Rocío G Urdinguio1, Gustavo F Bayón1, Marija Dmitrijeva1, Estela G Toraño1, Cristina Bravo1, Mario F Fraga2, Lluís Bassas3, Sara Larriba4, Agustín F Fernández5. 1. Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Oviedo 33006, Spain. 2. Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Oviedo 33006, Spain Department of Immunology and Oncology, National Center for Biotechnology, CNB-CSIC, Cantoblanco, Madrid 28049, Spain. 3. Laboratory of Seminology and Embryology, Andrology Service-Fundació Puigvert, Barcelona 08025, Spain. 4. Human Molecular Genetics Group-IDIBELL, L'Hospitalet de Llobregat, Barcelona 08908, Spain affernandez@hca.es slarriba@idibell.cat. 5. Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Oviedo 33006, Spain affernandez@hca.es slarriba@idibell.cat.
Abstract
STUDY QUESTION: Are there DNA methylation alterations in sperm that could explain the reduced biological fertility of male partners from couples with unexplained infertility? SUMMARY ANSWER: DNA methylation patterns, not only at specific loci but also at Alu Yb8 repetitive sequences, are altered in infertile individuals compared with fertile controls. WHAT IS KNOWN ALREADY: Aberrant DNA methylation of sperm has been associated with human male infertility in patients demonstrating either deficiencies in the process of spermatogenesis or low semen quality. STUDY DESIGN, SIZE, DURATION: Case and control prospective study. This study compares 46 sperm samples obtained from 17 normospermic fertile men and 29 normospermic infertile patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Illumina Infinium HD Human Methylation 450K arrays were used to identify genomic regions showing differences in sperm DNA methylation patterns between five fertile and seven infertile individuals. Additionally, global DNA methylation of sperm was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek) in 14 samples, and DNA methylation at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4) measured by bisulfite pyrosequencing in 44 sperm samples. A sperm-specific DNA methylation pattern was obtained by comparing the sperm methylomes with the DNA methylomes of differentiated somatic cells using data obtained from methylation arrays (Illumina 450 K) of blood, neural and glial cells deposited in public databases. MAIN RESULTS AND THE ROLE OF CHANCE: In this study we conduct, for the first time, a genome-wide study to identify alterations of sperm DNA methylation in individuals with unexplained infertility that may account for the differences in their biological fertility compared with fertile individuals. We have identified 2752 CpGs showing aberrant DNA methylation patterns, and more importantly, these differentially methylated CpGs were significantly associated with CpG sites which are specifically methylated in sperm when compared with somatic cells. We also found statistically significant (P < 0.001) associations between DNA hypomethylation and regions corresponding to those which, in somatic cells, are enriched in the repressive histone mark H3K9me3, and between DNA hypermethylation and regions enriched in H3K4me1 and CTCF, suggesting that the relationship between chromatin context and aberrant DNA methylation of sperm in infertile men could be locus-dependent. Finally, we also show that DNA methylation patterns, not only at specific loci but also at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4), were lower in sperm than in somatic cells. Interestingly, sperm samples at Alu Yb8 repetitive sequences of infertile patients showed significantly lower DNA methylation levels than controls. LIMITATIONS, REASONS FOR CAUTION: Our results are descriptive and further studies would be needed to elucidate the functional effects of aberrant DNA methylation on male fertility. WIDER IMPLICATIONS OF THE FINDINGS: Overall, our data suggest that aberrant sperm DNA methylation might contribute to fertility impairment in couples with unexplained infertility and they provide a promising basis for future research. STUDY FUNDING/COMPETING INTERESTS: This work has been financially supported by Fundación Cientifica de la AECC (to R.G.U.); IUOPA (to G.F.B.); FICYT (to E.G.T.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); Fundación Ramón Areces (to M.F.F); the Plan Nacional de I+D+I 2008-2011/2013-2016/FEDER (PI11/01728 to AF.F., PI12/01080 to M.F.F. and PI12/00361 to S.L.); the PN de I+D+I 2008-20011 and the Generalitat de Catalunya (2009SGR01490). A.F.F. is sponsored by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (CP11/00131). S.L. is sponsored by the Researchers Stabilization Program from the Spanish National Health System (CES09/020). The IUOPA is supported by the Obra Social Cajastur, Spain.
STUDY QUESTION: Are there DNA methylation alterations in sperm that could explain the reduced biological fertility of male partners from couples with unexplained infertility? SUMMARY ANSWER: DNA methylation patterns, not only at specific loci but also at Alu Yb8 repetitive sequences, are altered in infertile individuals compared with fertile controls. WHAT IS KNOWN ALREADY: Aberrant DNA methylation of sperm has been associated with human male infertility in patients demonstrating either deficiencies in the process of spermatogenesis or low semen quality. STUDY DESIGN, SIZE, DURATION: Case and control prospective study. This study compares 46 sperm samples obtained from 17 normospermic fertile men and 29 normospermic infertilepatients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Illumina Infinium HDHuman Methylation 450K arrays were used to identify genomic regions showing differences in sperm DNA methylation patterns between five fertile and seven infertile individuals. Additionally, global DNA methylation of sperm was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek) in 14 samples, and DNA methylation at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4) measured by bisulfite pyrosequencing in 44 sperm samples. A sperm-specific DNA methylation pattern was obtained by comparing the sperm methylomes with the DNA methylomes of differentiated somatic cells using data obtained from methylation arrays (Illumina 450 K) of blood, neural and glial cells deposited in public databases. MAIN RESULTS AND THE ROLE OF CHANCE: In this study we conduct, for the first time, a genome-wide study to identify alterations of sperm DNA methylation in individuals with unexplained infertility that may account for the differences in their biological fertility compared with fertile individuals. We have identified 2752 CpGs showing aberrant DNA methylation patterns, and more importantly, these differentially methylated CpGs were significantly associated with CpG sites which are specifically methylated in sperm when compared with somatic cells. We also found statistically significant (P < 0.001) associations between DNA hypomethylation and regions corresponding to those which, in somatic cells, are enriched in the repressive histone mark H3K9me3, and between DNA hypermethylation and regions enriched in H3K4me1 and CTCF, suggesting that the relationship between chromatin context and aberrant DNA methylation of sperm in infertilemen could be locus-dependent. Finally, we also show that DNA methylation patterns, not only at specific loci but also at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4), were lower in sperm than in somatic cells. Interestingly, sperm samples at Alu Yb8 repetitive sequences of infertilepatients showed significantly lower DNA methylation levels than controls. LIMITATIONS, REASONS FOR CAUTION: Our results are descriptive and further studies would be needed to elucidate the functional effects of aberrant DNA methylation on male fertility. WIDER IMPLICATIONS OF THE FINDINGS: Overall, our data suggest that aberrant sperm DNA methylation might contribute to fertility impairment in couples with unexplained infertility and they provide a promising basis for future research. STUDY FUNDING/COMPETING INTERESTS: This work has been financially supported by Fundación Cientifica de la AECC (to R.G.U.); IUOPA (to G.F.B.); FICYT (to E.G.T.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); Fundación Ramón Areces (to M.F.F); the Plan Nacional de I+D+I 2008-2011/2013-2016/FEDER (PI11/01728 to AF.F., PI12/01080 to M.F.F. and PI12/00361 to S.L.); the PN de I+D+I 2008-20011 and the Generalitat de Catalunya (2009SGR01490). A.F.F. is sponsored by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (CP11/00131). S.L. is sponsored by the Researchers Stabilization Program from the Spanish National Health System (CES09/020). The IUOPA is supported by the Obra Social Cajastur, Spain.