Overlapping sperm damages from vitamin B or D deficiency in mice: Insights into the role of clinical supplementations.

In this study, the effect of 14 weeks of standard diet (controls) or folate and vitamin B12-free diet (VBD group) or vitamin D-free diet (VDD group) were assessed on mice testicular function, and sperm function. Vitamin D deprivation caused increased body weight with no effect from VBD confirming the calcium-independent role of vitamin D on body weight homeostasis. The two deprivations caused convergent damages including decreased testosterone, worsened Johnson scores, tubular differentiation index and spermatogenesis index, and serious worsening of sperm parameters and of sperm functional tests (DNA methylation, protamination, DNA damage and lipid peroxidation). From a metabolic point of view, the damage from both models converged on the one carbon cycle (methylations) and the transsulfuration pathway (GSH and antioxidant defences) and increased circulating homocysteine, although with different mechanisms: VBD appeared to hamper methylations due to lower ability to regenerate homocysteine to methionine whereas VDD appeared to interfere with homocysteine transsulfuration to cysteine and, thereafter, GSH. VDD also caused a huge paradox increase of vitamin B12, which was likely in a non-functional form and warrants further investigation. These findings strongly endorse the potential benefit of combined folate/B12 and vitamin D supplementation in infertile patients.

Alpha-lipoic acid improves sperm motility in infertile men after varicocelectomy: a triple-blind randomized controlled trial.

RESEARCH QUESTION: Does supplementation with alpha-lipoic acid (ALA) enhance sperm parameters and/or the status of sperm lipid peroxidation and DNA fragmentation in men who have undergone microsurgical repair of a varicocele? DESIGN: Individuals with a varicocele who had undergone varicocelectomy were divided into two groups receiving either 600 mg of ALA or an identical placebo for 80 days. Semen samples obtained from the participants before surgery and after completion of the course of medication were analysed and compared. Participants, clinicians and data analysts were blinded to the randomization sequence. RESULTS: In the ALA group, total motility (P = 0.01) and progressive motility (P = 0.002) of the spermatozoa were significantly higher compared with the placebo group after surgery. Sperm lipid peroxidation and DNA damage (assessed by sperm chromatin structure assay) showed significant decreases in both the ALA and placebo groups (P ≤ 0.02) after treatment. CONCLUSIONS: An 80-day course of ALA medication after surgical repair improves total motility and progressive motility of the spermatozoa in individuals with a varicocele.

Alpha-lipoic acid minimises reactive oxygen species-induced damages during sperm processing.

Shearing forces during sperm preparation for assisted reproduction techniques may lead to excessive production of reactive oxygen species (ROS) which may have an unpleasant effect on embryonic development. In the current study, we assessed the effect of alpha-lipoic acid (ALA) on ROS-induced damages during sperm preparation process. Semen samples were collected from 15 normozoospermic men. Each semen sample was divided into two parts; one part was washed and centrifuged with sperm washing medium plus 0.02 mM ALA. Then, sperm pellet was diluted and incubated for 1 hr at 37°C in sperm washing media in the absence (ALA-) or presence of 0.02 mM ALA (ALA+). The second part was washed and centrifuged with sperm washing media in the absence of ALA, and then, sperm pellet was incubated for 1 hr at 37°C in sperm washing media in the absence (ALA-) or presence of 0.02 mM ALA (ALA+). Sperm viability, motility, intracellular oxidative stress and DNA fragmentation were assessed by eosin-nigrosin, computer-assisted sperm analysis system, H2 DCFDA staining and acridine orange staining respectively. Our results showed that addition of ALA as a fat- and water-soluble antioxidant to sperm washing media maintains sperm viability and motility by reduction in ROS production and can also protect sperm DNA integrity.

Zeta method: A noninvasive method based on membrane charge for selecting spermatozoa expressing high level of phospholipaseCζ.

