The study was conducted according to the Declaration of Helsinki and approved by the Ethical Committee of Saint-Joseph University and Hotel-Dieu de France Hospital, Beirut, Lebanon (Ref: HDF 917). Samples (sperm and AT) were collected after obtaining a written informed consent from all participants.

A total of 30 healthy men attending Azoury IVF Clinic (fertility center) at Mount Lebanon hospital were assessed in this study. All participants had a normal semen analysis based on sperm parameters evaluation. In-person interviews were conducted to complete a questionnaire about age, current or previous disease status including urogenital pathologies, and habits such as smoking and alcohol intake. The mean age of participants was 35±5 years.

Semen samples, obtained by masturbation and collected in sterile plastic containers, were incubated at 37°C for 30 minutes to allow liquefaction. Each sample was then evaluated for sperm concentration, motility, morphology, viability, DNA fragmentation and OS levels. The conventional semen profile for all samples was constructed according to the World Health Organization (WHO) manual for the examination of human semen (14).

Participants with a sperm concentration less than 14×10⁶ cells/ml were not included in the study. All patients presenting with abnormal sperm parameters, suffering from andrological disorders, known to have inflammatory or immune diseases or a recent fever, or undertaking any treatment that may alter spermatogenesis, were excluded from the study.

Sperm samples were prepared using density gradient technique according to manufacturer’s protocol (Sperm Gradient kit, COOK medical, Australia). Briefly, density gradient media was prepared by layering the 40% medium over the 80% medium, and two ml of the liquefied semen sample were added over the upper layer. After centrifugation (380 g for 10 minutes), most of the supernatant was gently removed and the pellet was resuspended in two ml of sperm medium (Sperm Medium, COOK medical, Australia) to remove the density gradient medium. At the end of the centrifugation (670 g for 5 minutes), the final pellet was resuspended in the sperm medium.

The resulting pellets (n=5) were cultured in duplicate in the presence of several concentrations of H₂O₂ ranging between 0 and 40 μM with different incubation time. Upon establishment of a dose-response curve for the effects of H₂O₂ on sperm parameters, the suitable concentration and incubation period for the induction of sperm OS were determined. Samples prepared were then left untreated or treated with 10 μM H₂O₂ for 1 hour (Supplementary Fig. S1).

Adipose tissue samples were obtained from healthy donors undergoing an elective liposuction procedure of the abdominal, hip or thigh areas at the department of plastic surgery, Hôtel-Dieu de France hospital, Beirut, Lebanon. Lipoaspirates were washed extensively with sterile Hank’s buffer (Sigma-Aldrich, USA) to remove red blood cells, and digested with a type I collagenase solution (Sigma-Aldrich, USA) at a concentration of 0.075% in Hank’s buffer for 2~3 hours at 37°C with gentle stirring. The collagenase was then inactivated with an equal volume of DMEM (Dulbecco’s modified eagle’s medium) containing 10% FBS (Sigma-Aldrich, USA), and the mixture was centrifuged for 10 min at 600 g at room temperature. The pellet obtained was resuspended in one volume of Hank’s buffer, and filtered through a 100 μm sieve to remove the debris. After centrifuging the filtrate for 10 min at 600 g, pellet was resuspended in one ml of red blood cell lysis buffer (Sigma-Aldrich, USA) for 1 min, then incubated with 19 ml of Hank’s buffer for 10 min at room temperature. Then, a centrifugation for 7 minutes at 300 g at room temperature was performed to remove lysed red cell debris as well as traces of the lysis buffer. The resulting stromal vascular fraction was plated in cell culture flasks at a density of 10,000 cells/cm² in DMEM-F12 containing 10% FBS and 1% penicillin-streptomycin-amphotericin (PSA) (Sigma-Aldrich, USA), then incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 48 hours, the non-adherent cells were removed and a fresh volume of DMEM-F12 was added and replaced every 2~3 days. Cells adherent to the plastic, and having a fibroblastic morphology were regarded as AT-MSCs. After 21 days, cells were detached by trypsinization, collected, and resuspended in a complete DMEM-F12 medium containing 20% FBS and 1% PS (Sigma-Aldrich, USA). These cells, considered in first-passage (P1), were used in subsequent experiments.

AD-MSCs were differentiated into adipocytes, osteocytes and chondrocytes using respectively the Human Mesenchymal Stem Cell (hMSC) Adipogenic Differentiation Medium and hMSC Adipogenic Maintenance Medium (Lonza, Walkersville, MD, USA), the hMSC Osteogenic Differentiation Medium (Lonza, Walkersville, MD, USA), and the hMSC Chondrogenic Differentiation Medium (Lonza, Walkersville, MD, USA) according to manufacturer’s instructions. Adipose cells, osteocytes and chondrocytes were identified using Oil Red O staining, Alizarin Red staining and Alcian Blue staining (Sigma-Aldrich, St. Louis, MO, USA) respectively.

