesity-related changes in sperm methylome translate into abnormal expression in the F1-placenta fathered by the obese male, presumably affecting placental and fetal development.

Varicocoele-associated male infertility: Cellular and molecular perspectives of pathophysiology.

BACKGROUND: Varicocoele is a common risk factor associated with reduced male fertility potential. The current understanding of varicocoele pathophysiology does not completely explain the clinical manifestation of infertility. The present treatment options such as antioxidant supplementation and varicocoelectomy only help ≈35% of men to achieve spontaneous pregnancy. OBJECTIVE: This review aims to summarize the available knowledge on cellular and molecular alterations implicated to varicocoele-associated male infertility and also highlights the new knowledge generated by "omics" technologies. MATERIALS AND METHODS: PubMed, MEDLINE, Cochrane and Google Scholar databases are searched using different combinations of keywords (varicocoele, infertile/fertile men with varicocoele, cellular changes, molecular mechanisms, proteome, epigenome, transcriptome and metabolome). A total of 229 relevant human and animal studies published till 2021 were included in this review. RESULTS: Current understanding advocates oxidative stress (OS) as a major contributory factor to varicocoele-associated male infertility. Excessive OS causes alteration in testicular microenvironment and sperm DNA fragmentation, which further contributes to infertility. Molecular and omics studies have identified several promising biomarkers such as AAMP, SPINT1, MKI67 (genetic markers), sperm quality and function related protein markers, global sperm DNA methylation level (epigenetic marker), Hspa2, Protamine, Gadd7, Dynlt1 and Beclin1 (mRNA markers), PRDX2, HSPA, APOA2, YKL40 (seminal protein markers), total choline and PHGDH (metabolic markers). DISCUSSION: Mature spermatozoa harbours a plethora of molecular information in form of proteome, epigenome and transcriptome, which could provide very important clues regarding pathophysiology of varicocoele-associated infertility. Recent molecular and omics studies in infertile men with varicocoele have identified several promising biomarkers. Upon further validation with larger and well-defined studies, some of these biomarkers could aid in varicocoele management. CONCLUSION: The present evidences suggest that inclusion of OS and sperm DNA fragmentation tests could be useful to the diagnostic workup for men with varicocoele. Furthermore, including precise molecular markers may assist in diagnostics and prognostics of varicocoele-associated male infertility.

Association of OPG and RANKL gene polymorphisms with bone mineral density in Indian women.

Osteoprotegerin (OPG) and receptor activator of the NF-kB ligand (RANKL) are key players in bone remodelling. Reports show that OPG and RANKL gene polymorphisms are associated with osteoporosis and fracture risk. The aim of this study was to examine the influence of 5 single nucleotide polymorphisms (SNPs) in OPG and RANKL gene on bone mineral density (BMD) in Indian women. The study included 374 healthy Indian women. Kompetitive Allele Specific PCR (KASP) was used for genotyping. There was a significant difference in the BMD at spine between genotypes of OPG rs2073618 (CC: 0.988 ± 0.167 CG: 1.023 ± 0.17 GG: 1.053 ± 0.155; p = 0.039) which was lost upon adjustment for age and BMI (p = 0.087). Multiple linear regression revealed that genotypes of OPG rs2073618 (ß = 0.098; p = 0.027) and rs3102735 (ß = 0.092; p = 0.038) are predictors of BMD at spine in Indian women. We did not observe any association of SNPs in RANKL gene with BMD. Thus, SNPs rs2073618 and rs3102735 in OPG gene may influence BMD at spine in Indian women.

Maternal cypermethrin exposure during perinatal period dysregulates gonadal steroidogenesis, gametogenesis and sperm epigenome in F1 rat offspring.

