This study designed as a double blind randomized controlled clinical trial that approved with Ethics Committee of the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. (Code of Ethics Committee: IR.AJUMS.REC.1396.621). A total of 52 infertile men were recruited who followed infertility treatment at department of urology, Imam Khomeini hospital, Ahvaz Jundishapur University of Medical Sciences between April 2018 and March 2019. This investigation was registered by the identification code of IRCT20141025019669N7 in clinical trials registry of Iran. Despite the approval of the research project and its registration on the IRCT website, the study began shortly after the date due to the late preparation of supplements. This work was financially supported by supported by a Grant (Number: NRC-9618) from Vice-Chancellor for Research Affairs of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Inclusion and exclusion criteria
The Inclusion criteria were: willing to cooperate, age range 20–45, oligosthenoterozospermia disease of unknown origin (idiopathic), normal levels of gonadotropins, testosterone and serum prolactin, and oligospermia 5–20 million per ml sperm count, asthenospermia with mobility less than 50% (idiopathic). Patients were excluded from the study if any of the following conditions existed: There was a known cause of infertility (such as hormonal disorders, epididymal duct obstruction, and epididymoorchitis), drugs, alcohol consumption, diabetes, kidney disease (creatinine more than doubled), chronic liver disease (more than twice the normal transaminase), varicocele, infectious diseases with fever and leukocytosis characterized by chromosomal abnormalities, debilitating diseases sperm and sexual system such as varicocele, drugs that stimulate sexual system or interfere with sex hormones, patients undergoing ICSI due to sperm quality severe impairment and the presence of other causes of infertility, contact pesticides, heavy metals and solvents, taking antioxidant supplements in the past three months and a body mass index (BMI) of 30 kg/m2 or greater.
At the beginning of the study, patients were randomized to group 1 who received 500 mg probiotic capsules daily and group 2 who took placebo for 10 weeks. The capsules used in this study were prepared by the Zist Takhmir Company, and the placebo capsules were completely similar to the probiotic appearance, using only Maltodextrin inside them. Each eligible patient received a randomization number, and were randomly divided into two groups of supplementation and placebo (n = 26). The investigator and patients were blinded to the treatment condition. The combination of probiotic capsules include: Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Bifidobacterium breve, Bifidobacterium longum, Streptococcus thermophiles, TVC: 2 * 1011 CFU. Participants were interviewed face to face by trained professional nutritionists. All participants were asked to avoid using probiotic yogurt for the duration of the study due to the presence of lactobacilli and any supplements to prevent inaccurate evaluation of the factors studied. In order to evaluate the diet of patients, at the beginning and the end of the study, 24-hour recall questionnaire, non-consecutive three-day, be completed through face-to-face interviews and telephone calls. The analysis of this questionnaire was done using Nutritionist IV (N4) nutritional software. Also, to evaluate the physical activity, we used the International Physical Activity Questionnaire (IPAQ). Data from the IPAQ were converted to metabolic equivalent-minutes/week using existing guidelines.
Sample size calculation
To determine the sample size we used the volume of the ejaculate (ml), before and after the probiotic with a prebiotic supplementation in C. Maretti study. Thus, with a sample size of 19 people in each group with probability 95% and a 99% confidence level can be ruled out to assume that the probiotic effect is equal before and after the study. Considering the drop-in participants during the study, 26 people were considered for each group.
N= [(Z1-α/2 + Z1-β) 2 (SD12 + SD22)] /∆2
Z1-α/2 = 2.58, Z1-β = 1.64
N = 19
Preparation of samples
After 3 days of sexual abstinence, semen samples were taken at urology unit of Imam Khomeini hospital, Ahvaz Jundishapur University of Medical Sciences (Ahvaz, Iran). All semen was carried at 37 °C to liquefy. The samples were analyzed according to the World Health Organization. Remnants of liquefied semen samples were immediately centrifuged at 300 × g for 10 min. Also, venous blood samples and centrifuged at 3,000 × g at 4 °C for 10 minutes and serum was aspirated out for hormone assays. The serum was stored at -80 C until further assays.
Assessment of Sperm Motility
Motility assessment of sperms was performed according to WHO criteria. Sperms were scored for motility evaluation expressed as grades a to d and progressive motility rate was calculated as the percentage of (a + b).
Turbidimetric immunoassay was used for measuring of C-reactive protein (CRP) levels (BioSystems Co, Barcelona, Spain). Also, enzyme-linked immunosorbent assay (ELISA) (DIAsource Co, Belgium) was used for determining serum levels of tumor necrosis factor a (TNF-α). Serum and seminal malondialdehyde (MDA) levels were measured by tiobarbituric acid method. Colorimetric method was used for analyzing serum and seminal total antioxidant capacity (TAC) (Randox Laboratories Ltd, UK). This method has completely been explained by Khosrowbeygi et al.
Reproductive hormones assay
Serum testosterone and prolactin were assayed using commercial radioimmunoassay kits. These commercial kits had been previously used with an inter-assay and intra-assay variation of less than 10%. The reference range for testosterone and PRL is 10 to 35 nmol/l and 92 to 697 pmol/l, respectively. Luteinizing hormone (LH) was measured by immunochemiluminometric assay, in which intra-assay and interassay coefficients of variation were 3.4% and 3.8%, respectively. The normal LH range is 1.5 to 9.3 IU/l. Follicle-stimulating hormone (FSH) was also measured using immunochemiluminometric assay with an intra-assay and interassay coefficient of variation of 3.2% and 6.7%, respectively. The normal FSH range is 1.4 to18.1 IU/l.
All data were presented as mean ± SD. The distribution of the data was evaluated by the Kolmogorov–Smirnov test. Due to normal distribution of variables, the independent sample t-test and the paired sample t-test were applied to analyses differences in variables between and within groups, respectively. Statistical computations were calculated using SPSS 16 for windows software (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.