Intracytoplasmic sperm injection (ICSI) is a technique developed to help attain successful fertilisation for couples with severe male factor. However, a small percentage of couples confront low or failed fertilisation, mainly due to failed oocyte activation. Several studies have introduced phospholipase Cζ (PLCζ) as the main sperm factor inducing oocyte activation and thereby has the potential to act as a biomarker for the prediction of ICSI fertilisation outcome. On the other hand, researchers have focused on novel sperm selection procedures based on cellular characteristics of spermatozoa such as surface electrical charge (Zeta potential) to isolate normal sperm subpopulation with intact chromatin. Therefore, we aimed to compare PLCζ between Zeta method and routine sperm preparation procedure: density gradient centrifugation (DGC). Our results showed that number of PLCζ-positive spermatozoa was significantly low in the Zeta method, but the intensity of PLCζ protein in such spermatozoa was significantly higher than DGC procedure. Therefore, the combination of DGC with Zeta procedure may allow selecting the population of spermatozoa with a high percentage of PLCζ which may also contain a high amount of PLCζ and with intact chromatin. This sperm selection procedure can open a new approach for infertile men with previously failed fertilisation.

Effects of branched-chain amino acid supplementation and/or aerobic exercise on mouse sperm quality and testosterone production.

Individuals who regularly exercise utilise dietary supplements to enhance their exercise routine and to increase lean mass. Branched-chain amino acids (BCAAs) are a popular supplement and have been shown to produce a number of beneficial effects in rodent and human models. Therefore, in the present study, the effect of exercise and/or BCAA on sperm parameters and testes tissue was assessed. C57BL6 male mice were divided to six groups; Control, Exercise (Exc), BCAA (consumes 20 mg BCAAs), BCAA+ (consumes 60 mg BCAAs), BCAA/Exc (consumes 20 mg BCAAs during aerobic training) and BCAA+/Exc (consumes 60 mg BCAAs during aerobic training). After 8 weeks of exercise and oral treatment with BCAA; testes and epididymides were dissected, and sperm function and plasma testosterone were assessed. Exercise significantly improved sperm motility and plasma testosterone in Exercise groups with or without BCAA. Percentage of sperm lipid peroxidation was significantly decreased in Exercise group, while intensity of lipid peroxidation at the same group has significantly increased. Epithelium diameters, meiotic index and Johnson' grade did not show any changes between groups. Unlike intensive exercise, endurance exercise along with modest supplementation of BCAAs, but not an overdose, may have some synergic effect on sperm function and testosterone production.

Hypoxia pathway has more impact than inflammation pathway on etiology of infertile men with varicocele.

This study aimed to compare main molecular markers of hypoxia (HIF1-α and P53) and inflammation (TLR-2, TLR-4 and TNF-α) pathways between infertile men with varicocele and fertile individuals. Sperm parameters such as sperm concentration, motility and morphology were assessed according to World Health Organization (Laboratory manual for the examination and processing of human semen. Geneva, Switzerland, 2010) guideline in 20 infertile men with grade II or III varicocele, and 20 fertile men candidate of family balancing. In addition, sperm DNA fragmentation and molecular markers involved in hypoxia and inflammation pathways were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and real-time PCR respectively. Mean of sperm parameters (concentration, motility and morphology) and DNA integrity were significantly lower in infertile men with varicocele compared to fertile individuals. Unlike markers involved in inflammation pathway, mean expression of markers of hypoxia pathway (HIF1-α and P53) was significantly higher in infertile men with varicocele compared to fertile individuals (p < 0.05), and also a significant correlation was observed between expression of HIF1-α and P53 (r = 0.461; p = 0.003). Overall, the result of this study suggests higher likelihood of involvement of hypoxia pathway, in comparison with inflammation pathway, in pathogenesis varicocele associated with male infertility.

One-carbon cycle support rescues sperm damage in experimentally induced varicocoele in rats.