Evidences suggest that maternal exposure to endocrine disruptor insecticide cypermethrin (CYP) affects the reproductive functions of offspring. However, its molecular effects are not well researched. This study elucidates the effects of CYP on gonadal steroidogenesis, gametogenesis and sperm epigenome of perinatally exposed F1 rat offspring. CYP (1, 10, 25 mg/kg bw/day) and Diethylestilbestrol (10 µg/kg bw/day; positive control) were gavaged once daily to pregnant dams from gestational day 6 to postnatal day 21 and their effects were assessed in F1 adults. Dysregulated steroidogenesis was observed in F1 testis; expression of Star, Cyp11a1 and Cyp19a1 were upregulated and Hsd3b1, Cyp17a1, Hsd17b3 downregulated. Expression of spermatogenesis cell-type markers Pcna, Plzf, Sohlh2 were upregulated and Ccna1, Sycp1, Ccnb1, Acrv, Amh downregulated. Upon epigenetic investigations of F1 spermatozoa; global hypermethylation, H19 DMR hypomethylation and increased expression of testicular Dnmts were observed. In F1 ovaries, expression of all studied steroidogenesis genes and oogenesis markers such as Amh, Gdf9, Ccnb1 and Pcna were altered. Amh was downregulated and Gdf9 was found to be upregulated. Overall, perinatal CYP exposure hampers molecular control of steroidogenesis and gametogenesis processes along with sperm epigenome in CYP exposed F1 offspring.

Age-related changes and reference intervals of RANKL, OPG, and bone turnover markers in Indian women.

RANKL and OPG are cytokines involved in bone remodeling that makes them potential bone biomarkers. The reference interval for these cytokines, their ratio, and bone turnover markers CTX and PINP were established in Indian women, which may serve in diagnosis and management of osteoporosis. PURPOSE: The aim of the study was to establish reference interval for RANKL, OPG, RANKL/OPG, and bone turnover markers CTX and PINP in healthy Indian women. METHODS: This was a cross-sectional study on 374 healthy Indian women in the age group of 20-65 years. Serum levels of total RANKL, OPG, CTX, PINP, and estradiol were determined by commercial ELISA kits. The reference intervals for these cytokines and bone turnover markers were based on the 95% centrally distributed data. RESULTS: Median RANKL (245.6 pmol/L vs. 149 pmol/L) and RANKL/OPG (38.7 vs. 20.4) were higher, while sCTX (380 ng/L vs. 551 ng/L) and OPG levels (6.1 pmol/L vs. 7.4 pmol/L) were lower in premenopausal women than those in postmenopausal women. PINP levels were comparable in both groups. Women were classified into 5 groups according to decades of age and the reference intervals for RANKL, OPG, RANKL/OPG ratio, and CTX and PINP in each group were reported. CONCLUSION: We reported menopausal status-based and age-related reference intervals for serum RANKL, OPG, RANKL/OPG ratio, and CTX and PINP in healthy Indian women.

Diverse role of endocannabinoid system in mammalian male reproduction.

Endocannabinoid system (ECS) is known for its modulatory role in numerous physiological processes in the body. Endocannabinoids (eCBs) are endogenous lipid molecules which function both centrally and peripherally. The ECS is best studied in the central nervous system (CNS), immune system as well as in the metabolic system. The role of ECS in male reproductive system is emerging and the presence of a complete enzymatic machinery to synthesize and metabolize eCBs has been demonstrated in male reproductive tract. Endocannabinoid concentrations and alterations in their levels have been reported to affect the functioning of spermatozoa. A dysfunctional ECS has also been linked to the development of prostate cancer, the leading cause of cancer related mortality among male population. This review is an attempt to provide an insight into the significant role of endocannabinoids in male reproduction and further summarize recent findings that demonstrate the manner in which the endocannabinoid system impacts male sexual behavior and fertility.

Diet-induced- and genetic-obesity differentially alters male germline histones.

