Thirty-three male partners participating IVF treatment during 2012 were recruited in this prospective study. We included the men who showed a total motile sperm count (TMC) ≥5 million for securing adequate amount of sperm to perform the assay. There were no exclusion criteria in the etiology of female infertility. All men were normozoospermic, and the conventional sperm parameters were presented in Table 1. An informed consent was obtained from all of the subjects, and the use of human semen for this study was approved by the Institutional Review Board of the Seoul National University Bundang Hospital (B-1112/141-005). All the male subjects had no history of genital inflammation or surgery. No subjective symptoms or self-reported medical risk factors were identified. Nobody had taken prescribed medications. Nineteen male partners were identified as smoker by self-report and/or urine cotinine measurement.

After avoiding coitus for at least 3 days, semen samples were obtained by masturbation at the time of oocyte pick-up. After liquefaction for 30 min at room temperature (RT), routine sperm quality was assessed by using computer-assisted semen analysis (SAIS-PLUS 10.1, Medical Supply Co., Seoul, Korea) and classified according to standard procedures of the World Health Organization (WHO, 2010). None had significant numbers of round cells or leukocytospermia as per WHO guidelines (<1 million/mL). Sperm morphology was evaluated according to the strict criteria proposed by Kruger/Tygerberg and adopted by the WHO. The semen was processed by conventional swim-up method and utilized for fertilization. The remaining sperm were used for the following experiments.

Nuclear DNA integrity was measured by the TUNEL assay as described before.25 The samples were smeared onto a silane-coated slide (DAKO, Glostrup, Denmark) and air-dried. Sperm were fixed with 4% neutral buffered formalin for 1 hr at 15-25℃, and then washed with phosphate buffered saline (PBS). Sperm were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (Sigma, St. Louis, MO, USA). A commercial apoptosis detection kit (In Situ Cell Death Detection Kit; Roche Diagnostics GmbH, Mannheim, Germany) was used. The remaining procedures were performed as directed by the instructions in the kits. Counterstaining was performed using a mounting medium with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). The sperm with fragmented DNA had their nuclei stained in green, whereas the nuclei of the other cells were blue. Sperm heads with >50% of the area stained green were considered positive. At least 500 sperm were counted per experimental set and the percentage of sperm with fragmented DNA was determined as DNA fragmentation index (DFI).

Semen was washed with PBS three times and smears were prepared on glass slides. The smears were fixed in 4% paraformaldehyde for 20 min and air-dried. Smears were rehydrated with PBS twice and decondensed in 5 mM dithiotreitol and 0.3 µg/mL heparin for 30-60 min. Smears were permeabilized with PBS containing 0.5% Triton X-100 (PBS-T) for 10 min at RT and then washed twice with PBS. Smears were blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at RT. After washing twice with PBS, the smears were incubated with the primary antibodies (1:100) for 2 hrs at RT. All of six primary antibodies (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac) were rabbit polyclonal antibodies purchased from Abcam (Cambridge, UK). Smears were then washed three times with PBS and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (1:100) for 1.5 hours at RT (Invitrogen, Carlsbad, CA, USA). After washing three times with PBS, smears were mounted with DAPI and observed under a Carl Zeiss Axiophot microscope (exciter filter BP450-490, emission filter BP520) at ×400 and ×1000 magnification. All sperm were stained with either 'weak' or 'strong' signal intensity (Fig. 1). We considered the strong signal as hypercetylated state. The signal intensity was judged by an independent investigator. On each slide, at least 400 sperm were counted, and the percentage of sperm exhibiting strong intensity was determined. Negative controls were performed in the absence of primary antibody.

A proportion of processed sperm was vitrified for the measurement of total HAT/HDAC activity. We performed a preliminary western blot assay from 5 men to verify the expression of HAT/HDAC in mature human sperm. For investigating the HAT/HDAC, polyclonal rabbit anti-HAT antibody (1:2000, orb128159; Biorbyt Ltd., Cambridge, UK) and polyclonal rabbit anti-HDAC1 antibody (1:1000, #2062; Cell Signaling Technology, Danvers, MA, USA) were used. A 50 kDa of HAT band and a 62 kDa of HDAC1 band were clearly identified in the mature human sperm lysate (Fig. 2). From the warmed semen samples, nuclear extracts were obtained using the Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. Pellets were re-suspended in 50 µL of Complete Lysis Buffer and centrifuged at 14000×g for 10 min at 4℃; supernatants were saved as the nuclear fractions. The protein concentration of each sample was measured (Bradford Bio-Rad Protein Assay kit, Bio Rad, San Diego, CA, USA), with BSA as a standard.

Total HAT activity was measured using a commercially available kit (Histone Acetyltransferase Activity Assay Kit, ab65352, Abcam) according to the detailed instructions provided by the manufacturer. Briefly, 30 µg of nuclear extracts was incubated with HAT substrates I/II and NADH-generating enzyme in HAT assay buffer for 4 hrs at 37℃. Absorbance was determined at 450 nm in an ELISA plate reader, with active nuclear extract used as a positive control and standard. The HAT activity was expressed as nmol/min/µg as indicated by the manufacturer. All experiments were performed in duplicate and the mean values were obtained.

Total HDAC activity was measured using the EpiQuik HDAC Activity/Inhibition Assay Kit (Epigentek, New York, USA). In brief, the 10 µg of sperm nuclear protein extract were incubated with a specific substrate for 1 hr at 37℃, followed by incubation with capture antibodies for 60 min and detection antibodies for 30 min at RT. Absorbance was determined using a microplate spectrophotometer at 450 nm. HDAC activity was calculated according to the manufacturer's instructions using the equation: activity (ng/h/mL)=[optical density (OD) (control-blank)-OD (sample-blank)]/slope×reaction time (0.5-1 hr)×sample dilution. All experiments were performed in duplicate and mean values were obtained. We arbitrary determined 'acetylation enzyme index' as HAT activity divided by HDAC activity.

Statistical analysis was performed using statistical software (MedCalc ver. 10.4, MedCalc Software, Mariakerke, Belgium). Data are always expressed as mean±SD. The Spearman correlation test was used to assess an association between different numerical parameters. Intergroup differences were assessed by nonparametric Wilcoxon test. A p-value of <0.05 (two-tailed) was considered statistically significant.