Subjects
One hundred and forty-two cases of male infertile patients who had visited the reproduction center of the International University of Health and Welfare Hospital (IUHW) from August 2012 to March 2013 were included in this study. All infertile couples had undergone treatments at IUHW. The policy of infertility treatment at IUHW, according to the semen condition of the male partner, is as follows: for normal findings or in the case of mild oligozoospermia and /or asthenozoospermia, natural pregnancy or intra-uterine insemination (IUI) using the timing method are considered as the first choice, followed by a “step-up” to IVF/ICSI when pregnancy is not achieved; for severe oligozoospermia and/or asthenozoospermia, IVF/ICSI is conducted from the beginning. Furthermore, partners of patients with psychogenic erectile dysfunction or intravaginal ejaculation disorder are initially treated with IUI.
As part of this study, a physical examination and evaluation of male infertility was performed for each subject. During the period of follow-up until the end of 2014, we sent questionnaires to all male infertile patients to obtain data of pregnancy outcomes and collected the answers from 96 of them, which were accompanied by semen analysis data and SEMG+ measurements taken at the first medical examination. Of these 96 partners of patients, 36 were confirmed to achieve pregnancy by any means of IUI, IVF, ICSI or spontaneous conception (“Pregnancy group”), while remaining 60 partners of patients kept unpregnant by the end of the follow-up period (“Non-pregnancy group”). Thirteen normal healthy male subjects whose wives were in the twentieth week or later of spontaneous pregnancy and who received prenatal examinations in the hospital were recruited to participate as “control” subjects in this study after it was confirmed that they did not have any types of intrascrotum abnormality, including varicocele. Informed consent was obtained from all participants for the use of the data obtained from a physical examination, semen, and blood samples. The study protocol was approved by the Institutional Review Board at IUHW and at Toin University of Yokohama in Japan.
Semen analysis and hormone measurements
Semen samples were obtained via masturbation following sexual abstinence for at least 48 h. Specimens were allowed to liquefy for up to 1 h after ejaculation at room temperature. Complete liquefaction was certified macroscopically according to the WHO manual by confirming that the semen becomes homogeneous and quite watery, and only small areas of coagulation remain. Manual semen analysis was performed according to the WHO manual to determine semen volume and sperm concentration. For analysis of sperm motility, the SMAS™ (Ditect, Tokyo, Japan) system was used. Additionally, the serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) were measured using a chemiluminescent immunoassay (SRL Inc., Tokyo, Japan). The reference ranges for LH, FSH, and T were 0.79–5.72 mIU/ml, 2.00–8.30 mIU/ml, and 1.31–8.71 ng/ml, respectively. The intra-assay coefficient of variation (CV) for LH, FSH, and T were 3.03%, 3.74%, and 5.13%, respectively. The inter-assay CV for LH, FSH, and T were 1.84%, 0.43%, and 3.99%, respectively.
Indirect immunofluorescence assay
Following semen analysis, semen samples were refrigerated until use. Pretreatment and staining methods were the same as described in the previous study [9]. Briefly, semen samples were layered onto 65% Percoll/HEPES-buffered saline (HBS; 130 mM NaCl, 4 mM KCl, 1 mM, CaCl2, 0.5 mM MgCl2, 14 mM fructose and 10 mM HEPES, pH 8.0) and centrifuged at 1200 g for 30 min at 25 °C. The resulting pellet was fixed with 2% paraformaldehyde phosphate buffer, pH 7.4. The fixed sperm were then washed twice with HBS, mixed with 25% block-Ace (Dainippon Sumitomo Pharma, Osaka, Japan) and then incubated at 37 °C for 1 h, then washed with HBS at 3300 g for 10 min at 25 °C. An aliquot of the washed sperm was incubated with 1 μg/mL anti-seminal plasma motility inhibitor (SPMI) mouse immunoglobulin G (IgG) that recognized a part of the SPMI region (138–154: GTQNPSQDSGNSPSGKG) of SEMG (monoclonal antibody, previously described as anti-Sg antibody F11) or mouse IgG isotype control (DAKO Japan, Tokyo, Japan) at 37 °C for 1 h [10]. Sperm were then washed twice with HBS at 3300 g for 10 min at 25 °C. Washed sperm were incubated with Alexa 488-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR, USA) at 37 °C for 1 h. The samples were then analyzed using a flow cytometer.
Flow cytometric analysis
Flow cytometric analysis was performed as described in the previous study [9]. Briefly, samples were analyzed with flow cytometry using a Gallios™ flow cytometer (Beckman Coulter, Tokyo, Japan) equipped with standard optics. For each cell, forward light scatter, orthogonal light scatter, and Alexa 488 (FL1) were evaluated using the Kaluza® software (Beckman Coulter, Tokyo, Japan). The sperm population of each sample was identified using the side scatter (SSC) and FL1 fluorescence intensities. Debris was gated out by establishing a region around the population of interest, based on FL1/ SSC 2-dimensional histogram. Ten thousand spermatozoa per sample were analyzed. The spermatozoa were labeled with anti-SPMI mouse IgG (SEMG-positive spermatozoa) and positive and negative populations were determined by comparing the population of control staining using mouse IgG isotype control, instead of the antibody (Additional file 1: Figure S1). The proportion of SEMG-positive spermatozoa (SEMG+) was then calculated by dividing the number of labeled spermatozoa by the number of spermatozoa analyzed, and the proportion of SEMG-negative spermatozoa (SEMG−) was determined by subtracting the percentage of SEMG+ from 100. Total SEMG- count was calculated using the proportion of SEMG- and total sperm count.
Statistical analysis
Statistical analyses comparing mean values of each parameter for patients and control subjects were performed with JMP software (version 10.0.0, SAS Institute Inc., Cary, NC, USA) using Wilcoxon rank sum test. Additionally, the Spearman’s rank correlation coefficient was applied between the results for the proportion of Sg + and the results of standard semen analysis. Statistical analyses comparing differences in the continuous variables were performed with the “R” statistical software system (www.cran.r-project.org) [11] using analysis of variance, followed by Dunnett’s test using the “multcomp” package [12].