Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction.

BACKGROUND: Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development. In some mammals, like pigs, ejaculates are emitted in three separate fractions: pre-sperm, sperm-rich (SRF) and post sperm-rich (PSRF). These fractions are known to vary in volume, sperm concentration and quality, as well as in the origin and composition of seminal plasma (SP), with differences being also observed within the SRF one. Yet, whether disparities in the DNA integrity and chromatin condensation and protamination of their sperm exist has not been interrogated. RESULTS: This study determined chromatin protamination (Chromomycin A3 test, CMA3), condensation (Dibromobimane test, DBB), and DNA integrity (Comet assay) in the pig sperm contained in the first 10 mL of the SRF (SRF-P1), the remaining portion of the sperm-rich fraction (SRF-P2), and the post sperm-rich fraction (PSRF). While chromatin protamination was found to be similar between the different ejaculate fractions (P > 0.05), chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF (P = 0.018 and P = 0.004, respectively). Regarding DNA integrity, no differences between fractions were observed (P > 0.05). As the SRF-P1 has the highest sperm concentration and ejaculate fractions are known to differ in antioxidant composition, the oxidative stress index (OSi) in SP, calculated as total oxidant activity divided by total antioxidant capacity, was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF (0.42 ± 0.06 vs. 0.23 ± 0.09 and 0.08 ± 0.00, respectively; P < 0.01); this index, in addition, was observed to be correlated to the sperm concentration of each fraction (Rs = 0.973; P < 0.001). CONCLUSION: While sperm DNA integrity was not found to differ between ejaculate fractions, SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF. This could be related to the OSi of each fraction.

Sperm degradation after vasectomy follows a sperm chromatin fragmentation-dependent mechanism causing DNA breaks in the toroid linker regions.

Vasectomy is a widely used surgical technique creating an obstructive azoospermia. Although sperm cannot be ejaculated, the testis maintains sperm production in vasectomized males. The continuous accumulation of sperm deposited in the epididymis and the vas deferens fraction necessarily need to be degraded and eliminated. While the elimination process is carried out by granulomas that form after vasectomy, the detailed mechanisms of sperm degradation are still not known. The aim was to assess whether sperm chromatin fragmentation (SCF), a mechanism that degrades the entire sperm genome at the toroid linker regions (TLRs), is activated after vasectomy in sperm cells. We vasectomized mice and evaluated the presence of TLR-specific double-strand breaks through pulsed-field gel electrophoresis and the Comet assay at 1, 2 and 3 weeks after surgery. Results for DNA damage (Olive tail moment) at single-cell level showed an increase of double-strand breaks after vasectomy for vas deferens sperm after 1, 2 and 3 weeks postvasectomy (21.78 ± 2.29; 19.71 ± 1.79 and 32.59 ± 1.81, respectively), compared to mock surgery (7.04 ± 1.03; 10.10 ± 1.29 and 8.64 ± 0.85, respectively; P < 0.001). Similar findings were obtained for cauda epididymis sperm (P < 0.001), but not for caput epididymis (P > 0.05). Pulsed-field gel electrophoresis showed the presence of double-stranded breaks between 15 and 145 kb, indicating that DNA breaks were produced mainly in the sperm TLRs. Results presented here suggest that SCF is a mechanism activated in vas deferens after vasectomy to degrade sperm DNA when they cannot be ejaculated, preventing their function.

Women's and men's intake of omega-3 fatty acids and their food sources and assisted reproductive technology outcomes.