OBJECTIVES: To investigate whether micronutrients in support of the one-carbon cycle and glutathione synthesis are effective in improving sperm damage after surgical varicocoele induction in rats and whether any effect is achieved without a rebound reductive stress as seen with oral antioxidants. MATERIALS AND METHODS: Surgical varicocoele was induced in adult male Wistar rats and resulted in significant damage to the testis and sperm cells measured at 2 and 4 months after surgery. At 2 months after surgery, rats received a 2-month oral supplementation in support of the one-carbon cycle containing B vitamins (B2, B3, B6, folic acid and B12), N-acetyl-cysteine, zinc, small amounts of vitamin E, and a natural source of betalains and quercetine (Condensyl® ; Parthenogen SAGL, Lugano, Switzerland and Nurilia SARL, Lyon, France). RESULTS: One-carbon cycle supplementation, compared to untreated controls, significantly improved the morphometric characteristics of testis (P < 0.05), sperm concentration, motility and abnormal morphology (P < 0.001), sperm chromatin condensation (aniline blue staining, P < 0.05), sperm DNA damage (acridine orange staining, P < 0.05) and sperm lipid peroxidation (BODIPY C11, P < 0.001). The improvement in both nuclear condensation and DNA damage and the lack of excessive inhibition of lipid peroxidation confirmed that no reductive stress had occurred. CONCLUSIONS: Micronutrients in support of the one-carbon cycle are effective in the treatment of surgically induced varicocoele in rats, probably by activating natural antioxidant defences and epigenetics. These results support the idea that essential micronutrients including B vitamins may also have a positive influence in clinical varicocoele, which should be tested in prospective clinical trials.

Alpha-Lipoic Acid improves the testicular dysfunction in rats induced by varicocele.

Alpha-lipoic acid (ALA) is a disulphide compound with multifunctional antioxidant properties and is soluble in both water and lipid. Several recent studies evaluated efficacy of ALA in various diseases related to oxidative damage such as diabetes, multiple sclerosis and Alzheimer and concluded that ALA can reduce oxidative stress by quenching reactive oxygen and nitrogen species, restoring antioxidants such as glutathione, vitamins C and E, and/or improving activity of antioxidant enzymes. Varicocele, an enlargement of the veins in scrotum, is considered as the most common repairable cause of male infertility and is associated with high levels of oxidative stress. In this study, surgical varicocele was induced in 30 adult male Wistar rats with other 20 rats serving as sham-operated and nonoperated control. Varicocele caused significant worsening of sperm parameters, DNA damage and lipid peroxidation 2 and 4 months after surgery. A 2-month ALA administration after surgery was able to revert these effects. These results clearly showed that ALA can reduce the negative side effects of elevated testicular temperature and increased oxidative stress in varicocelised rats. This study warrants future clinical research to assess whether ALA is of help in the treatment of infertile men with varicocele.

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose.

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose-free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization-competent and are able to produce good-quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.

An association between sperm PLCζ levels and varicocele?

PURPOSE: We aimed to compare the expression of phospholipase C ζ (PLCζ), as one of the main sperm factors involved in oocyte activation, at both RNA and protein levels in fertile men and those with varicocele. METHODS: This study included 35 individuals with male factor infertility presenting primary infertility with grade II and III unilateral varicocele and 20 fertile men without varicocele. Semen parameters were assessed according to WHO 2010. Sperm DNA fragmentation, relative expression of PLCζ at messenger RNA, and protein levels were evaluated by sperm chromatin structure assay (SCSA), real-time PCR, and Western blot analysis, respectively. RESULTS: The results of this study reveal that the mean relative expression of PLCζ was significantly lower in individuals with varicocele compared to fertile men at both transcription and translation levels. In addition, the percentage of DNA fragmentation was significantly higher in infertile men with varicocele compared to fertile men. CONCLUSIONS: The findings of the present study illustrate that one of the etiologies of reduced fertility associated with varicocele is the low expression of PLCζ. This effect could subsequently reduce the sperm ability to induce oocyte activation. Therefore, these results hold promise to modify our understanding of reproductive physiology of varicocele state.

Safety, efficacy and pitfalls of fibrocyte application in the treatment of diabetic foot ulcer.

