Semen samples were collected by masturbation after 2–5 days of sexual abstinence. Semen analysis was assessed in fresh semen samples according the 2010 World Health Organization’s criteria19 . After the semen analysis, semen samples were frozen following a well-stablished slow freeze protocol20 (link). Briefly, semen samples were mixed in a 1:1 ratio with test yolk buffer (Irvine Scientific, CA, USA) and placed in liquid nitrogen vapors and finally maintained in liquid nitrogen until further analysis.
After thawing the sperm samples a sperm purification protocol that included stringent somatic cell lysis was performed as described previously21 (link). This purification was performed by incubating sperm samples with 0.1% SDS and 0.5% Triton X-100 (in Milli-Q® water), on ice followed by two high volume wash steps and a final optical microscopic examination to verify the somatic cell elimination. All samples were also epigenetically screened (DLK1 locus) to ensure no somatic cell contamination according to a previously published method of our group14 (link).
Total sperm DNA was isolated using a sperm-specific modification to the Qiagen DNeasy (QIAGEN, CA, USA) manufacturer protocol5 (link), and DNA concentration and purity were determined using a Nanodrop-1000 spectrophotometer (Thermo Fisher Scientific, MA, USA).
After thawing the sperm samples a sperm purification protocol that included stringent somatic cell lysis was performed as described previously21 (link). This purification was performed by incubating sperm samples with 0.1% SDS and 0.5% Triton X-100 (in Milli-Q® water), on ice followed by two high volume wash steps and a final optical microscopic examination to verify the somatic cell elimination. All samples were also epigenetically screened (DLK1 locus) to ensure no somatic cell contamination according to a previously published method of our group14 (link).
Total sperm DNA was isolated using a sperm-specific modification to the Qiagen DNeasy (QIAGEN, CA, USA) manufacturer protocol5 (link), and DNA concentration and purity were determined using a Nanodrop-1000 spectrophotometer (Thermo Fisher Scientific, MA, USA).
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