Sperm chromatin structure assay (SCSA®) and flow cytometry-assisted TUNEL assay provide a concordant assessment of sperm DNA fragmentation as a function of age in a large cohort of approximately 10,000 patients.

BACKGROUND: Sperm DNA integrity is increasingly seen as a critical characteristic determining reproductive success, both in natural reproduction and in assisted reproductive technologies (ART). Despite this awareness, sperm DNA and nuclear integrity tests are still not part of routine examinations for either infertile men or fertile men wishing to assess their reproductive capacity. This is not due to the unavailability of DNA and sperm nuclear integrity tests. On the contrary, several relevant but distinct tests are available and have been used in many clinical trials, which has led to conflicting results and confusion. The reasons for this are mainly the lack of standardization between different clinics and between the tests themselves. In addition, the small number of samples analyzed in these trials has often weakened the value of the analyses performed. In the present work, we used a large cohort of semen samples, covering a wide age range, which were simultaneously evaluated for sperm DNA fragmentation (SDF) using two of the most frequently used SDF assays, namely the TUNEL assay and the sperm chromatin structure assay (SCSA®). At the same time, as standard seminal parameters (sperm motility, sperm morphology, sperm count) were available for these samples, correlations between age, SDF and conventional seminal parameters were analyzed. RESULTS: We show that the SCSA® and TUNEL assessments of SDF produce concordant data. However, the SDF assessed by TUNEL is systematically lower than that assessed by SCSA®. Regardless of the test used, the SDF increases steadily during aging, while the HDS parameter (High DNA stainability assessed via SCSA®) remains unchanged. In the cohort analyzed, conventional sperm parameters do not seem to discriminate with aging. Only sperm volume and motility were significantly lower in the oldest age group analyzed [50-59 years of age]. CONCLUSIONS: In the large cohort analyzed, SDF is an age-dependent parameter, increasing linearly with aging. The SCSA® assessment of SDF and the flow cytometry-assisted TUNEL assessment are well correlated, although TUNEL is less sensitive than SCSA®. This difference in sensitivity should be taken into account in the final assessment of the true level of fragmentation of the sperm nucleus of a given sample. The classical sperm parameters (motility, morphology, sperm count) do not change dramatically with age, making them inadequate to assess the fertility potential of an individual.

RéSUMé: CONTEXTE: l'intégrité de l'ADN des spermatozoïdes est de plus en plus considérée comme une caractéristique essentielle déterminant le succès de la reproduction, tant dans la reproduction naturelle que dans les techniques de reproduction assistée (AMP). Malgré cette prise de conscience, les tests d'intégrité nucléaire des spermatozoïdes ne font toujours pas partie des examens de routine pour les hommes infertiles ou fertiles souhaitant évaluer leur capacité de reproduction. Cette situation n'est pas due à l'indisponibilité des tests. Au contraire, plusieurs tests pertinents mais distincts sont disponibles et ont été utilisés dans de nombreux essais cliniques, ce qui a donné lieu à des résultats contradictoires et à une certaine confusion. Les raisons en sont principalement le manque de normalisation entre les différentes cliniques et entre les tests eux-mêmes. En outre, le petit nombre d'échantillons analysés dans ces essais a souvent affaibli la valeur des analyses effectuées. Dans le présent travail, nous avons utilisé une vaste cohorte d'échantillons, couvrant une large tranche d'âge, évalués simultanément pour la fragmentation de l'ADN des spermatozoïdes à l'aide de deux des tests les plus fréquemment utilisés, à savoir le test TUNEL et le test de la structure de la chromatine des spermatozoïdes (SCSA®). Parallèlement, comme les paramètres séminaux standard (motilité, morphologie, numération) étaient disponibles pour ces échantillons, les corrélations entre l'âge, le niveau de fragmentation et les paramètres séminaux conventionnels ont été analysées. RéSULTATS: Nous montrons que les évaluations SCSA® et TUNEL produisent des données concordantes. Cependant, le SDF évalué par TUNEL est systématiquement plus faible que celui évalué par SCSA®. Quel que soit le test utilisé, la fragmentation augmente régulièrement au cours du vieillissement, alors que le paramètre HDS (« High DNA stainability¼ évalué par le test SCSA®) reste inchangé. Dans la cohorte analysée, les paramètres spermatiques conventionnels ne semblent pas varier avec le vieillissement. Seuls le volume et la mobilité des spermatozoïdes étaient significativement plus faibles dans le groupe d'âge le plus élevé analysé [50­59 ans]. CONCLUSIONS: Dans la grande cohorte analysée, la fragmentation de l'ADN spermatique est un paramètre dépendant de l'âge, augmentant linéairement avec le vieillissement. L'évaluation du SDF par SCSA® et l'évaluation via le test TUNEL assistée par cytométrie de flux sont bien corrélées, bien que le TUNEL soit moins sensible que le SCSA®. Cette différence de sensibilité doit être prise en compte dans l'évaluation finale du niveau réel de fragmentation du noyau des spermatozoïdes d'un échantillon donné. Les paramètres classiques du sperme (motilité, morphologie, nombre de spermatozoïdes) ne changent pas de façon spectaculaire avec l'âge, ce qui les rend inadéquats pour évaluer le potentiel de fertilité d'un individu.