Obesity, an established risk factor for male subfertility or infertility, is primarily due to genetic and environmental causes. Our earlier studies have shown differential effects of high-fat diet-induced- (DIO) and genetically inherited- (GIO) obesity on DNA methylation in male germline and its subsequent effect on fertility. Here, we hypothesized that the effects of DIO and GIO on histone modifications in male germline could also contribute to fertility defects. We observed that DIO affected both active (H3K4me3, H3ac, and H4ac) and repressive (H3K9me3 and H3K27me3) histone marks in testis and their cell types, whereas GIO solely altered acetylated histones. This correlated with the deregulation of histone-modifying enzymes in the testis of both obese groups. Further, we also observed a decrease in chromatin remodelers in the testis of the DIO group, which were increased in the GIO group. Besides, there was an increase in core histones and a decrease in histone marks along with protamine deficiency in spermatozoa of the DIO group, whereas only H3K4me3 levels were increased in spermatozoa of the GIO group. Moreover, we observed alterations in the expression and enrichment patterns of a few developmental genes harbored by the active histone mark in resorbed embryos and spermatozoa of DIO rats. Together these epigenetic defects in the male germline could alter sperm quality and cause fertility defects in these obese groups. Differential changes in two obese groups could also be attributed to differences in their pathophysiological variations. Our study highlights epigenetic differences between DIO and GIO in the male germline and their subsequent impact on male fertility.

High fat diet-induced- and genetically inherited- obesity differential alters DNA demethylation pathways in the germline of adult male rats.

Obesity is a multifactorial condition with predominantly genetic and environmental causes and is an emerging risk factor for male infertility/subfertility. Epigenetic mechanisms are vulnerable to genetic and environmental changes. Our earlier studies have shown differential effects of genetically inherited (GIO) - and diet-induced- obesity (DIO) on DNA methylation in male germline. Contrary to DNA methylation is DNA demethylation, which also regulates the gene expression by activating transcription. The present study aimed to delineate the effects of obesity on the DNA demethylation pathway using two rat models: GIO (WNIN/Ob) and DIO (high-fat diet). We observed differential alterations in enzymes involved in DNA demethylation by oxidation (Tet1-3) pathway in testis in both groups. An increase in Tets in DIO group and a decrease in GIO group were noted. Analysis of oxidation pathway intermediates (5-hmC, 5-fC, and 5-caC) did not show any effect on testis in DIO group but an increase in 5-hmC and decrease in 5-caC levels in GIO group was observed. Analysis of transcript levels of enzymes related to deamination pathway in testis showed an increase (Gadd45a, Aicda, and Tdg) in DIO group and a decrease (Gadd45a, Aicda, and Tdg) in GIO group. Also, 5-hmC levels were differentially altered in the spermatozoa of both groups without any changes in Tet enzyme levels. These findings highlight differences in effects of GIO and DIO on DNA demethylation mechanisms in male germline, which could be due to differences in endocrine and metabolic profile as well as white fat distribution observed earlier in two groups.

Sex hormones regulate lipid metabolism in adult Sertoli cells: A genome-wide study of estrogen and androgen receptor binding sites.

Optimal functioning of Sertoli cells is crucial for spermatogenesis which is under tight regulation of sex hormones, estrogen and androgen. Adult rat Sertoli cells expresses estrogen receptor beta (ERß) and androgen receptor (AR), both of which regulate gene transcription by binding to the DNA. The present study is aimed to acquire a genome-wide map of estrogen- and androgen-regulated genes in adult Sertoli cells. ChIP-Seq was performed for ERß and AR in Sertoli cells under physiological conditions. 30,859 peaks in ERß and 9,594 peaks in AR were identified with a fold enrichment >2 fold. Pathway analysis for the genes revealed metabolic pathways to be significantly enriched. Since Sertoli cells have supportive functions and provide energy substrates to germ cells during spermatogenesis, significantly enriched metabolic pathways were explored further. Peaks of the genes involved in lipid metabolism, like fatty acid, glyceride, leucine, and sphingosine metabolism were validated. Motif analysis confirmed the presence of estrogen- and androgen-response elements (EREs and AREs). Moreover, transcript levels of enzymes involved in the lipid metabolic pathways were significantly altered in cultured Sertoli cells treated with estrogen and androgen receptor agonists, demonstrating functional significance of these binding sites. This study elucidates a mechanism by which sex hormones regulate lipid metabolism in Sertoli cells by transcriptionally controlling the expression of these genes, thereby shedding light on the roles of these hormones in male fertility.

High, not low-dose of stanozolol (Anabolic - androgenic steroid) impedes embryo implantation by attenuating endometrial receptivity in the mouse, Mus musculus.

