Effect of Antioxidant Probiotic Administration on Seminal Quality and Reproductive Outcomes.

This is a prospective randomized, unicentric, intervention study aimed at evaluating the effect of the combination of two probiotics on seminal quality and subsequent reproductive outcomes of infertile patients. As it is a food supplement, we will be subject to the Biomedicine Regulation and not to the Clinical Trials Regulation, as already classified by the Spanish Drug Agency. The study will be conducted on infertile patients at the IVIRMA Valencia clinic for two years to complete planned interventions and data collection and analysis. The reference population includes infertile patients coming to the clinic for IVF/ICSI assisted reproduction treatment with own or donated oocytes.

Patients will come to the clinic to undergo the treatment. If their characteristics are in accord with the inclusion criteria, the study will be presented and explained to them and their partners so they can evaluate their participation in it. If they wish to participate, they will sign an informed consent form, and they will be randomized using a computer-generated randomisation list into the control group (who will receive placebo) or the treatment group (who will receive the probiotic compound).

The success rate in patients with these characteristics, confirmed by the evaluation of historical information from the clinic's own database, is approximately 35% of the cycles using own oocytes, which can be used as a valid approximation. In a unilateral contrast test, assuming a risk of an alpha error of 0.05 and a beta (statistical power) of 80%, to demonstrate a difference of 15% thanks to the intervention, we need a number of 134 cases per arm and, assuming losses of 5% during the follow-up, this number would be rounded to 140 cases per study group, which makes a total of 280 couples to be included in the study.

The trial is double-blind: the patient, the investigator and all site personnel (with the exception of the trial nurse/coordinator and/or pharmacy assistant) will remain blinded throughout the course of the trial as to which treatment each subject received, including the assessing physician, the embryologist and other auxiliary members of the team.

The male assigned to the experimental group will start taking a 50% combination of the two probiotics (Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347) at a dose of 109 CFU/day on the same day of the first visit. The probiotics will be supplied by the company ADM Biopolis S.L. (Valencia, Spain). The carrier of lyophilized probiotics is maltodextrin and the mixture is encapsulated in hypromellose capsules. The patient will take one capsule daily corresponding to the administration of 109 cfu/day. The patient should take these probiotics for at least 21 days before undergoing an assisted reproduction cycle. Patients receiving the placebo will be following the same instructions. For both groups of patients, a seminal sample will be collected on their first visit to evaluate the baseline quality of the semen, as well as a microbiome analysis will be carried out using 16S rRNA sequencing (by ADM Biopolis) to determine the status of the semen at the starting point of the study. After 21 days, the patients will be appointed to deposit a second semen sample in order to perform the IVF/ICSI cycle. This sample will also be analysed, both for semen parameters and microbiome. Values obtained from both of the samples collected will be compared to observe the effect of the administration of the probiotic on the quality of sperm and its microbial population.

Fresh semen samples will be evaluated by means of a spermiogram following the standard operating procedures, in which data will be collected on volume, pH, sperm concentration, sperm mobility, cell viability and morphology. In addition, a DNA fragmentation analysis will be carried out using the Halosperm G2 kit (Halotech) based on the sperm chromatin dispersion technique. A part of the fresh sample will be frozen by submersion into liquid nitrogen and then storage in a -80ºC freezer, and sent to ADM Biopolis to perform the taxonomical identification of the bacterial populations in the semen samples using 16S rRNA sequencing with the Ion Torrent platform (Lifetechnologies).

Once the IVF/ICSI has been performed and oocytes have been correctly fertilised, embryo selection and blastocyst transfer will be carried out according to the usual medical practice to the woman's uterus, so that they can continue their development in the womb.

During recruitment, the couple will also sign a consent form allowing the team to collect data from the results of the cycle, both during embryo development and during the pregnancy and the delivery.

A database will be defined with the variables destined to be analysed according to the objectives set: semen quality analysis, sperm DNA fragmentation, microbial analysis, embryo development, gestation and delivery. The exported data will be duly codified in order to protect the clinical and personal information of the patients according to the Spanish data protection law. Finally, and prior to the statistical study, an exploratory data analysis will be carried out to review the quality of the information extracted.

The main variable of the study is the live birth rate per cycle defined as the percentage of cases in which a complete treatment of assisted reproduction is carried out with control or experimental semen, which manage to obtain offspring for each cycle of ovarian stimulation included in the study.