Ferulago angulata extract alleviates testicular toxicity in male mice exposed to diazinon and lead.

In this study, we investigated the protective effects of Ferulago angulata extract (FAE) against the reproductive toxicants Diazinon (DZN) and Lead (Pb) in mice. These pollutants are known to induce oxidative stress (OS), while FAE acts as a natural antioxidant. Adult male NMRI mice were exposed to DZN, Pb, and DZN+Pb, with or without FAE treatment for six weeks. We evaluated OS markers, testicular histology, and expression of mRNA related to enzymatic antioxidants. Exposure to DZN and Pb led to increased levels of thiobarbituric acid reactive substance (TBARS) and nitric oxide (NO) in the testes, along with a decrease in the total antioxidant capacity (TAC). Furthermore, the mRNA expression of antioxidant enzymes such as superoxide dismutase 1 (SOD1) and glutathione peroxidase 4 (GPX4) was altered. However, when FAE was administered concurrently, it restored the biochemical parameters to normal levels, reduced the toxic effects of DZN and Pb, and provided protection against testicular histopathological injury. These findings suggest that FAE has the potential to serve as a protective agent against oxidative damage caused by contaminants in reproductive organs, specifically in the testes.

Aberrant Expression of TET2 Accounts for DNA Hypomethylation in Varicocele.

OBJECTIVE: Epigenetic modifications such as DNA methylation play a key role in male infertility etiology. This study aimed to explore the global DNA methylation status in testicular spermatogenic cells of varicocele-induced rats and consider their semen quality, with a focus on key epigenetic marks, namely 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), as well as the mRNA and proteins of ten-eleven translocation (TET) methylcytosine dioxygenases 1-3. MATERIALS AND METHODS: In this experimental study, 24 mature male Wistar rats (8 in each group) were assigned amongst the control, sham, and varicocele groups. Sperm quality was assessed, and DNA methylation patterns of testicular spermatogenic cells were investigated using reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunofluorescence techniques. RESULTS: Sperm parameters, chromatin and DNA integrity were significantly lower, and sperm lipid peroxidation significantly increased in varicocele-induced rats in comparison with control rats. During spermatogenesis in rat testis, 5-mC and 5-hmC epigenetic marks, and TET1-3 mRNA and proteins were expressed. In contrast to the 5-mC fluorescent signal which was presented in all testicular cells, the 5-hmC fluorescent signal was presented exclusively in spermatogonia and a few spermatids. In varicocele-induced rats, the 5-mC signal decreased in all cells within the tubules, whereas a strong signal of 5-hmC was detected in seminiferous tubules compared to the control group. As well, the levels of TET2 mRNA and protein expression were significantly upregulated in varicocele-induced rats in comparison with the control group. Also, our results showed that the varicocele-induced animals exhibited strong fluorescent signals of TET1-3 in testicular cells, whereas weak fluorescent signals were identified in the seminiferous tubules of the control animals. CONCLUSION: Consequently, we showed TET2 upregulation and the 5-hmC gain at testicular levels are associated with varicocele and sperm quality decline, and therefore they can be exploited as potential biomarkers of spermatogenesis.

