Study question: We investigated the DNA Fragmentation Index (DFI) in motile normal morphologically spermatozoa comparing samples with total DFI < 15% Vs ≥ 15% collected after pellet swim up Summary answer: In the case of DFI ≥15% the percentage of normal morphologically spermatozoa with fragmented DNA is significantly higher than the population with DFI < 15% What is known already: Intracytoplasmic sperm injection (ICSI) is widely used in the treatment of male infertility. Only morphologically normal spermatozoa are mainly used by embryologists to fertilize an oocyte. Different papers have reported that spermatozoa with apparently normal morphology may have DNA fragmentation. These evaluations suggest that it is possible that normal- shaped spermatozoa but with DNA fragmentation could be easily selected to fertilize oocytes during ICSI. It is known that the presence of an increased proportion of normal spermatozoa with damaged DNA is negatively associated with embryo quality affecting both pregnancy and implantation outcomes after ICSI. Study design, size, duration: We designed an observational study on 70 male patients. We speculated that the examination of DNA integrity in motile and morphologically normal sperm, collected after pellet swim up, could provide useful information concerning sperm competence, rather than the DFI evaluation in the raw seminal sample. We analyzed data from January 2019 to December 2019. 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 semen parameters (WHO, 2010), were evaluated in all patients. DFI was calculated using in situ TUNEL assay in at least 250 spermatozoa. By means of NIS-Elements BR 3.10 image analyzer software (Nikon) using images of the same field (light, fluorescence and “merged”) it was possible to evaluate sperm morphology associated with DNA fragmentation. Data were analyzed using the Kruskal-Wallis test, a non-parametric ANOVA, confirmed by restrictive Bonferroni correction using the Dunn’s test. Main results and the role of chance: In this observational study we included 70 oligoasthenospermic patients undergoing ICSI. The patients were classified in 2 groups according to the sperm DFI: Group A (n=35) included those who had a DFI < 15% in the population of sperm collected after swim up. In group B (n= 35) patients with a DFI ≥ 15%. We did not find any statistical difference between the two groups in the traditional sperm parameters like density, motility and morphology. We observed that, in Group A, the average value of the total of sperm DFI was 9.32% while in Group B was 24.71 % (p< 0.0001). When the analysis was restricted only to spermatozoa with normal morphology, it was observed that among patients of Group B the DFI value was 13.6%, while in A Group the average DFI value was 2.2%, with a strong statistical difference (p<0.0001). DFI calculated on motile, normal morphologically spermatozoa can provide an important information on the probability and risk of injecting, during ICSI procedure, a sperm with normal morphology but with fragmented DNA. This risk is higher if the sperm population collected after pellet swim up has a DFI higher than 15%. Limitations, reasons for caution: This type of analysis only provides a prediction to select a sperm with fragmented DNA, but does not allow the selection of single spermatozoa with intact DNA to be used for ICSI. Further studies are needed to correlate these data with the clinical outcome. Wider implications of the findings: Our results suggest that the evaluation of DFI in morphologically motile normal sperm selected after pellet swim up appears to be a more accurate strategy to evaluate the sperm competence, with the aim to improve the ICSI outcomes, than the traditional evaluation of sperm DFI in the whole seminal sample. Trial registration number: not applicable

G. Ruvolo, A. Ferrigno, M.C. Roccheri, E. Cittadini, A. Pane, C. Simonaro, et al. (2020). In spermatozoa collected after pellet swim up, when total dna fragmentation is higher than 15%, the normal morphologically spermatozoa population shows an increased dna damage. HUMAN REPRODUCTION, 35(Suppl1), 159-160.

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

M. C. Roccheri
Membro del Collaboration Group
;
E. Cittadini
Membro del Collaboration Group
;
L. Bosco
Project Administration
2020-01-01

Abstract

Study question: We investigated the DNA Fragmentation Index (DFI) in motile normal morphologically spermatozoa comparing samples with total DFI < 15% Vs ≥ 15% collected after pellet swim up Summary answer: In the case of DFI ≥15% the percentage of normal morphologically spermatozoa with fragmented DNA is significantly higher than the population with DFI < 15% What is known already: Intracytoplasmic sperm injection (ICSI) is widely used in the treatment of male infertility. Only morphologically normal spermatozoa are mainly used by embryologists to fertilize an oocyte. Different papers have reported that spermatozoa with apparently normal morphology may have DNA fragmentation. These evaluations suggest that it is possible that normal- shaped spermatozoa but with DNA fragmentation could be easily selected to fertilize oocytes during ICSI. It is known that the presence of an increased proportion of normal spermatozoa with damaged DNA is negatively associated with embryo quality affecting both pregnancy and implantation outcomes after ICSI. Study design, size, duration: We designed an observational study on 70 male patients. We speculated that the examination of DNA integrity in motile and morphologically normal sperm, collected after pellet swim up, could provide useful information concerning sperm competence, rather than the DFI evaluation in the raw seminal sample. We analyzed data from January 2019 to December 2019. 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 semen parameters (WHO, 2010), were evaluated in all patients. DFI was calculated using in situ TUNEL assay in at least 250 spermatozoa. By means of NIS-Elements BR 3.10 image analyzer software (Nikon) using images of the same field (light, fluorescence and “merged”) it was possible to evaluate sperm morphology associated with DNA fragmentation. Data were analyzed using the Kruskal-Wallis test, a non-parametric ANOVA, confirmed by restrictive Bonferroni correction using the Dunn’s test. Main results and the role of chance: In this observational study we included 70 oligoasthenospermic patients undergoing ICSI. The patients were classified in 2 groups according to the sperm DFI: Group A (n=35) included those who had a DFI < 15% in the population of sperm collected after swim up. In group B (n= 35) patients with a DFI ≥ 15%. We did not find any statistical difference between the two groups in the traditional sperm parameters like density, motility and morphology. We observed that, in Group A, the average value of the total of sperm DFI was 9.32% while in Group B was 24.71 % (p< 0.0001). When the analysis was restricted only to spermatozoa with normal morphology, it was observed that among patients of Group B the DFI value was 13.6%, while in A Group the average DFI value was 2.2%, with a strong statistical difference (p<0.0001). DFI calculated on motile, normal morphologically spermatozoa can provide an important information on the probability and risk of injecting, during ICSI procedure, a sperm with normal morphology but with fragmented DNA. This risk is higher if the sperm population collected after pellet swim up has a DFI higher than 15%. Limitations, reasons for caution: This type of analysis only provides a prediction to select a sperm with fragmented DNA, but does not allow the selection of single spermatozoa with intact DNA to be used for ICSI. Further studies are needed to correlate these data with the clinical outcome. Wider implications of the findings: Our results suggest that the evaluation of DFI in morphologically motile normal sperm selected after pellet swim up appears to be a more accurate strategy to evaluate the sperm competence, with the aim to improve the ICSI outcomes, than the traditional evaluation of sperm DFI in the whole seminal sample. Trial registration number: not applicable
2020
Settore BIO/06 - Anatomia Comparata E Citologia
G. Ruvolo, A. Ferrigno, M.C. Roccheri, E. Cittadini, A. Pane, C. Simonaro, et al. (2020). In spermatozoa collected after pellet swim up, when total dna fragmentation is higher than 15%, the normal morphologically spermatozoa population shows an increased dna damage. HUMAN REPRODUCTION, 35(Suppl1), 159-160.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10447/484326
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