AD-MSCs in P1 were characterized by flow cytometric quantification of the surface markers CD73, CD44, CD90, CD29, CD105, CD45, CD31, CD106 and CD34 by specific antibodies (Becton, Dickinson, USA). Cells in culture were detached by trypsin, washed with a DMEM-F12 medium containing 10% FBS and 1% PS, and incubated in the presence of the appropriate monoclonal antibodies in the absence of light for 20 min at 4°C. After incubation, cells were washed twice with cold PBS containing 0.5% bovine serum albumin, centrifuged at 300 g for 5 min, and fixed with a 1% formaldehyde solution. Labeled and unlabeled cells were subsequently analyzed by CellQuest software (Becton Dickinson, USA). A minimum of 10,000 events has been characterized and reported.

One million AD-MSCs in P1, cultured in 75 cm² flasks (Sarstedt, USA) at 37°C using DMEM-F12 medium containing 10% FBS and 1% PS, were used to obtain the CM. At 70% confluence, the media was removed, cells were washed twice with PBS, and re-incubated with serum-free medium. After 24 h, 48 h and 72 h, the CM containing AD-MSCs secretions was collected and centrifuged at 380 g for 10 min to remove cell debris. The CM was stored at −80°C until further use.

The effect of CM on sperm parameters was assessed by incubating stressed and non-stressed sperm in 24-well plates in the presence or absence of 100% CM collected after 24 h, 48 h and 72 h of AD-MSCs confluence. Sperm cultured with DMEM-F12 were considered as controls. Sperm parameters were evaluated, after 1, 2, 4, 8, 12, 24 and 48-hour incubation at 37°C.

Sperm H₂O₂-induced OS was evaluated through measurement of ROS in sperm cells. ROS status was assessed using nitroblue tetrazolium (NBT) test (Sigma-Aldrich, Japan) as described previously (15). Briefly, NBT staining was done by mixing equal volumes of 0.1% of NBT solution and sample (10 μl) and incubation for 30 minutes at 37°C. A smear was prepared from the mix, then let to dry and finally counter stained with Wright stain solution (Sigma-Aldrich, Japan). A total of 200 sperm cells were evaluated under 100× oil immersion with a bright field microscope. NBT, an electron acceptor, reacts with cellular superoxide ions to form a blue-black compound formazan. ROS positive sperm cells were scored as NBT+ when solid deposits of formazan were found in cytoplasm and/or head.

Sperm motility was determined under light microscopy using 40× magnification by counting sperm cells in 6 microscopic fields to determine the average percentage, and noted as progressive, non-progressive and immotile, according to WHO 2010 criteria (14). Each sample was evaluated by two technicians, in duplicate, and readings were averaged between the two evaluations after calculation of error.

Halosperm kit (halotech DNA, Spain) was used as an improved sperm chromatin dispersion test to assess sperm DNA fragmentation (SDF) according to manufacturer’s instructions. A minimum of 300 sperm cells per sample were scored under microscope, using the objective 100. Spermatozoa with large or medium sized halos, characteristic of dispersed DNA loops, were considered to have intact DNA. Sperm cells with small, degraded or absent halos were considered to have fragmented DNA. SDF was determined for each specimen, using the following formula: SDF (%)=100×(number of spermatozoa with fragmented DNA)/(number of spermatozoa counted).

Sperm morphology and vacuolization were evaluated using the Spermoscan Kit (RAL diagnostics, Australia) according to manufacturer’s instruction. Briefly, the smears were fixed in the spermo FIX-RAL solution. The sperm cytoplasm and nucleus were then stained using the spermo EOSIN-RAL and the spermo BLUE-RAL respectively. After drying, a minimum of 200 sperm were counted and assessed for morphology and vacuolization using the optic microscope according to Kruger’s strict criteria: head defects: large or small, tapered, amorphous, vacuolated (more than two vacuoles or >20% of the head occupied by unstained vacuolar areas), small or large acrosomal areas; mid-piece defects: bent, cytoplasmic residues; tail defects: coiled, multiple (14).

Sperm viability was evaluated using eosin-nigrosin staining. Ten microliters of each sample was mixed with equal volume of eosin-nigrosin solution. After 30 seconds, the samples were smeared and let to air dry. At least 200 sperm were evaluated under 100× oil immersion with a bright field microscope. Unstained sperm were classified as alive, whereas dead ones with altered membranes were stained pink or red (eosin) with a dark background (high concentration of nigrosin) (14).

Means and standard deviations were calculated for all variables using the GraphPad Prism software version 6 (GraphPad Software, Inc., USA). The analysis of variance (ANOVA) for nonparametric data was used to assess the variables between groups, followed by Tukey’s post hoc test. p-values less than 0.05 were considered statistically significant.