Distribution of Plasma One-Carbon Metabolism Factors and Amino Acids Profile in Depression State Treated with Paroxetine: A Model Study.

OBJECTIVE: Stress may have an important role in the origin and progress of depression and can impair metabolic homeostasis. The one-carbon cycle (1-CC) metabolism and amino acid (AA) profile are some of the consequences related to stress. In this study, we investigated the Paroxetine treatment effect on the plasma metabolite alterations induced by forced swim stress-induced depression in mice. MATERIALS AND METHODS: In this experimental study that was carried out in 2021, thirty male NMRI mice (6-8 weeks age, 30 ± 5 g) were divided into five groups: control, sham, paroxetine treatment only (7 mg/kg BW/day), depression induction, and Paroxetine+depression. Mice were subjected to a forced swim test (FST) to induce depression and then were treated with Paroxetine, for 35 consecutive days. The swimming and immobility times were recorded during the interventions. Then, animals were sacrificed, plasma was prepared and the concentration of 1-CC factors and twenty AAs was measured by spectrophotometry and high-performance liquid chromatography system (HPLC) techniques. Data were analyzed by SPSS, using One-Way ANOVA and Pearson Correlation, and P<0.05 was considered significant. RESULTS: Plasma concentrations of phenylalanine, glutamate, aspartate, arginine, ornithine, citrulline, threonine, histidine, and alanine were significantly reduced in the depression group in comparison with the control group. The Homocysteine (Hcy) plasma level was increased in the Paroxetine group which can be associated with hyperhomocysteinemia. Moreover, vitamin B12, phenylalanine, glutamate, ornithine, citrulline, and glycine plasma levels were significantly reduced in the depression group after Paroxetine treatment. CONCLUSION: This study has demonstrated an impairment in the plasma metabolites' homeostasis in depression and normal conditions after Paroxetine treatment, although, further studies are required.

Assisted Oocyte Activation following Intracytoplasmic Sperm Injection: A Sensible Option for Infertile Couples with Severe Teratozoospermia.

The intracytoplasmic sperm injection (ICSI) has significantly improved male factor infertility treatment; however, complete fertilization failure still occurs in 1-5% of ICSI treatment cycles mainly due to oocyte activation failure. It is estimated that around 40-70% of oocyte activation failure is associated with sperm factors after ICSI. Assisted oocyte activation (AOA) as an effective approach to avoid total fertilization failure (TFF) has been proposed following ICSI. In the literature, several procedures have been described to overcome failed oocyte activation. These include mechanical, electrical, or chemical stimuli initiating artificial Ca2+ rises in the cytoplasm of oocytes. AOA in couples with previous failed fertilization and those with globozoospermia has resulted in varying degrees of success. The aim of this review is to examine the available literature on AOA in teratozoospermic men undergoing ICSI-AOA and determine whether the ICSI-AOA should be considered as an adjunct fertility procedure for these patients.

The effect of alpha-lipoic acid on sperm functions in rodent models for male infertility: A systematic review.

