Low levels of mouse sperm chromatin fragmentation delay embryo development.

We previously demonstrated that MnCl2 induces double-stranded DNA breaks in sperm in a process that we term as sperm chromatin fragmentation. Here, we tested if the levels of double-stranded DNA breaks were corelated to the concentration of MnCl2, and we compared this to another agent that causes single-stranded DNA breaks, H2O2. We found that both methods have the advantage of inducing DNA breaks in a concentration-dependent manner. Mouse sperm were treated with varying concentrations of either H2O2 or MnCl2, and the DNA damage was assessed by pulse-field gel electrophoresis, and the alkaline and neutral comet assays. Oocytes were injected with either treated sperm and the resulting embryos analyzed with an embryoscope to detect subtle changes in embryonic development. We confirmed that H2O2 treatment induced primarily single-stranded DNA breaks and MnCl2 induced primarily double-stranded DNA breaks, indicating different mechanisms of damage. These sperm were injected into oocytes, and the development of the resulting embryos followed with an embryoscope equipped with time lapse recording. We found that aberrations in early embryonic development by day 2 with even the lowest levels of DNA damage and that the levels of embryonic aberrations correlated to the concentration of either H2O2 or MnCl2. Low levels of H2O2 caused significantly more aberrations in embryonic development than low levels of MnCl2 even though the levels of DNA damage as measured by comet assays were similar. These data demonstrate that even low levels of sperm DNA damage cause delays and arrests in embryonic development.

Condensation and protamination of sperm chromatin affect ICSI outcomes when gametes from healthy individuals are used.

STUDY QUESTION: Do defects in sperm chromatin protamination and condensation have an impact on ICSI outcomes? SUMMARY ANSWER: Sperm protamination is related to fertilization rates in healthy donors, and the in vitro capacity of sperm to condense their chromatin is linked to blastocyst rates, both associations being more apparent in women <33 years of age. WHAT IS KNOWN ALREADY: Previous data on how sperm chromatin damage affects ICSI outcomes are inconsistent. Revealing which sperm factors influence embryo development is necessary to understand the male contribution to ICSI success and to develop novel sperm selection techniques or male-based treatments. Sperm chromatin is mainly condensed in protamines, which are cross-linked through disulphide bridges. This study aimed to determine whether sperm protamination and the integrity of disulphide bonds (condensation) are related to embryo development after ICSI. STUDY DESIGN, SIZE, DURATION: The design was a retrospective study with a blind analysis of sperm chromatin. Gametes were divided into two groups: double donation (DD) cohort and single donation (SD) cohort. Samples from 45 semen donors used in 55 ICSI cycles with oocyte donors (age range 19-33 years), generating 491 embryos, were included in the DD cohort. The SD cohort consisted of samples from 34 semen donors used in 41 ICSI cycles with oocytes from healthy females (single-parent families or lesbian couples, age range 20-44 years), generating a total of 378 embryos. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Donor sperm samples from DD and SD cohorts were used for standard ICSI, and embryo development was observed by time-lapse imaging. The incidence of thiol reduction (dibromobimane, DBB) and the degree of chromatin protamination (chromomycin A3, CMA3, indicating non-protaminated regions) in sperm were determined by flow cytometry at 0 and 4 h post-thawing. MAIN RESULTS AND THE ROLE OF CHANCE: Percentages ± standard deviation of CMA3 were 21.08 ± 9.09 and 35.01 ± 14.68 at 0 and 4 h post-thawing, respectively, in the DD cohort and 22.57 ± 9.48 and 35.79 ± 12.58, at 0 and 4 h post-thawing, respectively, in the SD cohort. Percentages of DBB+ were 16.57 ± 11.10 and 10.51 ± 8.40 at 0 and 4 h post-thawing (P < 0.0001), respectively, in the DD cohort and 17.98 ± 10.19 and 12.72 ± 8.76 at 0 and 4 h post-thawing (P < 0.0001), respectively, in the SD cohort. Female age correlated with fertilization rates, and the relation between sperm chromatin and embryo development was determined through multiple linear regression. While CMA3 was associated with fertilization rates, with no influence of female age, in the DD cohort (ß1 = -1.036, P < 0.001 for CMA3; ß2 = 0.667, P = 0.304 for female age), this was not observed in the SD cohort, where female age had a significant effect, masking the effects of CMA3 (ß1 = -0.066, P = 0.804 for CMA3; ß 2 = -1.451, P = 0.003 for female age). The in vitro capacity of sperm to condense their chromatin after 4 h of incubation was associated with blastocyst rates, independent of female age (DD cohort: ß1 = -0.238, P = 0.008 for %DBB+ variation; ß2 = 0.404, P = 0.638 for female age; SD cohort: ß1 = -0.278, P = 0.010 for %DBB+ variation; ß2 = -0.292, P = 0.594 for female age). The in vitro capacity of sperm to condense their chromatin was also related to the time required for the embryo to reach blastocyst stage in the DD cohort (P = 0.007). Finally, multiple logistic regression showed that both chromatin protamination and condensation, together with the age of the oocyte donors and the embryo recipients, had an impact on pregnancy achievement (P < 0.01) and on live birth rates (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: The main limitation was the restrictive selection of couples, which led to a relatively small sample size and could influence the observed outcomes. For this reason, and to reduce Type I error, the level of significance was set at P ≤ 0.01. On the other hand, the use of cryopreserved samples could also be a limitation. WIDER IMPLICATIONS OF THE FINDINGS: This research demonstrated that protamination and condensation of sperm chromatin are related to embryo development after ICSI, but female age could be a confounding factor when oocytes from older females are used. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the European Union's Horizon 2020 Research and Innovation scheme under the Marie Sklodowska-Curie grant agreement No 801342 (Tecniospring INDUSTRY; TECSPR-19-1-0003); La Marató de TV3 Foundation (214/857-202039); the Ministry of Science and Innovation, Spain (IJC2019-039615-I); the Catalan Agency for Management of University and Research Grants, Regional Government of Catalonia, Spain (2017-SGR-1229); and the Catalan Institution for Research and Advanced Studies, Spain (ICREA). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm.

