Background: Suboptimal human semen handling in vitro may induce sperm damage.However, the effects of semen swim-up, pellet swim-up, density gradient, and density gradient followed by SU on sperm motility, morphology, DNA fragmentation, acrosome reaction, intracellular reactive oxygen species, and mitochondrial activity were not fully understood.Objectives: To study the impact of four sperm preparation techniques on sperm functional parameters. Materials and methods:This study was conducted on 60 infertile men with a minimum sperm concentration of 20 × 10 6 /ml and total sperm motility of ≥30%. Each raw semen sample was divided into four aliquots. Each aliquot was prepared by one of the tested techniques. Various sperm characteristics were assessed before and after sperm preparation.Results: Density gradient and density gradient followed by SU resulted in significantly higher DNA fragmentation percentages compared with semen swim-up (p < 0.001 and p < 0.001, respectively) and pellet swim-up (p < 0.001 and p < 0.001, respectively). Significantly higher percentages of spermatozoa with intact acrosome were detected in semen swim-up (p < 0.001) and pellet swim-up (p < 0.001) compared with raw semen. The percentage of reactive oxygen species-positive spermatozoa was significantly higher after pellet swim-up (p < 0.001), density gradient (p < 0.001), and density gradient followed by SU (p < 0.001) than raw semen. In addition, the percentages of 100% stained midpiece (active mitochondria) were significantly higher in semen swim-up (p < 0.001) and pellet swim-up (p < 0.001) compared with raw semen. Discussion and conclusion:To the best of our knowledge, this is the first report comparing the impact of these techniques on various sperm functional parameters.Semen swim-up was more effective than density gradient in selecting better spermatozoa in terms of DNA integrity, reactive oxygen species levels, acrosome status, and

Study question Do alterations in mitochondrial DNA levels in luteal granulosa cells (LGCs) affect the quality of mature oocytes in infertile women? Summary answer Alterations in the mitochondrial DNA levels in LGCs may modify the characteristics of the first polar body in oocytes and subsequently decrease their fertilization potential. What is known already Assisted reproductive treatment outcomes are closely linked to the oocyte quality as shown in several systematic reviews and meta-analyses. Particularly, the oocyte quality is highly affected by the surrounding luteal granulosa cells (LGCs). This is mainly due to the crucial role of the mitochondria in the LGCs in fueling the metabolic processes required for oocyte maturation. Therefore, modifications in the quality of LGCs may have direct effects on the developmental competence of oocytes. However, the exact mechanisms by which this could happen are not fully understood. Study design, size, duration A retrospective multicenter study was conducted on 303 mature oocytes retrieved from 51 women undergoing intracytoplasmic sperm injection (ICSI). It was conducted in Lebanon at Al Hadi IVF center and Azoury IVF clinic, between January 2019 and January 2020. G-Power 3.1 was used to determine the sample size for Generalized Linear Mixed Models through a one-sample t-test power analysis with alpha 0.05, power 0.8, medium effect size (f2 = 0.15), and 5 predictors. Resulting sample size: 43. Participants/materials, setting, methods This study excluded cases of premature ovarian failure, severe oligozoospermia (&lt;2 x 102/ml), or cases using frozen gametes. Mature oocytes, from young women (&lt; 36 years old), were injected and cultured in the Embryoscope where morphometric measurements were performed. LGCs vitality and mitochondrial DNA (mtDNA) levels were analyzed using trypan blue exclusion dye and next-generation sequencing, respectively. Possible associations between these independent variables and the size and integrity of the first polar body were evaluated. Main results and the role of chance The included women presented with primary infertility. Their mean age was 29.98 ± 5.5 years old and their mean body mass index was 23.33 ± 3.32 Kg/m2. 10% of the included women had polycystic ovary syndrome, and 48% were smokers. Regarding the LGCs parameters, a statistically significant negative correlation was found between the mtDNA levels in LGCs and their vitality percentage (r = −0.313, p = 0.029). Interestingly, the average size of the first polar body (PBI) was 346.67 µm. Here we found that the PBI size decreased by 15.05 units when the mtDNA level in LGCs increased by one unit above its average. In parallel, the median percentage of oocytes having a fragmented PBI was 33.33 (0-100). This percentage increased by 4.26% for every one unit increase in the mtDNA level in LGCs (p &lt; 0.0001). In contrast, this percentage decreased by 0.86% for every unit increase in the percentage of LGCs vitality (p &lt; 0.0001). Moreover, the mean percentage of fertilization rate was found to be 70.19 ± 26.7. A higher percentage of fragmented PBI (38.3%) was found among oocytes that did not fertilize compared to those that did successfully fertilize (24%) (p = 0.019). Limitations, reasons for caution This study only analyzed the effects of alterations in mtDNA levels in LGCs on oocytes. It would be worthwhile to also analyze the effects on foetal development and offspring health. Furthermore, it would be interesting to study the relationship between alterations in LGCs and oocyte quality within individual follicles. Wider implications of the findings Several studies have suggested that abnormal size and fragmentation of the PBI may impair embryo development. Therefore, treating factors that affect LGCs, such as obesity and polycystic ovary syndrome, may benefit infertile women. This highlights the need for new clinical trials in this context. Trial registration number Not applicable

BackgroundSemen cryopreservation is a widely used procedure for fertility preservation, despite some level of cryodamage that may occur in spermatozoa after thawing. However, there is some evidence that lactobacilli, one of the bacteria found in semen, might benefit sperm quality.ObjectivesThis study aims to determine whether the addition of Lactobacillus plantarum secretions to sperm freezing medium has an impact on sperm motility, morphology, and DNA fragmentation.Materials and methodsThis is a prospective auto‐controlled study. It was conducted on 30 raw semen samples from 30 infertile men attending a fertility center for semen analysis. Before freezing, all the samples were analyzed for motility, morphology, and DNA fragmentation percentages. Each sample was then divided equally into three aliquots. Cryopreservation was performed on each aliquot using one of the following three media: without Lactobacillus plantarum secretions (control group) or with 107 or 108 colony‐forming units/mL Lactobacillus plantarum secretions. Sperm motility, morphology, and DNA integrity were evaluated after the cryopreservation media were added and after semen thawing.ResultsThe results of this study indicated that after thawing, no statistically significant decrease in progressive motility and non‐progressive percentages were detected in the sperm freezing medium supplemented with 108 colony‐forming units/mL Lactobacillus plantarum secretions than the fresh raw semen. Moreover, multivariate linear regression model analyses showed that the progressive motility (p = 0.02), non‐progressive motility (p = 0.016), and non‐motile spermatozoa (p = 0.012) percentages were significantly decreased in the freezing medium (without Lactobacillus plantarum secretions) compared to the fresh raw semen.Discussion and conclusionTo the best of our knowledge, this is the first study showing that Lactobacillus plantarum secretions had a cryoprotective effect on sperm motility when added to the sperm freezing medium. Furthermore, Lactobacillus plantarum secretions were found to protect sperm DNA integrity more effectively than the freezing medium without Lactobacillus plantarum secretions in non‐normozoospermia group. Cryopreservation procedures must therefore be optimized to minimize any iatrogenically induced sperm DNA damage, given the correlation between sperm DNA damage and increased mutation loads in progeny.

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