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Array-based DNA methylation profiling reveals peripheral blood differential methylation in male infertility

      Objective

      To study peripheral blood DNA differential methylation in oligozoospermic infertile men in comparison with normozoospermic fertile controls.

      Design

      Case-control study.

      Setting

      Reproductive biology laboratory.

      Patients(s)

      Azoospermic and oligozoospermic infertile patients (n = 6) and normozoospermic fertile controls (n = 6) in the discovery phase, and oligo/asthenozoospermic infertile men (n = 11) and normozoospermic fertile controls (n = 10) in the validation phase.

      Intervention(s)

      Blood samples drawn from all participants, DNA isolation and methylation analysis.

      Main Outcome Measure(s)

      DNA methylation values analyzed using genomewide methylation 450K BeadChip array, followed by deep sequencing of selected regions for methylation analysis in the neighborhood regions of differentially methylated CpGs.

      Result(s)

      We found 329 differentially methylated CpG spots, out of which 245 referred to the genes, representing 170 genes. Deep-sequencing analysis confirmed the methylation pattern suggested by 450K array. A thorough literature search suggested that 38 genes play roles in spermatogenesis (PDHA2, PARP12, FHIT, RPTOR, GSTM1, GSTM5, MAGI2, BCAN, DDB2, KDM4C, AGPAT3, CAMTA1, CCR6, CUX1, DNAH17, ELMO1, FNDC3B, GNRHR, HDAC4, IRS2, LIF, SMAD3, SOD3, TALDO1, TRIM27, GAA, PAX8, RNF39, HLA-C, HLA-DRB6), are testis enriched (NFATC1, NMNAT3, PIAS2, SRPK2, WDR36, WWP2), or show methylation differences between infertile cases and controls (PTPRN2, RPH3AL).

      Conclusion(s)

      We found a statistically significant correlation between peripheral blood DNA methylation and male infertility, raising the hope that epigenome-based blood markers can be used for screening male infertility risk. The study also identified new candidates for spermatogenesis and fertility.
      El análisis de metilación mediante arrays en sangre periférica revela perfiles de metilación diferencial en infertilidad masculina

      Objetivo

      Estudiar la metilación diferencial de ADN en sangre periférica en pacientes infértiles oligozoospérmicos en comparación con pacientes control normozoospérmicos fértiles.

      Diseño

      Estudio de casos y controles.

      Lugar

      Laboratorio de biología reproductiva.

      Pacientes(s

      ): Pacientes azoospérmicos y oligozoospérmicos infértiles (n= 6) y controles fértiles normozoospérmicos (n= 6) para la fase de investigación, y pacientes oligo/astenozoospérmicos (n=11) y normozoospérmicos controles fértiles (n=10) para la fase de validación.

      Intervención(es)

      Muestras de sangre tomadas de todos los participantes, aislamiento de ADN y análisis de metilación.

      Medida(s) principal(es) de resultado

      Los valores de metilación del ADN se analizaron mediante el uso del arrays de metilación BeadCheap 450K, seguido de secuenciación especifica de regiones seleccionadas para el análisis de metilación en las regiones vecinas a las regiones de CpGs diferencialmente metiladas.

      Resultado(s)

      Se encontraron 329 regiones de CpG diferentemente metiladas, de las cuales 245 se referían a genes, lo que representa 170 genes. Los análisis de secuenciación específica confirmaron los perfiles de metilación obtenidos por el arrays de 450K. Una búsqueda bibliográfica exhaustiva sugirió que 38 de los genes juegan un papel en la espermatogénesis (PDHA2, PARP12, FHIT, RPTOR, GSTM1, GSTM5, MAGI2, BCAN, DDB2, KDM4C, AGPAT3, CAMTA1, CCR6, CUX1, DNAH17, ELMO1, FNDC3B, GNRHR, HDAC4, IRS2, LIF, SMAD3, SOD3, TALDO1, TRIM27, GAA, PAX8, RNF39, HLA-C, HLA-DRB6), están presentes en el testículo (NFATC1,NMNAT3, PIAS2, SRPK2,WDR36,WWP2), o muestran diferencias de metilación entre los controles infértiles y fértiles.

      Conclusión(es)

      Se encontró una correlación estadísticamente significativa entre la metilación del ADN de la sangre periférica y la infertilidad masculina, lo que realza la esperanza de que los marcadores sanguíneos basados en el epigenoma puedan utilizarse para detectar el riesgo de infertilidad masculina. El estudio también identificó nuevos candidatos para la espermatogénesis y la fertilidad.

      Key Words

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