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Intracytoplasmic sperm injection (ICSI) using surgically retrieved spermatozoa outside the classic context of azoospermia has been increasingly used to overcome infertility. The primary indications include high levels of sperm DNA damage in ejaculated spermatozoa and severe oligozoospermia or cryptozoospermia, particularly in couples with ICSI failure for no apparent reason. Current evidence suggests that surgically retrieved spermatozoa for ICSI in the above context improves  outcomes, mainly concerning pregnancy and miscarriage rates. The reasons are not fully understood but may be related to the lower levels of DNA damage in spermatozoa retrieved from the testis compared with ejaculated counterparts. These findings are consistent with the notion that excessive sperm DNA damage  can be a limiting factor responsible for the failure to conceive. Using testicular in preference of low-quality ejaculated spermatozoa bypasses post-testicular sperm DNA damage caused primarily by oxidative stress, thus increasing the likelihood of oocyte fertilization by genomically intact spermatozoa. Despite the overall favorable results, data remain limited, and mainly concern males with confirmed sperm DNA damage in the ejaculate. Additionally, information regarding the health of ICSI offspring resulting from the use of surgically retrieved spermatoa of non-azoospermic males is still lacking. Efforts should be made to improve the male partner's reproductive health for safer ICSI utilization. A comprehensive andrological evaluation aiming to identify and treat the underlying male infertility factor contributing to sperm DNA damage is essential for achieving this goal.

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BACKGROUND: The assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays. RESULTS: The results showed that global DNA damage negatively correlates (P < 0.05) with normal sperm morphology (R = - 0.460) and progressive motility (R = - 0.419), and positively with the percentage of non-viable sperm (R = 0.507). Multiple regression analyses showed that non-viable sperm were related to SSB (ß = - 0.754). In addition, while fertilisation did not seem to be affected by sperm DNA integrity, global DNA damage, DSB and SSB were found to be correlated to embryo development outcomes. Specifically, whereas global DNA damage and DSB negatively affected (P < 0.05) the later preimplantation embryo stages (percentage of early blastocyst/blastocyst D6: for global DNA damage, R = - 0.458, and for DSB, R = - 0.551; and percentage of hatching/hatched blastocyst D6: for global DNA damage, R = - 0.505, and for DSB, R = - 0.447), global DNA damage and SSB had a negative impact (P < 0.05) on the developmental competency of fertilised embryos (R = - 0.532 and R = - 0.515, respectively). Remarkably, multiple regression analyses supported the associations found in correlation analyses. Finally, the present work also found that the inclusion of Comet assays to the conventional sperm quality tests improves the prediction of blastocyst formation (AUC = 0.9021, P < 0.05), but not fertilisation rates (P > 0.05). CONCLUSION: Considering all these findings, this work sets a useful model to study how SDF negatively influences fertility.

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BACKGROUND: The assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays. RESULTS: The results showed that global DNA damage negatively correlates (P 0.05). CONCLUSION: Considering all these findings, this work sets a useful model to study how SDF negatively influences fertility.

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Methyl parathion (Me-Pa) is an extremely toxic organophosphorus pesticide still used in developing countries. It has been associated with decreased sperm function and fertility and with oxidative and DNA damage. The blood-testis barrier (BTB) is a structure formed by tight junction (TJ) proteins in Sertoli cells and has a critical role in spermatogenesis. We assessed the effect of repeated doses of Me-Pa (3-12 mg/kg/day for 5 days, i.p.) on sperm quality, lipid oxidation, DNA integrity, and BTB permeability in adult male mice and explored oxidation as a mechanism of toxicity. Me-Pa caused dose-dependent effects on sperm quality, lipoperoxidation, and DNA integrity. Testis histology results showed the disruption of spermatogenesis progression and atrophy of seminiferous tubules. The pesticide opened the BTB, as evidenced by the presence of a biotin tracer in the adluminal compartment of the seminiferous tubules. This effect was not observed after 45 days of exposure when a spermatogenic cycle had completed. The coadministration of the antioxidant α-tocopherol (50 mg/kg/day for 5 days, oral) prevented the effects of Me-Pa on sperm quality, DNA and the BTB, indicating the importance of oxidative stress in the damage generated by Me-Pa. As evidenced by immunochemistry, no changes were found in the localization of the TJ proteins of the BTB, although oxidation (carbonylation) of total proteins in testis homogenates was detected. Our results show that Me-Pa disturbs the BTB and that oxidation is involved in the observed toxic effects on sperm cells.

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Varicocele, the leading cause of male infertility, can impair sperm quality and fertility via various oxidative stress mechanisms. An imbalance between excessive reactive oxygen species production and antioxidant protection causes alterations in nuclear and mitochondrial sperm DNA, thus rendering a subset of varicocele men less fertile. In particular, sperm DNA fragmentation is usually elevated in men with clinical varicocele in both abnormal and normal semen parameters by the current World Health Organization criteria. In this review, we discuss the evidence concerning the association between varicocele, oxidative stress, and SDF, and the possible mechanisms involved in infertility. Furthermore, we summarize the role of varicocele repair as a means of alleviating SDF and improving fertility. Lastly, we critically appraise the evidence-based algorithm recently issued by the Society for Translational Medicine aimed at guiding urologists on the use of SDF testing in men with varicocele seeking fertility. Current evidence based on careful review of published studies confirms the effectiveness of varicocelectomy as a means of both reducing oxidatively induced sperm DNA damage and potentially improving fertility. Varicocele repair should be offered as part of treatment option for male partners of infertile couples presenting with palpable varicoceles.

