Gene Expression and Apoptosis Levels in Cumulus Cells of Patients with Polymorphisms of FSHR and LHB Undergoing in Vitro Fertilization Program

Abstract

Background/Aims: FSH receptor (FSHR) Ala307Thr and Asn680Ser and LHβ chain (LHB) Trp28Arg and Ile35Thr polymorphisms affect the response to pharmacological ovarian stimulation with r-FSH in women undergoing assisted reproductive treatment (ART). Here, we evaluated the expression level of selected genes involved in follicle maturation and the possible onset of apoptosis in cumulus cells of patients with single and double FSHR and LHB polymorphisms, as potential markers of oocyte competence. Methods: Cumulus cells from 36 stimulated patients were collected and SNP genotyping performed by PCR. Gene expression was evaluated through real-time PCR, and apoptosis estimated via TUNEL assay, and cleaved caspase-3 and pAKT immunostaining. Results: The cumulative data show significant correlations indicating that the genetic alteration of FSHR and/or LHB genes may lead to perturbations of the signaling network programmed to granulosa cell survival and follicle development. Notably, when double heterozygotes were compared to the rest of the patients, a higher level of apoptosis in terms of both DNA fragmentation index and amount of active caspase-3 was observed in cumulus cells. Conclusions: These results may help to define personalized stimulation protocols in ART programs, to increase the success rate of ICSI procedures in accordance with the polymorphic condition of the individual patient.

DNA fragmentation index, pAKT and pERK1/2 in cumulus cells are related to oocyte competence in patients undergoing in vitro fertilization programme

“…Sample preparation was performed as reported by Bosco et al (2017c). Essentially, after hyaluronidase treatment of the COCs, cumulus cells were fixed in 3.7% paraformaldehyde for 60 min, harvested by centrifugation at 2000 rpm for 7 min and resuspended in phosphate-buffered saline (PBS).…”

Quantification of hormone membrane receptor FSHR, GPER and LHCGR transcripts in human primary granulosa lutein cells by real-time quantitative PCR and digital droplet PCR

“…Although these data are not surprising, since it is well-established that ddPCR has potentially higher sensitivity than qPCR (Hayden et al, 2013;Zhao et al, 2016), they suggest the use of ddPCR as a preferential method for gene expression screening of samples with low cell number or poor yield of DNA extraction. These data may be relevant for approaching personalized human infertility treatments, where the characterization of hormone receptor expression levels may be useful for predicting patient-specific response to the clinical hormonal treatment (Bosco et al, 2017;Simoni and Casarini, 2014). ddPCR and qPCR differ for the conditions in which primers work, resulting in different amplification efficiency of probe pairs.…”

Hormonal treatments for endometriosis: The endocrine background

“…Besides, the SS (680 Ser/Ser) or AA (307 Ala/Ala) genotype are associated with a reduced risk to develop stage 3–4 endometriosis compared to the stage 1–2 endometriosis [ 30 ]. FSHR 680Asn/Asn induces aromatase activity resulting in higher estrogens levels and proliferation of endometriotic lesions [ 31 ]. Among LHR SNPs, a polymorphic insertion in exon 1 of LH receptor (LHR) gene (insLQ) is common in women with endometriosis and infertility, and it is thought to boost LHR activity, by decreasing the half maximal effective concentration and increasing the cell surface expression [ 32 ].…”