BACKGROUND: In vitro incubation of epididymal and vas deferens sperm with Mn2+ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn2+ and Mg2+) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry. RESULTS: Incubation with Mn2+/Ca2+ activated SCF in a dose-dependent (P < 0.05) albeit not time-dependent manner (P > 0.05); in contrast, Mg2+/Ca2+ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn2+/Ca2+ or Mg2+/Ca2+, SCF generated DNA fragments of 33-194 Kb, compatible with the size of one or multiple toroids. Besides, Mn2+/Ca2+ affected sperm motility in a dose-dependent manner (P < 0.05), whereas Mg2+/Ca2+ only impaired this variable at high concentrations (P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn2+/Ca2+ or Mg2+/Ca2+ treatments. CONCLUSION: Mn2+/Ca2+ and Mn2+/Ca2+ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death.

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.

Involvement of extracellular vesicle-encapsulated miRNAs in human reproductive disorders: a systematic review.

In recent years, extracellular vesicles (EVs) have emerged as essential players in cell-to-cell communication, particularly having an active regulating role in biological systems. Because reproductive-associated processes are not exempt of this communication, multiple studies have been devoted to this realm, focusing on gamete maturation, embryo implantation or fetal development. The aim of the present review was to comprehensively and systematically collect evidence about the function of the microRNA (miRNA) encapsulated in EVs isolated from different reproductive tissues or fluids in reproductive-related diseases. Following PRISMA guidelines, we conducted a systematic search of the literature published in MEDLINE-PubMed until the end of February 2021. After selection, 32 studies were included in the qualitative review comparing the miRNA expression profile in EVs between different pathological disorders. Most reports showed the potential of the miRNAs carried by EVs to be used as putative biomarkers of reproductive disorders, including pregnancy affections, disease progression and quality of preimplantation embryos. The most relevant miRNAs were found to be highly heterogeneous among studies, with some conflicting results. Further research is thus warranted to address whether cofounding factors, such as the methods to isolate EVs and miRNAs, the subset of EVs, the criteria of patient selection, the timing of sample retrieval, or any other factor, may explain the inconsistencies between studies.

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) 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.

Sperm DNA integrity does play a crucial role for embryo development after ICSI, notably when good-quality oocytes from young donors are used.

Based on the inconsistent literature published thus far involving infertile patients, whether intracytoplasmic sperm injection (ICSI) allows overcoming total fertilization failure due to sperm DNA fragmentation is still unclear. Related to this, female factors, which may have a significant impact on assisted reproduction outcomes, can mask male infertility. In this scenario, evaluating ICSI outcomes following cycles using healthy donor gametes could shed light on this realm, as it would avoid the influence of (un)known confounding factors present in infertile individuals. The present work, therefore, aimed to address whether single- and double-stranded sperm DNA fragmentation leads to impaired ICSI outcomes in double gamete donation cycles. The study also compared these double-gamete donation cycles to cycles in which only sperm were donated and oocytes were obtained from infertile patients. Two cohorts were included: (a) the Donor-Donor (DD) cohort, which included 27 semen donor samples used in 49 ICSI cycles with young healthy oocyte donors; and (b) the Donor-Infertile (DI) cohort, which involved 34 semen donor samples used in 57 ICSI cycles with oocytes from patients. Single- and double-stranded sperm DNA breaks were determined with alkaline and neutral Comet assays, respectively; ICSI was conducted following standard protocols and embryos were monitored through time-lapse microscopy. In the DD cohort, the percentage of sperm with high overall DNA damage correlated with fertilization rates (Rs = - 0.666; P < 0.001) and with the percentage of blastocysts per injected oocyte (Rs = - 0.414; P = 0.040). In addition, sperm DNA damage delayed the first embryo division (Rs = 0.421; P = 0.036), and development from the 8-cell to the morula stage (Rs = 0.424; P = 0.034). In contrast, double-stranded DNA breaks had no effect in this cohort. As far as the DI cohort is concerned, while overall sperm DNA damage was not found to be correlated to fertilization or blastocyst rates, pronuclei formation following ICSI was delayed when the incidence of double-stranded DNA breaks was high (Rs = 0.485; P = 0.005). In conclusion, this study, which is the first involving double donation cycles (i.e., a donor-donor cohort), supports that sperm DNA damage has a detrimental impact on fertilization rates after ICSI, and delays embryo development. Moreover, the use of oocytes from infertile individuals is suggested to hide the male-factor effect.