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Resumen OBJETIVO: Determinar las repercusiones del daño al ADN espermático en los parámetros seminales más estudiados en diagnóstico clínico de varones infértiles. MATERIALES Y MÉTODOS: Estudio de casos y controles, prospectivo y comparativo efectuado en pacientes masculinos atendidos en el Centro Integral de la Mujer y Reproducción Asistida de Puebla, México. Parámetros de estudio: edad, movilidad, morfología, diagnóstico seminal, leucocitos y factor de infertilidad. Los resultados se analizaron con Graphpad Prisma 5.0 y se consideraron estadísticamente significativos con p < 0.005. RESULTADOS: Se estudiaron 110 pacientes: 33 con mala integridad del ADN espermático (grupo 1) y 77 con buena integridad (grupo 2). La concentración espermática y la movilidad tipo A+B en el grupo 2 fue significativamente más alta que en el grupo 1 (p < 0.0001) en donde se registró mayor número de móviles no progresivos e inmóviles. La morfología normal fue más alta en el grupo 2 (p = 0.0063). En los varones menores de 40 años se observó un número significativamente mayor de casos de buena integridad espermática (p = 0.013). El diagnóstico seminal demostró que los varones con mala integridad tuvieron alteraciones espermáticas más severas. Los factores de infertilidad más frecuentes implicados en ambos grupos fueron: aborto de repetición, edad de la pareja, falla previa en la técnica de reproducción asistida, factor masculino severo y factor tubárico. CONCLUSIONES: La mala integridad del ADN espermático tiene repercusiones en la concentración espermática, movilidad y morfología, además de alterar el diagnóstico seminal, pues los varones tuvieron trastornos más severos cuando no hubo algún factor de infertilidad que describiera un comportamiento específico relacionado con la mala integridad espermática.

Abstract OBJECTIVE: To determine the impact of sperm DNA damage on the most studied seminal parameters in clinical diagnosis of infertile males. MATERIALS AND METHODS: Prospective and comparative case-control study that included male patients seen at Centro Integral de la Mujer y Reproducción Asistida de Puebla, Mexico. Study parameters: age, mobility, morphology, seminal diagnosis, leukocytes and infertility factor. The results were analyzed with Graphpad Prism 5.0 and were considered statistically significant with p < 0.005. RESULTS: 110 male patients were studied: 33 patients with poor sperm DNA integrity (group 1) and 77 patients with good integrity (group 2). Sperm concentration and type A + B mobility in group 2 was significantly higher than in group 1 (p <0.0001), where a greater number of non-progressive and immobile mobiles was recorded. The normal morphology was higher in group 2 (p = 0.0063). In men under 40 years of age, a significantly higher number of cases of good sperm integrity was observed (p = 0.013). The seminal diagnosis showed that males with poor integrity had more severe sperm alterations. The most frequent infertility factors involved in both groups were: repeat abortion, age of the couple, previous failure in the technique of assisted reproduction, severe male factor and tubal factor. CONCLUSIONS: The poor integrity of the sperm DNA has repercussions on sperm concentration, mobility and morphology, alters the seminal diagnosis, since males had more severe alterations when there was no infertility factor that described a specific behavior related to poor sperm integrity.

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OBJECTIVE: To compare the sperm chromatin dispersion (SCD) test and the terminal uridine nick-end labeling (TUNEL) assay for assessment of sperm DNA damage. DESIGN: Prospective comparative experimental study. SETTING: Andrology laboratory. PATIENT(S): Twenty subfertile men with unexplained infertility. INTERVENTION(S): Sperm DNA damage was determined in the same semen samples using the TUNEL assay with fluorescence microscopy and the SCD test with bright-field microscopy. MAIN OUTCOME MEASURE(S): Correlation coefficient and receiver operating characteristic analysis outcomes. The TUNEL assay was used as the reference standard to identify optimal cutoff points for assessing DNA damage by SCD. RESULT(S): The SCD test detected a significantly higher proportion of sperm with damaged DNA (20.6% ± 14.0%) than the TUNEL assay (11.5% ± 7.3%). Spearman's rank correlation showed that the methods were not comparable (r = 0.29). Receiver operating characteristic analysis revealed that 15% was the best SCD cutoff point to classify patients within the same levels of DNA fragmentation, normal or abnormal, as determined by the TUNEL assay, with an accuracy of 69%. CONCLUSION(S): The SCD test is more sensitive than the TUNEL assay for the assessment of DNA damage in men with unexplained infertility. Although the methods are poorly correlated, SCD may discriminate men with normal and abnormal sperm DNA damage with moderate accuracy when compared with TUNEL. It is important to distinguish between the methods because they differently evaluate sperm DNA damage.

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