Subjects
In this study, all the participants were recruited during July 2020 to September 2020 from the outpatient department of reproductive center in the International Peace Maternity & Child Health Hospital (IPMCH), Shanghai Jiao Tong University School of Medicine. Males were included when they met the criteria: 1). 20-50 years of age; 2). without AZF gene microdeletions; 3). without mycoplasma or chlamydia infections; 4). without medication taken within three months. Semen samples were collected by masturbation after 3-7 days of sexual abstinence. Written consent forms were obtained from all participants. This study was approved by the IPMCH Ethics Review Committee and performed according to the Declaration of Helsinki.
Semen analysis and sperm chromatin structure assay
After liquefaction, sperm parameters, including sperm morphology, concentration, and motility, were assessed by the computer assisted sperm analysis (CASA) system according to the World Health Organization (WHO) laboratory manual[16]. Sperm DFI was determined by sperm chromatin structure assay[8]. In detail, sperm samples were diluted to a concentration of 2 x 106/ml with TNE buffer (0.01 M Tris–HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA) firstly. Then 100 µl diluted sperm suspension was mixed with 200 µl acid-detergent solution (pH 1.2, 0.08 N HCl, 0.15 M NaCl, 0.1% Triton X-100) and incubated for 30 s on ice. After adding 600 µl acridine orange solution into the sample, it was incubated for 3 min, and tested by NovoCyte Flow Cytometer (Agilent, California, USA).
Sperm DNA extraction
For sperm DNA isolation, samples were washed three times with 1 x PBS, centrifuged at 200g for 5 min. Somatic cells were eliminated with 0.1 % sodium dodecyl sulfate (3250GR500, BioFroxx) and 0.5 % TritonTM-X100 (X100, Sigma) in DEPC-treated water (AM9920, Invitrogen) at 4℃ for 15 min. Then the spermatozoa were homogenized with 1mm beads in ALT buffer (69504, Qiagen) containing 10 mg/ml Proteinase K and 150 mM DL-Dithiothreitol (A100281, Sangon Biotech, China). Total DNA was extracted using the DNeasy Blood & Tissue Kit (69504, Qiagen) following the manufacturer's instructions.
Quantification of mtDNA copy number
The mtDNA-CN was measured by a quantitative polymerase chain reaction (qPCR) method using a QuantStudioTM 7 Flex real-time PCR machine (4485701, Applied Biosystems)[17]. Briefly, the primers of the Taqman assay were designed in a stable segment in the minor arc of mtDNA (mtMinArc) and RNAse P (4403326, ThermoFisher, USA) was chosen as the genomic DNA reference. Detailed primer sequence was shown in Supplementary Table S1. Real-time PCR with three technical replicates were performed as previously described[17]. The mtDNA-CN was calculated using the following formulae: mtDNA-CN = 2△CT (mtDNA-CN), where △CT (mtDNA-CN) = CTRNase P − CTmtMinArc.
Determination of sperm ROS levels
Sperm ROS content was measured by Reactive Oxygen Species Assay Kit (S0033M, Beyotime Biotech, Shanghai, China) following the manufacturer's instructions. Collected sperm samples were washed three times with 1 x PBS (200g, 5min) and then incubated with 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μmol/L) at 37℃ for 20 mins. Then the samples were washed three times with 1 x PBS (200g, 5min), and the fluorescent signals of DCFH-DA oxidized products (2',7'-dichlorofluorescein, DCF) were detected using SynergyTM H1 Microplate Reader (BioTek Instruments, Inc., Winooski, USA) under 488 nm excitation. To normalize the ROS level, ROS per million sperms (ROS/MS) was represented the average ROS content in each seminal sample.
Outcome assessment
In terms of ART, only those IVF/ICSI cycles performed within three months after the semen analysis were included. The embryonic outcomes included fertilization rate, cleavage rate and top-quality embryo rate. The fertilization rate was the percentage of fertilized embryos in retrieved oocytes. The cleavage rate was the percentage of 2-cell embryos in all fertilized embryos. The cleaving embryos on Day 3 was classified into Grade 1 to 5 according to the numbers and sizes of blastomeres and the percentage of cytoplasmic fragments[18]. The top-quality embryo rate was the percentage of embryo evaluated as Grade 1 or 2 in all fertilized embryos. When embryos were transferred, pregnancy outcomes were observed in every transfer cycle. Clinical pregnancy was defined as the presence of an intrauterine gestational sac by ultrasound examination at a gestational age of 7 weeks.
Statistical analysis
Characteristics of participants were summarized. Continuous variables were expressed using the median (inter-quartile range) and compared with the Mann-Whitney U test. Categorical variables were described with percentages and compared using the Chi-square test. The correlations between male age, mtDNA-CN, ROS/MS, DFI and sperm parameters were analyzed with Spearman's rank correlation. To further investigate their relationships, seminal quality was categorized as normal or abnormal sperm group according to sperm parameters and analyzed with a binary logistic regression. The sample that at least one parameter among sperm concentration, motility and morphology lowered than the criteria was categorized as abnormal sperm group. The associations between mtDNA-CN, ROS/MS, DFI and embryonic outcomes were analyzed with linear regression, respectively. The generalized linear model (GLM) was performed to adjust the covariates, including male age, female age, seminal quality, and ART strategy. Pregnancy outcomes were analyzed with generalized estimating equations (GEE) to address the correlation of different transfer cycles in the same patient. Variates in the model were yielded with odds ratios (OR) and 95% confidence intervals (CIs). All statistical analyses were conducted with SPSS statistics 24 (IBM, Chicago, USA).