Flow cytometry was used to characterize MSCs isolated from adipose tissues. Analysis of results showed that these cells expressed the specific surface markers of mesenchymal stem cells, namely CD73, CD29, CD44, CD90 and CD105, and lacked the expression of endothelial and hematopoietic cell markers CD34, CD31, CD45 and CD106 (Fig. 1).

Undifferentiated AD-MSCs have a fibroblast-like morphology. To test their multilineage differentiation capacity, cells were incubated for 21 days in adipogenic, osteogenic and chondrogenic media. The formation of intra-cellular lipid droplets revealed the differentiation of AD-MSCs into adipocytes, which was further confirmed by the presence of a red orange color using “Oil Red O” staining. The presence of calcium deposits, characterizing AD-MSCs differentiation into osteocytes, was visualized by red alizarin stain. AD-MSCs differentiated into chondrocyte-like cells were identified by the development of a blue color using Alcian Blue staining (Fig. 1).

The optimum incubation period to improve sperm quality and function was determined by culturing sperm cells for two days with or without AD-MSCs-CM collected at 24, 48 and 72 h of conditioning, and assessing sperm parameters at different time points of culture (1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h). Results revealed that the total motility (%) at 24 h was still stable in stressed and non-stressed sperm treated with AD-MSCs-CM compared to the starting point (T=0 h) while it decreased significantly and in a steady fashion in the control group of sperm cultured with DMEM-F12 medium as of the starting point at T=0 h, hence showing the ability of AD-MSCs-CM to maintain the motility for 24 h before dropping thereafter (Fig. 2A, 2B). Regarding dead sperm, the percentage started to increase progressively in all conditions after 1 h of culture, with levels reaching their maximum at 48 h incubation time. However, in both stressed and non-stressed sperm treated with CM, the percentage of dead sperm was always lower when compared to the control group at all-time points, showing the ability of the CM to withhold sperm death and keep cell viability. Moreover, it is important to note that the turning point at which the curve of dead sperm starts increasing rapidly appears after 24 h incubation time in both group (Fig. 2C, 2D). As for sperm vacuolization (Fig. 2E, 2F), sperm DNA fragmentation (Fig. 2G, 2H) and ROS positive cells (Fig. 2I, 2J), the most significant improvement was noted at 24 h in comparison to other culture conditions and all other incubation timings. Finally, stressed and non-stressed sperm showed no difference in morphology at all-time points of culture, and irrespective of culture conditions (Fig. 2K, 2L). Taking all tested parameters into consideration, the 24 h incubation time was established for having the best effect on sperm vacuolization, DNA fragmentation and oxidative stress while motility and viability were preserved, and thus regarded in our study as the optimum period to study sperm parameters.

In order to determine how the temporal profiles of CM collection affect sperm quality, stressed and non-stressed sperm were cultured with DMEM-F12 medium or AD-MSCs-CM collected at 24, 48 and 72 h, and parameters were compared after 24 hours of incubation time, defined previously as the optimum period of incubation. Interestingly, incubation with CM significantly improved motility (Fig. 3A) and vitality (Fig. 3B) of stressed and non-stressed sperm for all the time points, but without noticing any difference in the impact with regards to the time of CM collection. Furthermore, a significant decrease in sperm DNA fragmentation (Fig. 3C), sperm vacuolization (Fig. 3D) and ROS positive cells (Fig. 3E) was detected in stressed and non-stressed sperm treated with CM compared to controls. Interestingly, both non-stressed and stressed sperm showed better DNA fragmentation and vacuolization rates when treated with CM 24 h as compared to CM 48 or 72 h; the percentage of ROS positive cells being on the other hand comparable in all CM treated groups, irrespectively of the collection time. Finally, no significant difference in sperm morphology was observed between the treated and control groups, hence indicating no effect of CM on the appearance of the spermatozoa (Fig. 3F).

After establishing the CM 24 h as the most suitable condition to improve sperm quality and function, parameters were compared to those of fresh semen (T0) usually collected immediately after processing in couples undergoing fertility treatment.

Stressed and non-stressed sperm showed comparable motility (Fig. 4A) and vitality (Fig. 4B) profiles in CM-treated and fresh sperm, thus pointing out the ability of CM to relatively maintain these two parameters over time for about 24 hours. In contrast, CM treatment significantly reduced the percentage of sperm DNA fragmentation (Fig. 4C), sperm vacuolization (Fig. 4D) and ROS positive cells (Fig. 4E) in comparison to the non-treated fresh sperm. These results show that infertile patients with a high degree of sperm vacuolization and DNA fragmentation can benefit from a 24-hour CM treatment of their fresh semen while keeping the other parameters stable, which can improve sperm quality and subsequently IVF outcome.