Genomic evidence of Y chromosome microchimerism in the endometrium during endometriosis and in cases of infertility

Muzaffer A Bhat,Debabrata Ghosh,Jai B Sharma,Kallol K Roy,Jayasree Sengupta

doi:10.1186/s12958-019-0465-z

Muzaffer A Bhat, Debabrata Ghosh + Show 3 more

Open Access

https://doi.org/10.1186/s12958-019-0465-z

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Abstract

BackgroundPrevious studies, which were primarily based on the fluorescent in-situ hybridisation (FISH) technique, revealed conflicting evidence regarding male foetal microchimerism in endometriosis. FISH is a relatively less sensitive technique, as it is performed on a small portion of the sample. Additionally, the probes used in the previous studies specifically detected centromeric and telomeric regions of Y chromosome, which are gene-sparse heterochromatised regions. In the present study, a panel of molecular biology tools such as qPCR, expression microarray, RNA-seq and qRT-PCR were employed to examine the Y chromosome microchimerism in the endometrium using secretory phase samples from fertile and infertile patients with severe (stage IV) ovarian endometriosis (OE) and without endometriosis.MethodsMicroarray expression analysis followed by validation using RNA-seq and qRT-PCR experiments at the RNA levels and further validation at the DNA level by qPCR of target inserts for selected targets in eutopic endometrium samples obtained from control (CON) and stage IV ovarian endometriosis (OE), either from fertile (FCON and FOE; n = 30/each) or infertile (ICON and IOE; n = 30/each) women, were performed.ResultsSix coding (AMELY, PCDH11, SRY, TGIF2LY, TSPY3, and USP9Y) and 10 non-coding (TTTY2, TTTY4C, TTTY5, TTTYY6, TTTY8, TTTY10, TTTY14, TTTY21, TTTY22, and TTTY23) genes exhibited a bimodal pattern of expression characterised by low expression in samples from fertile patients and high expression in samples from infertile patients. Seven coding MSY-linked genes (BAGE, CD24, EIF1AY, NLGN4Y, PRKY, VCY and ZFY) exhibited differential regulation in microarray analysis, and this change was validated by RNA-seq or qRT-PCR. DNA inserts for 7 genes in various samples were validated by qPCR. The prevalence and concentration of PCR-positive target inserts for BAGE, PRKY, TTTY9A and ZFY displayed higher values in the fertile, control (FCON) patients compared with the fertile, endometriosis patients (FOE).ConclusionSeveral coding and non-coding MSY-linked genes displayed microchimerism as evidenced by the presence of their respective DNA inserts, along with their differential transcript expression, in the endometrium during endometriosis and in cases of infertility.

Highlights

Previous studies, which were primarily based on the fluorescent in-situ hybridisation (FISH) technique, revealed conflicting evidence regarding male foetal microchimerism in endometriosis
PCR detection for target DNA DNA specimens extracted as previously described [22] were used for quantitative PCR to quantify the DNA copy numbers of the following genes: BAGE, EIF1AY, KDM5D, NLGN4Y, PRKY, TTTY9A, TTTY14, SRY and ZFY using SYBR-green chemistry with the abovementioned PCR system
Further functional studies are required in order to gather large scale dynamic sets of data yielding knowledge in this regard, which may be of significance for understanding endometriosis and infertility. In conclusion, this is the first report with a general observation that several coding and non-coding genes of male-specific regions of the Y chromosome (MSY) origin displayed microchimerism in the form of presence of their respective DNA inserts along with their microarraydetectable expression

Summary

Introduction

Previous studies, which were primarily based on the fluorescent in-situ hybridisation (FISH) technique, revealed conflicting evidence regarding male foetal microchimerism in endometriosis. Several autoimmune diseases are reportedly associated with male microchimerism [9,10,11] Taken together, it appears that male microchimerism in the eutopic endometrium may be a supplementary factor towards the histogenesis of endometriosis at ectopic sites. We have examined this issue of Y-chromosomal microchimerism in fertile and infertile women with ovarian endometriosis (OE), without any reported uterine pathology. This was achieved using a sequence of experiments that included microarray expression followed by RNA-seq and quantitative RT-PCR experiments at the RNA levels and further validation at the DNA level by quantitative PCR of target inserts for selected targets (see Fig. 1 for the study design)

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