In spermatozoa collected after pellet swim up, when totaldna fragmentation is higher than 15%, the normal morphologicallyspermatozoa population shows an increased dna damage

Study question: We investigated the DNA Fragmentation Index (DFI) in motilenormal morphologically spermatozoa comparing samples with total DFI < 15%Vs ≥ 15% collected after pellet swim upSummary answer: In the case of DFI ≥15% the percentage of normal morphologicallyspermatozoa with fragmented DNA is significantly higher than thepopulation with DFI < 15%What is known already: Intracytoplasmic sperm injection (ICSI) is widelyused in the treatment of male infertility. Only morphologically normal spermatozoaare mainly used by embryologists to fertilize an oocyte. Different papershave reported that spermatozoa with apparently normal morphology may haveDNA fragmentation. These evaluations suggest that it is possible that normal-shaped spermatozoa but with DNA fragmentation could be easily selectedto fertilize oocytes during ICSI. It is known that the presence of an increasedproportion of normal spermatozoa with damaged DNA is negatively associatedwith embryo quality affecting both pregnancy and implantation outcomesafter ICSI.Study design, size, duration: We designed an observational study on 70male patients. We speculated that the examination of DNA integrity in motileand morphologically normal sperm, collected after pellet swim up, could provideuseful information concerning sperm competence, rather than the DFI evaluationin the raw seminal sample. We analyzed data from January 2019 to December2019. The aim is to demonstrate that DFI in normal morphologically spermatozoa,could be indicated as predictive parameter of ICSI success.Participants/materials, setting, methods: DFI and traditional semenparameters (WHO, 2010), were evaluated in all patients. DFI was calculatedusing in situ TUNEL assay in at least 250 spermatozoa. By means of NIS-ElementsBR 3.10 image analyzer software (Nikon) using images of the same field (light, fluorescence and “merged”) it was possible to evaluate sperm morphologyassociated with DNA fragmentation. Data were analyzed using the Kruskal-Wallis test, a non-parametric ANOVA, confirmed by restrictive Bonferroni correctionusing the Dunn’s test.Main results and the role of chance: In this observational study we included70 oligoasthenospermic patients undergoing ICSI. The patients were classifiedin 2 groups according to the sperm DFI: Group A (n=35) included those whohad a DFI < 15% in the population of sperm collected after swim up. In groupB (n= 35) patients with a DFI ≥ 15%. We did not find any statistical differencebetween the two groups in the traditional sperm parameters like density, motilityand morphology.We observed that, in Group A, the average value of the total of sperm DFIwas 9.32% while in Group B was 24.71 % (p< 0.0001). When the analysis wasrestricted only to spermatozoa with normal morphology, it was observed thatamong patients of Group B the DFI value was 13.6%, while in A Group theaverage DFI value was 2.2%, with a strong statistical difference (p<0.0001). DFIcalculated on motile, normal morphologically spermatozoa can provide animportant information on the probability and risk of injecting, during ICSI procedure,a sperm with normal morphology but with fragmented DNA. This riskis higher if the sperm population collected after pellet swim up has a DFI higherthan 15%.Limitations, reasons for caution: This type of analysis only provides aprediction to select a sperm with fragmented DNA, but does not allow theselection of single spermatozoa with intact DNA to be used for ICSI. Furtherstudies are needed to correlate these data with the clinical outcome.Wider implications of the findings: Ou