Fibrocytes are unique bone marrow-derived cells with great potential in wound healing. Hence, the aim of this study was to determine the safety and efficacy of the applied circulating fibrocytes in the treatment of non healing diabetic foot ulcers. Peripheral blood mononuclear cells were isolated by centrifugation through Ficoll-Paque method. After 3 days, the non adherent cells were removed by a single, gentle aspiration. Adherent cells were cultured in the same medium for 10 days. The cells were characterised using mouse anti-human-CD45-fluorescein isothiocyanate (FITC) and mouse anti-human-collagen I, and also characterised by immunofluorescence microscopy using the above mentioned antibodies. Sterility measures were applied for clinical evaluation. Based on the literature review, cell transplantation generally requires at least 3 × 10(6) cells regarding efficacy measures. As fibrocytes are non proliferating cells, 350 ml patient's blood is required to prepare patient-specific serum before cell isolation and culture, and 85 ml patient's blood is needed for cell isolation and differentiation on cell transplantation applications. In our survey, no diabetic patient was inclined to be donor of such blood volume, mainly because of their pre-assumption that they are anaemic. It is concluded that fibrocytes do not seem to be candidate cells for cell therapy in the treatment of diabetic foot ulcers because of the rarity of this cell population in circulation.

Globozoospermia is mainly due to DPY19L2 deletion via non-allelic homologous recombination involving two recombination hotspots.

To date, mutations in two genes, SPATA16 and DPY19L2, have been identified as responsible for a severe teratozoospermia, namely globozoospermia. The two initial descriptions of the DPY19L2 deletion lead to a very different rate of occurrence of this mutation among globospermic patients. In order to better estimate the contribution of DPY19L2 in globozoospermia, we screened a larger cohort including 64 globozoospermic patients. Twenty of the new patients were homozygous for the DPY19L2 deletion, and 7 were compound heterozygous for both this deletion and a point mutation. We also identified four additional mutated patients. The final mutation load in our cohort is 66.7% (36 out of 54). Out of 36 mutated patients, 69.4% are homozygous deleted, 19.4% heterozygous composite and 11.1% showed a homozygous point mutation. The mechanism underlying the deletion is a non-allelic homologous recombination (NAHR) between the flanking low-copy repeats. Here, we characterized a total of nine breakpoints for the DPY19L2 NAHR-driven deletion that clustered in two recombination hotspots, both containing direct repeat elements (AluSq2 in hotspot 1, THE1B in hotspot 2). Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene. This is a major finding and should contribute to the development of an efficient molecular diagnosis strategy for globozoospermia.

Peptide functionalized polyhydroxyalkanoate nanofibrous scaffolds enhance Schwann cells activity.

Interactions between Schwann cells (SCs) and scaffolds are important for tissue development during nerve regeneration, because SCs physiologically assist in directing the growth of regenerating axons. In this study, we prepared electrospun scaffolds combining poly (3-hydroxybutyrate) (PHB) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) functionalized with either collagen I, H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS), H-Tyr-Ile-Gly-Ser-Arg-NH2 (YIGSR), or H-Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile-OH (p20) neuromimetic peptides to mimic naturally occurring ECM motifs for nerve regeneration. Cells cultured on fibrous mats presenting these biomolecules showed a significant increase in metabolic activity and proliferation while exhibiting unidirectional orientation along the orientation of the fibers. Real-time PCR showed cells cultured on peptide-modified scaffolds had a significantly higher neurotrophin expression compared to those on untreated nanofibers. Our study suggests that biofunctionalized aligned PHB/PHBV nanofibrous scaffolds may elicit essential cues for SCs activity and could serve as a potential scaffold for nerve regeneration. From the clinical editor: Nanotechnology-based functionalized scaffolds represent one of the most promising approaches in peripheral nerve recovery, as well as spinal cord recovery. In this study, bio-functionalized and aligned PHB/PHBV nanofibrous scaffolds were found to elicit essential cues for Schwann cell activity, therefore could serve as a potential scaffold for nerve regeneration.

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos.

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

Short-term in-vitro culture of goat enriched spermatogonial stem cells using different serum concentrations.

PURPOSE: To investigate the effect of serum supplementing on short-term culture, fate determination and gene expression of goat spermatogonial stem cells (SSCs). METHODS: Crude testicular cells were plated over Datura-Stramonium Agglutinin (DSA) for 1 h, and non-adhering cells were cultured in the presence of different serum concentrations (1, 5, 10, and 15%) for 7 days in a highly enriched medium initially developed in mice. Colonies developed in each group were used for the assessment of morphology, immunocytochemistry, and gene expression. RESULTS: Brief incubation of testicular cells with DSA resulted in a significant increase in the number of cells that expressed the germ cell marker (VASA). The expression of THY1, a specific marker of undifferentiated spermatogonia, was significantly higher in colonies developed in the presence of 1% rather than 5, 10 and 15% serum. CONCLUSION: Goat SSCs could proliferate and maintain in SSC culture media for 1 week at serum concentrations as low as 1%, while higher concentrations had detrimental effects on SSC culture/expansion.

Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent.

In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.

Is ferroptosis involved in ROS-induced testicular lesions in a varicocele rat model?

BACKGROUND: Ferroptosis is an iron-dependent cell death that is distinct from apoptosis. Based on excessive amounts of iron and reactive oxygen species in varicocele (VCL) rats, we hypothesize that ferroptosis might be involved in VCL. In addition, since alpha-lipoic acid (ALA) was shown to have both antioxidant and anti-ferroptotic activity we assessed in the present work the status of ferroptosis in our varicocele model and the protective effect of ALA. To this end, 70 male Wistar rats were divided into 7 groups: control, sham and varicocele groups which were initially sacrificed 2 months after the operation to verify the induction of varicocele. A second batch of the same 3 groups were sacrificed 4 months after varicocele induction to evaluate the effect of ALA supplementation. The parameters measured were chromatin integrity (aniline blue and acridine orange staining), lipid peroxidation (BODIPY staining), testicular morphometry and iron content. In addition, redox (GSH and NADPH) and ferroptosis (Nrf2, Slc7a11, P53 and p-Jnk) markers were evaluated at 2 and 4 months post-operation. RESULT: The alteration of the spermatic parameters made it possible to verify the induction of the varicocele. Iron accumulated well in the testicles during varicocele and decreased significantly following ALA treatment. Ferroptotic molecular markers at the mRNA and protein levels were not significantly altered. ALA supplementation did not alter NADPH values, but increased GSH levels. CONCLUSION: Despite the increased accumulation of iron in the testes 2 and 4 months after surgical induction of varicocele, molecular evidence did not demonstrate the involvement of ferroptosis. This could be explained by the mosaic nature of the varicocele affecting some seminiferous tubules and not others which could mask variations in molecular markers. In parallel, our study confirms that ALA stimulates the NRF2 pathway.

RéSUMé: CONTEXTE: La ferroptose est une mort cellulaire dépendante du fer qui est distincte de l'apoptose. Sur la base de quantités excessives de fer et d'espèces réactives de l'oxygène chez les rats varicocèles (VCL), nous avons fait l'hypothèse que la ferroptose pourrait être impliquée dans la VCL. Comme l'acide alpha-lipoïque (ALA) s'est avéré avoir des activités antioxydante et anti-ferroptotique nous avons dans ce travail testé le statut ferroptose dans notre modèle de rats VCL et évalué l'effet protecteur de ALA. Dans ce but, 70 rats mâles Wistar ont été divisés en plusieurs groupes: témoin, fictif et groupes varicocèle qui ont été initialement sacrifiés deux mois après l'opération pour vérifier l'induction de la varicocèle. Un deuxième lot des 3 mêmes groupes a été sacrifié 4 mois après l'induction de la varicocèle pour évaluer l'effet de la supplémentation en ALA. Les paramètres mesurés étaient l'intégrité de la chromatine (via les tests au bleu d'aniline et à l'orange acridine), la peroxydation lipidique (via le test BODIPY), la morphométrie testiculaire et la teneur en fer. De plus, les marqueurs redox (GSH et NADPH) et ferroptotique (Nrf2, Slc7a11, P53 et p-Jnk) ont été évalués 2 et 4 mois après l'opération. RéSULTATS: L'altération des paramètres spermatiques a permis de vérifier l'induction de la VCL. Le fer s'est bien accumulé dans les testicules pendant la VCL et a diminué de manière significative après le traitement ALA. Les marqueurs moléculaires ferroptotiques n'ont pas été modifiés de manière significative que ce soit en quantité d'ARNm et de protéines. La supplémentation en ALA n'a pas modifié la teneur en NADPH, mais a augmenté les niveaux de GSH. CONCLUSIONS: Malgré l'accumulation accrue de fer dans les testicules 2 et 4 mois après l'induction chirurgicale de la VCL, les investigations au niveau moléculaire n'ont pas clairement démontré l'implication de la ferroptose. Cela pourrait s'expliquer par la nature mosaïque de la VCL qui affecte certains tubules séminifères et pas d'autres, ce qui pourrait atténuer les évaluations réalisées sur organe entier. En parallèle, notre étude confirme que l'ALA stimule la voie NRF2 stimulant la réponse anti-oxydante.