In this systematic review, we assessed different studies to evaluate the protective effect of alpha-lipoic acid (ALA), as a multifaceted antioxidant, on sperm functions in rodent models. Four databases were searched to find papers reporting the effect of ALA treatment on animal models of male infertility. Up to December 2022, 11,787 articles were identified to explain the ALA protective effects. The included studies were evaluated for eligibility and risk of bias (CRD42022341370). Finally, we identified 23 studies that explain the effect of ALA on sperm functions in rodents. Among them, 15 studies indicated that ALA could restore sperm parameters. Six studies showed a significant reduction in sperm DNA damage by ALA treatment. Seventeen papers displayed the ALA antioxidant ability, and four studies indicated the ALA anti-inflammatory effect. Besides, thirteen studies displayed that ALA could modulate androgenesis. Also, eighteen studies revealed that ALA restored the testicular architecture to normal, and was also effective in restoring reproductive performance in two included studies. This systematic review provided cogent evidence for the protective effect of ALA in rodent models for male infertility by re-establishing spermatogenesis and steroidogenesis and maintaining redox and immune systems homeostasis.

Ferulago angulata extract improves the quality of buck spermatozoa post-thaw and counteracts the harmful effects of diazinon and lead.

During cryopreservation, spermatozoa are exposed to chemical or physical stress that has adverse effects on the quality of mammalian spermatozoa. Recently, much attention has been paid to environmental contaminants (ECs) in livestock, because of their detrimental effects on livestock productivity and fertility. ECs like diazinon (DZN) and lead acetate (LA) are considered ubiquitous and induced oxidative stress, which decreases spermatozoa quality. Since Ferulago angulata extract (FAE) has antioxidant properties, the present study investigated the effect of FAE supplementation in a freezing extender, in the presence or absence of DZN + LA, during cryopreservation, on the quality and fertility ability of buck spermatozoa after thawing. Pooled ejaculates were diluted with a freezing extender and supplemented with FAE (0.002%, w/v) in the presence or absence of DZN (100 µM) + LA (12.5 µM). Post-thaw spermatozoa parameters, ROS production, fertilization ability, and developmental competence of oocytes inseminated with FAE/DZN + LA treated spermatozoa were calculated. The results demonstrated that FAE improves cryopreserved spermatozoa motility, viability, membrane integrity, fertilizability, and developmental competence, and reduced spermatozoa ROS production in the presence or absence of DZN + LA. Besides, FAE significantly restored the adverse effects of DZN + LA exposure during cryopreservation on inner cell mass (ICM) count, trophectoderm (TE) cell count, total cell number (TCN), and the ratio between ICM to TCN. In conclusion, FAE on its own resulted in an improvement in the buck spermatozoa's quality and fertility. Therefore, the addition of FAE, as a natural antioxidant to buck semen extender, can increase spermatozoa cryotolerance and post-thaw resistance even when exposed to ECs.

Ferulago angulata extract ameliorates epididymal sperm toxicity in mice induced by lead and diazinon.

BACKGROUND: The potential toxicity that results from environmental xenobiotics is not completely known. Increasing levels of heavy metals and the use of organophosphate pesticides (OPs) and their co-existence in the environment could be associated with an increasing incidence of male reproductive system disorders in humans and animals. Ferulago angulata is a dietary source of phenolic compounds with reported health benefits. OBJECTIVE: This study was conducted to investigate whether an extract of Ferulago angulata could protect adult male NMRI mice against reproductive toxicity induced by lead acetate (PbAc), diazinon (DZN), or PbAc + DZN. MATERIALS AND METHODS: Adult male NMRI mice were exposed to either 0.5% PbAc in drinking water, DZN (3 mg/kg/day, intraperitoneal [i.p.] injection), or PbAc + DZN in the presence or absence of 400 mg/kg/day Ferulago angulata hydroalcoholic extract (FAE) that was administered via gavage for 6 weeks. RESULTS: Chronic exposure to PbAc, DZN, and PbAc + DZN decreased sperm quality, sperm chromatin maturity and integrity, increased oxidative stress and lipid peroxidation, and could reduce male fertility indices. Co-administration of FAE could reduce these negative effects. CONCLUSION: The Ferulago angulata extract should be considered as a useful natural extract for the treatment of male infertility, especially in males exposed to conditions which induce reproductive toxicity.