BACKGROUND: Long-chain omega-3 fatty acids and their food sources have garnered interest as a potential nutrient with wide-range health benefits, including fertility. OBJECTIVE: This study aimed to investigate the association of women's and men's intake of omega-3 fatty acids and omega-3 rich-foods with semen quality and outcomes of infertility treatment with assisted reproductive technologies. STUDY DESIGN: Couples presenting to the Massachusetts General Hospital were invited to enroll in a prospective cohort study (2007-2020). Male and female diets were assessed using a validated 131-item food frequency questionnaire. The primary outcomes were implantation, clinical pregnancy, and live birth probabilities. The secondary outcomes included total and clinical pregnancy loss and conventional semen parameters, for males only. We estimated the relationship between intakes of omega-3 fatty acids, nuts, and fish and the probability (95% confidence interval) of study outcomes using generalized linear mixed models to account for repeated treatment cycles per participant while simultaneously adjusting for age, body mass index, smoking status, education, dietary patterns, total energy intake, and male partner diet. RESULTS: A total of 229 couples and 410 assisted reproductive technology cycles were analyzed for primary and secondary outcomes. Of note, 343 men contributing 896 semen samples were included in analyses for semen quality measures. Women's docosahexaenoic acid + eicosapentaenoic acid intake was positively associated with live birth. The multivariable-adjusted probabilities of live birth for women in the bottom and top quartiles of eicosapentaenoic acid + docosahexaenoic acid intake were 0.36 (95% confidence interval, 0.26-0.48) and 0.54 (95% confidence interval, 0.42-0.66) (P trend=.02). Eicosapentaenoic acid + docosahexaenoic acid intake was inversely related to the risk of pregnancy loss, which was 0.53 among women in the lowest quartile of eicosapentaenoic acid + docosahexaenoic acid intake and 0.05 among women in the highest quartile (P trend=.01). Men's intake of total omega-3 fatty acids was positively related to sperm count, concentration, and motility, but unrelated to any assisted reproductive technology outcomes. Similar associations were observed when evaluating the intake of primary food sources of these fatty acids. CONCLUSION: Women's consumption of omega-3 fatty acids and omega-3-rich foods may improve the probability of conception by decreasing the risk of pregnancy loss. In addition, men's intake of omega-3 fatty acids may influence semen quality.

Microsurgical varicocelectomy effect on sperm telomere length, DNA fragmentation and seminal parameters.

Varicocele is one of the main causes of male infertility and microsurgical varicocelectomy (MV) seems to be the best procedure for its repair and to reduce testicular oxidative stress (ROS). As ROS causes guanine modifications, we postulated that DNA damage could be more intense in telomeres due to their G-rich nature. We studied the effect of MV on sperm telomere length (TL), single- and double-strand DNA fragmentation (ssSDF and dsSDF) and seminal parameters. Sperm telomeres from 12 fertile donors and 20 varicocele patients before and nine months after MV were labelled using FITC-PNA qFISH (a new method to obtain absolute TL from relative fluorescence intensity using FITC-fluorescent spheres). Both ssSDF and dsSDF were analysed using the alkaline and neutral Comet assays, respectively. The results showed that varicocele and MV had no effect on TL. Seminal parameters, ssSDF and dsSDF of varicocele patients were altered. Although these parameters improved after MV, values did not reach those seen in fertile donors. A good estimation of absolute TL was developed based on FITC-fluorescent spheres. The results showed that TL is not affected by varicocele or surgery. However, MV is able to partially reduce altered seminal parameters, ssSDF and dsSDF values in varicocele patients.

Sperm DNA damage compromises embryo development, but not oocyte fertilisation in pigs

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.

Oral antioxidant treatment partly improves integrity of human sperm DNA in infertile grade I varicocele patients.

Infertile males with varicocele have the highest percentage of sperm cells with damaged DNA, compared to other infertile groups. Antioxidant treatment is known to enhance the integrity of sperm DNA; however, there are no data on the effects in varicocele patients. We thus investigated the potential benefits of antioxidant treatment specifically in grade I varicocele males. Twenty infertile patients with grade I varicocele were given multivitamins (1500 mg L-Carnitine, 60 mg vitamin C, 20 mg coenzyme Q10, 10 mg vitamin E, 200 µg vitamin B9, 1 µg vitamin B12, 10 mg zinc, 50 µg selenium) daily for three months. Semen parameters including total sperm count, concentration, progressive motility, vitality, and morphology were determined before and after treatment. In addition, sperm DNA fragmentation and the amount of highly degraded sperm cells were analyzed by Sperm Chromatin Dispersion. After treatment, patients showed an average relative reduction of 22.1% in sperm DNA fragmentation (p = 0.02) and had 31.3% fewer highly degraded sperm cells (p = 0.07). Total numbers of sperm cells were increased (p = 0.04), but other semen parameters were unaffected. These data suggest that sperm DNA integrity in grade I varicocele patients may be improved by oral antioxidant treatment.