Single-blind clinical trial: Sperm selection based on capacity to pass through cumulus oophorous column improves ICSI outcomes.

BACKGROUND: Sperm selection procedures for future strategies that aim to select normal spermatozoa with intact DNA to improve intracytoplasmic sperm injection (ICSI) outcomes are in early developing stage. OBJECTIVES: The objective is to find out whether the sperm selection procedure based on the ability of spermatozoa to traverse the cumulus cells could improve clinical outcomes of ICSI technique in infertile couples with male factor etiology. MATERIALS AND METHODS: For this single-blind clinical trial, mature metaphase II oocytes were retrieved from 150 couples with male factor infertility, male age lower than 45 years and female age under 38 years. These couples were divided into two groups. In control group (n = 75), spermatozoa processed by density gradient centrifugation (DGC) were used to inject the oocytes. In the study group (n = 75), the oocytes were divided into sibling groups. In one sibling group (DGC), the oocytes were inseminated with DGC-processed spermatozoa while in the other group (DGC-CC), they were inseminated with DGC-processed spermatozoa that passed cumulus oophorous column. RESULTS: Mean fertilization and embryo quality were significantly higher in DGC-CC group compared to DGC and control group. In addition, mean of chemical pregnancy (52.27% vs. 34.14%; p = 0.05), clinical pregnancy based on sac (52.27% vs. 32.92%; p = 0.03), clinical pregnancy with heart beat (52.27% vs. 25.60%; p = 0.003) and ongoing pregnancy (43.18% vs. 21.95%; p = 0.02) rates were significantly higher in DGC-CC group compared to control group. CONCLUSION: Sperm selection based on integrated systems such as DGC and ability to pass through cumulus oophorous column could improve clinical outcomes of ICSI in couples with male factor infertility.

Signs of ROS-Associated Autophagy in Testis and Sperm in a Rat Model of Varicocele.

Since autophagy was suspected to occur in the pathological situation of varicocele (VCL), we have attempted to confirm it here using a surgical model of varicocele-induced rats. Thirty Wistar rats were divided into three groups (varicocele/sham/control) and analyzed two months after the induction of varicocele. Testicular tissue sections and epididymal mature sperm were then monitored for classic features of varicocele, including disturbance of spermatogenesis, impaired testicular carbohydrate and lipid homeostasis, decreased sperm count, increased sperm nuclear immaturity and DNA damage, oxidative stress, and lipid peroxidation. At the same time, we evaluated the Atg7 protein content and LC3-II/LC3-1 protein ratio in testis and mature sperm cells, two typical markers of early and late cellular autophagy, respectively. We report here that testis and mature sperm show higher signs of autophagy in the varicocele group than in the control and sham groups, probably to try to mitigate the consequences of VCL on the testis and germ cells.

Endoplasmic Reticulum Stress (ER Stress) and Unfolded Protein Response (UPR) Occur in a Rat Varicocele Testis Model.

Using a surgically induced varicocele rat model, we show here strong evidence that the misfolded/unfolded protein response that is part of the stress response of the endoplasmic reticulum (ER) is activated in the varicocele testis (VCL), leading to the induction of apoptosis. To support this hypothesis, it is observed that the spliced variant of the X-box protein 1 (XBP1s), resulting from the activation of the inositol-requiring enzyme 1 (IRE1) membrane sensor, is significantly more represented in VCL testicular extracts. The activation of the IRE1/XBP1s pathway is also supported by the observation that the VCL testes show an increase phosphorylation of the c-Jun-kinase (JNK) known to be one intermediate of this pathway and an increased level of caspase-3, the terminal apoptotic effector, partly explaining the apoptotic status of the VCL testis.