ALTERED BLASTOCYST METHYLOME AND IMPRINTING DYSREGULATION ASSOCIATED WITH THE PROLONGED DISEASE STATE OF INFERTILITY

Abstract

The increased incidence of rare imprinting disorders in children born following infertility treatments has led to questions regarding the origin of this observed epigenetic dysregulation. Epidemiological studies suggest that the underlying disease state and severity of infertility plays a significant role. Imprinting disorders frequently arise from DNA methylation errors at imprinting control regions (ICRs), leading to our investigation of the euploid blastocyst methylome in association with duration of infertility (time to pregnancy; TTP). Research study. Surplus cryopreserved euploid blastocysts of transferrable quality (grade ≥3BB; n=104) were donated with IRB approval and patient consent. Blastocysts were grouped based on TTP; months of reported primary infertility prior to oocyte retrieval that resulted in a live birth (Fertile Control: 0 months, donor oocyte/donor sperm; Infertile Short: 12-24 months; Infertile Intermediate: 36-48 months; Infertile Long: ≥60 months). Patient demographics and total gonadotropin doses were comparable between groups. Individual blastocyst DNA underwent whole genome bisulfite sequencing (Methyl-MaxiSeq; Zymo Research) with significant methylated CpGs determined by Student’s t-test adjusted using Benjamini-Hochberg FDR; q<0.05. Methylation validation of ICRs was performed by pyrosequencing (PyroMark Q24 Advanced; Qiagen) with significance determined by Student’s t-test and one-way ANOVA where appropriate; p<0.05. Quantitative RT-PCR was utilized on individual blastocysts for gene expression analysis with REST 2009 Software (Qiagen) and significance determined by a pairwise fixed reallocation randomization test; p<0.05. Prolonged TTP (Infertile Long ≥60 months) resulted in 6,609 significantly altered CpGs compared with blastocysts from Fertile Controls (0 months; q<0.05). Significant CpGs were situated at five ICRs, including KvDMR and MEST, and 36 imprinted genes, with NFAT signaling identified as the top canonical pathway (62/221 genes, q=1.28E-08). Overall, extended TTP ≥36 months (mean=65 months) exhibited significant hypomethylation compared to shorter TTP ≤24 months (mean=10 months) for KvDMR (39% Extended vs. 48% Shorter; p<0.05), MEST (40% Extended vs. 49% Shorter; p<0.05), and H19 (29% Extended vs. 41% Shorter; p<0.05) while SNRPN trended downward without significance. Significant decreases in expression were observed for maternally imprinted genes (KCNQ1OT1, MEST, COPG2, DNMT1; p<0.05), and significant increases in expression for paternally imprinted genes (H19, CDKN1C; p<0.05). This novel study is the first to report evidence that an extended duration of infertility correlates with altered methylation in euploid blastocysts, with a particular emphasis on genomic imprinting. Our results provide evidence to support an association between imprinting dysregulation and infertility as a prolonged disease.