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.

Distribution of killer immunoglobulin-like receptor genes in HIV infected long-term non-progressors from Mumbai, India.

BACKGROUND: Few reports suggest the association of killer immunoglobulin-like receptors of natural killer cells with human immunodeficiency virus infection. India with world's third largest population of human immunodeficiency virus / acquired immunodeficiency syndrome, offers scope to study such association. OBJECTIVE: Current study (2010-2015) was designed to evaluate if killer immunoglobulin-like receptors gene polymorphisms are associated with HIV infection outcomes specifically, with long term non progressors. METHODS: Killer immunoglobulin-like receptors genotyping was done using polymerase chain reaction - sequence-specific primer method. Viral load was measured by Cobas Taqman HIV-1 test. Estimation of CD4 counts was done using BD FACS CD4 count reagent. RESULTS: The activating gene frequencies identified were 3DS1 (53.8%), 2DS3 (69.2%), 2DS4 (76.9%), 2DS5 (69.2%), 2DS1 (76.9%) and 2DS2 (92.3%). The inhibitory gene frequencies were 2DL2 (92.3%), 2DL5 (76.9%), 2DL3 (69.5%), 3DL1 (84.6%), 3DL2 (92.3%) and 2DL1 (100%). The results highlight high frequency of 3DS1/3DL1 heterozygote and killer immunoglobulin-like receptor 2DS1, among these long term non progressors indicating their possible association with slow progression. Genotype analysis shows total 13 genotypes, of which 8 genotypes were identified for the first time from India. Two genotypes were unique/novel, which were unreported. All genotypes observed in this study were considered to be Bx genotype (100 %). LIMITATIONS: A small sample size (n=13, due to a rare cohort) and the absence of control group were the limitations of this study. CONCLUSIONS: The present study highlights the distribution of killer immunoglobulin-like receptor genes in a very rare group of human immunodeficiency virus -1 infected individuals - long term non progressors. All the long term non progressors tested show the presence of Bx haplotype and each long term non progressors has a different killer immunoglobulin-like receptor genotype.

Diversity in KIR gene repertoire in HIV-1 exposed infected and uninfected infants: A study from India.

Natural killer (NK) cells have antiviral activity mediated through killer immunoglobulin receptors (KIRs). Studies have shown the importance of KIR receptors in HIV infection. However reports on association of KIR genes in HIV infection from Indian population are limited, not a single study is reported in HIV exposed uninfected (EU) and infected infants. This study compared the KIR gene repertoire of HIV-1 positive (n = 29) with EU (n = 76) infants to elucidate its association with transmission. KIR genotyping was analysed using the PCR-SSP method. Viral load of mothers, CD4 count of both mothers and infected infants were done using commercial kits. The data was analysed using SPSS software. Results revealed presence of significantly high frequencies of activating gene KIR 2DS5 (P = 0.040) and inhibitory gene KIR 2DL3 (P = 0.013) in EU infants as compared to HIV-1 positive infants, confirmed with multivariable linear regression modelling. Fifty-nine KIR genotypes were identified in these 105 infants. Nine genotypes were unique, reported for the first time. Twenty six genotypes were shared with the World populations. Twenty four genotypes were reported for the first time from India. Specific KIR genotype combinations (GIDs) were exclusively present either in HIV-1 positive (n = 19) or in EU infants (n = 30). The Linkage disequilibrium (LD) analysis shows a strong linkage between four pairs of genes in HIV-1 positive and three pairs of genes in EU infants. In conclusion, this study revealed that, besides maternal confounding factors such as ART and viral load, specific KIR genes are associated independently with perinatal HIV infection.

Variations in KIR genes: a study in HIV-1 serodiscordant couples.

BACKGROUND: NK cells have anti-HIV activity mediated through killer cell immunoglobulin-like receptors (KIRs). The current prospective cohort study evaluated whether variation in KIR genes is associated with HIV infection in discordant couples (DCs), where one spouse remains seronegative (HSN) despite repeated exposure to the HIV. METHODS: KIR was genotyped using PCR SSP. Viral load and CD4 counts were estimated using commercially available reagents. Data were analyzed using SPSS software. RESULTS: Among the 47 DCs, HSN spouses had significantly (P = 0.006) higher frequencies of KIR3DS1. Regression analysis revealed significant (P = 0.009) association of KIR2DS1 with low viral load. KIR2DS4 variant was associated (P = 0.032) with high viral load. Three pairs of KIR genes were in strong LD in HSNs and two pairs in HSPs. There were 60 KIR genotypes, and 16 are reported the first time in the Indian population. Exclusive genotypes were present either in HSPs (N = 22, 11 unique genotypes) or in HSNs (n = 27, 9 unique genotypes). CONCLUSIONS: This study highlights for the first time in the Indian population an association of KIR genes in HIV infection where presence of exclusive and unique genotypes indicates possible association with either HIV infection or with protection.