The present investigation is aimed at evaluating the efficacy of one of the anabolic -androgenic steroids, stanozolol (ST), on establishment and maintenance of pregnancy in mice. A total of 40 female mice were assigned to three experimental groups. Stanozolol was dosed subcutaneously (low-dose, 0.5 mg/kg bwt; high-dose, 5.0 mg/kg bwt or 1% alcohol-baseline control) for 30 consecutive days. On the 31st day, treatment was withdrawn. The estrous cycle was disrupted in both treatment groups and its resumption was dose dependent. Following estrous resumption, mice were allowed to mate. Results reveal that the low-dose ST-treated mice maintained gestation until term with reduced litter size, while high-dose-treated mice divulged vaginal plug at frequent intervals, indicating conception failure. Because pregnancy failure was noticed in high-dose-treated mice, they were autopsied on GD1.5 and 4.5. Interestingly, neither dose of stanozolol affected early embryonic development or blastocyst hatching. A decrease in the number of corpora lutea in both treated groups suggests it affects either ovulation or recruitment of follicles that occurs in each cycle for maturation. In high-dose-treated mice, decreased serum levels of estradiol, progesterone and increased testosterone along with downregulated endometrial expression of ERα and PR suggest the deficiency of steroid hormones and their respective receptors. Decreased ovarian expression of ERα, hyperexpression of PRLR, AR and abated progesterone secretion led to luteal dysfunction, consequently attenuating endometrial receptivity. Therefore, in high-dose-treated mice, decreased maternal estradiol and progesterone levels and their receptors during implantation hindered signaling to LIF and Hoxa-10, resulting in pragmatic implantation failure.

DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss.

STUDY QUESTION: What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? SUMMARY ANSWER: Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST), zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. WHAT IS KNOWN ALREADY: RPL is defined as loss of two or more pregnancies, affecting 1-2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative paternal factors that could be contributing towards pregnancy loss in cases of idiopathic RPL. STUDY DESIGN, SIZE, DURATION: In this case-control study, 112 couples undergoing RPL with no identifiable cause were recruited from September 2015 to May 2018. The control group comprised of 106 healthy proven fertile couples with no history of infertility or miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we investigated the paternal genetic and epigenetic factors that could be associated with RPL. We studied DNA methylation, by pyrosequencing, of selected imprinted genes implicated in embryo development, such as IGF2-H19 DMR, IG-DMR, MEST, ZAC, KvDMR, PEG3, PEG10 and small nuclear ribonucleoprotein polypeptide N (SNRPN) in sperm of men whose partners present RPL. Global DNA methylation in sperm was evaluated by studying 5mC content and long interspersed nuclear element 1 (LINE1) promoter methylation. We also studied polymorphisms by pyrosequencing in the IGF2-H19 DMR as well in the IGF2 promoter in both groups. MAIN RESULTS AND THE ROLE OF CHANCE: In the RPL group, we found a significant decrease in the global sperm 5mC levels and significant decrease in DNA methylation at three CpG sites in LINE1 promoter. For IGF2-H19 DMR and IG-DMR, a significant decrease in sperm DNA methylation at specific CpG sites was observed in RPL group. For maternally imprinted genes like MEST, ZAC, KvDMR, PEG3 and PEG10 hypermethylation was noted. Polymorphism studies for IGF2-H19 DMR and IGF2 revealed significant differences in the genotypic frequencies in males. LIMITATIONS, REASONS FOR CAUTION: In this study, we analysed the methylation levels of selected candidate imprinted genes implicated in embryo development. Detection of methylation changes occurring at the genome-wide level may reveal further candidate genes having a better distinction between the control and study groups. WIDER IMPLICATIONS OF THE FINDINGS: Our study demonstrates that certain polymorphisms and aberrant sperm methylation status in imprinted genes are associated with RPL and could contribute to the aetiology of RPL. This study suggests that investigation of paternal genetic and epigenetic factors could be useful in identification of possible causes of idiopathic RPL. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Department of Science and Technology-Science and Engineering Research Board (EMR/2014/000145) and National Institute for Research in Reproductive Health intramural funds (RA/872/01-2020). All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.