Subjects
From 2018 to 2019 we collected 78 male semen samples from the Reproductive Center of the Fujian Provincial Maternity and Children’s Hospital. This study was approved by the ethics committee of Fujian Provincial Maternity and Children’s Hospital, under number 2019KY105. All donors provided informed consent in accordance with the regulations of the ethics committee. All methods were carried out in accordance with relevant guidelines and national regulations.
All participants were 20 to 35 years old. During each of their physical examinations, no organic lesions were found in reproductive organs such as the testis, epididymis, and prostate. Genital erection and sperm excretion were normal. No varicocele or endocrine disease was present in the participants, and each participant had a normal karyotype and a normal reproductive history. The semen was negative for mycoplasma, chlamydia, and gonorrhea. Other related factors affecting semen quality were excluded (BMI, orchitis, tobacco use, alcohol use, drug use, and poor health habits such as regularly staying up late).
Sample collection
Patients were asked to abstain from sexual activity for 2 to 7 days before semen extraction; after that, the masturbation method was used to obtain semen. The sample was collected in a sterile sperm collection cup and immediately placed in a 37 °C constant temperature incubator for liquefaction. We recorded the semen color, volume, liquefaction time, pH value, viscosity, and other physical and chemical parameters. We also recorded each patient’s fertility history and physical examination findings.
Analysis of macrophages
The samples were stained with Pap staining according to the WHO guidelines,5 and the morphology of the Mφ in the semen was observed with standard macrophages (Cell Bank of Chinese Academy of Sciences). The concentrations of macrophages were detected by flow cytometry (BDCantoII, USA) with CD14 (Biolegend, USA). Three specific test tubes were used: Tube 1 contained upstream sperm as a blank control; tube 2 had a standard macrophage cell line as a positive control; and tube 3 contained a specimen. Following the instructions, we added CD14 antibody fluorescein isothiocyanate (CD14-FITC) in sequence and kept it in a dark room at the appropriate temperature for 20 minutes. Then we added 1000 μl of PBS, shook it well, washed it twice at 1500 r/min, centrifuged it for 5 minutes, shook it well, and tested it on the machine. Next we used the software to make a dot plot of all the collected cells through CD14-FITC, and we used the FITC positive area as gate P1. According to the Solis[7], the samples were divided into a low concentration group (Mφ<6×105/ml) and a high concentration group (Mφ>6×105/ml).
Sperm kinetic analysis
According to the standards of the WHO guidelines[5], sperm kinetic analysis was performed using a Makler counting chamber (Haifa, Israel). The sperm concentration, progressive motility (PR%), non-progressive motility (NP%), and immotility (IM%) were detected, and the results were reviewed by computer automatic semen analyzer.
Sperm morphology analysis
According to the standards of the WHO guidelines[5], the samples were stained by Pap staining (Anhui Anke Biotechnology Co, Ltd, Anhui, China), and at least 200 sperm were observed in each sample. Normal sperm shape rate, head deformity rate, neck and middle segment deformity rate, major segment deformity rate, cytoplasmic residual droplet rate, the sperm malformation index (SDI), the abnormal sperm index (TZI) were evaluated according to the WHO criteria[5]. See Figure 1.
Sperm DFI analysis
The samples were stained by Sperm chromatin diffusion (SCD) staining. According to methods described in the literature[8-9]. we established that the semen had undergone fixation, acid denaturation, lysis, diffusion, dehydration, and staining, which are in keeping with the references in the manual for specific operations (Anhui Anke Biotechnology Co, Ltd, Anhui, China). At least 500 sperm were observed in each sample. A large halo on the sperm head indicated that the sperm DNA was intact, and no or only a small halo indicated that the sperm DNA was incomplete. We then calculated the DNA fragmetation index (DFI) of the sperm (DFI=number of sperm with positive DNA fragmentation rate/total number of observed sperm×100%). See Figure 1.
Sperm cell membrane integrity analysis
The samples were subjected to Eosin-aniline black staining5. We mixed the staining solution (BRED Life Science, Shenzhen, China) and semen for 30 s and observed it under a microscope. At least 200 sperm were observed and the sperm survival rate was calculated. The head turned black or dark red to indicate abnormal sperm; they were colorless or light red to indicate normal sperm (sperm survival rate=number of spermatozoa without damage to the cell membrane/total number of observed sperm×100%). See Figure 1.
Anti-sperm antibody analysis
An mixed antiglobulin response (MAR) reagent was used to detect anti-sperm antibodies on the surfaces of the sperm. Relevant operations were carried out in strict accordance with the instructions (BRED Life Science, Shenzhen, China). Magnetic beads attached to the surfaces of the sperm represented a positive sperm antibody. When the number of immobilized sperm accounted for more than 50% of the total sperm, semen AsAb was positive[5]. as shown in Figure 1.
Cytokines analysis
According to the instructions of the ELISA kit (American Standard Biotechnology Co, Ltd, Jiangsu, China), the semen was centrifuged in a centrifuge (5000g/30min); the seminal plasma was sucked out; samples were added (calibrator, specimen); a wash plate was used; IL-10, IL-12 enzyme conjugate was added; color developing solution and stop solution were used; samples were colorimetric at 450 nm, and the concentration of IL-10 and IL-12 in semen was detected.
Statistical analysis
Statistical analysis of the data was performed by using the SPSS17.0 ( IBM, Armonk, NY, USA). The measurement data were expressed as mean±standard deviation; the means were compared by use of an independent sample t-test; and the counting data were expressed by rate (%) and compared by a χ2 test. The linear relationship between macrophage concentration and sperm quality was drawn by GraphPad Prism 6.07( San Diego, CA, USA), and the linear relationship was expressed as R2. The significance level was 0.05.