Advanced Sperm Selection Strategies as a Treatment for Infertile Couples: A Systematic Review.

Assisted reproductive technology (ART) is an essential tool to overcome infertility, and is a worldwide disease that affects millions of couples at reproductive age. Sperm selection is a crucial step in ART treatment, as it ensures the use of the highest quality sperm for fertilization, thus increasing the chances of a positive outcome. In recent years, advanced sperm selection strategies for ART have been developed with the aim of mimicking the physiological sperm selection that occurs in the female genital tract. This systematic review sought to evaluate whether advanced sperm selection techniques could improve ART outcomes and sperm quality/functionality parameters compared to traditional sperm selection methods (swim-up or density gradients) in infertile couples. According to preferred reporting items for systematic reviews and meta-analyses (PRISMA guidelines), the inclusion and exclusion criteria were defined in a PICOS (population, intervention, comparator, outcome, study) table. A systematic search of the available literature published in MEDLINE-PubMed until December 2021 was subsequently conducted. Although 4237 articles were recorded after an initial search, only 47 studies were finally included. Most reports (30/47; 63.8%) revealed an improvement in ART outcomes after conducting advanced vs. traditional sperm selection methods. Among those that also assessed sperm quality/functionality parameters (12/47), there was a consensus (10/12; 83.3%) about the beneficial effect of advanced sperm selection methods on these variables. In conclusion, the application of advanced sperm selection methods improves ART outcomes. In spite of this, as no differences in the reproductive efficiency between advanced methods has been reported, none can be pointed out as a gold standard to be conducted routinely. Further research addressing whether the efficiency of each method relies on the etiology of infertility is warranted.

Sperm selection during ICSI treatments reduces single- but not double-strand DNA break values compared to the semen sample.

PURPOSE: To detect a possible bias in sperm DNA fragmentation (SDF) testing when performed on semen samples or on those few spermatozoa selected for Intracytoplasmic Sperm Injection (ICSI) treatments. METHODS: A multimethodological analysis of Single- and Double-Strand DNA Breaks (SSB and DSB, respectively) was performed through the Neutral Comet, the Alkaline Comet, the Sperm Chromatin Dispersion (SCD) and the Terminal deoxynucleotidyl transferase dUTP Nick End Labelling (TUNEL) assays. SDF was evaluated in (i) semen samples from 23 infertile patients (not achieving pregnancy or suffering recurrent miscarriage); (ii) samples after a Swim-up and (iii) spermatozoa microselected for ICSI (ICSI-S). RESULTS: The analysis of 3217 ICSI-S revealed a significant reduction of SSB values compared to the Ejaculate and the Swim-up samples. On the contrary, DSB values were not reduced after any sperm selection method. The No-pregnancy group presented poorer semen parameters and higher SSB values. The Recurrent miscarriage group presented better semen parameters but also higher DSB values. CONCLUSION: The analysis of SDF on semen samples may not be fully representative of those few spermatozoa selected for ICSI. Since oxidative stress impairs sperm motility and causes SSB, selecting a motile sperm may intrinsically imply choosing a sperm not affected by this damage. DSB have an enzymatic origin which does not affect motility, making it difficult to select a sperm without this damage. Therefore, ICSI treatments could be effective in patients presenting high SSB values. Patients presenting high DSB values should expect bad ICSI results if this damage is not reduced through other specific methods.

Sperm chromatin condensation and single- and double-stranded DNA damage as important parameters to define male factor related recurrent miscarriage.

The aim of the present work is to characterize the relationship between sperm protamine deficiency and single- and double-stranded DNA damage and to assess the diagnostic potential of chromomycin A3 (CMA3). For that purpose, semen samples from 90 human males with different clinical features were included (fertile donors, patients with recurrent pregnancy loss [RPL], and infertile patients). DNA condensation was analyzed by CMA3 and different types of DNA fragmentation were analyzed through the comet assay. A positive correlation between DNA condensation and single-stranded DNA fragmentation was found (Rs = .456; p = .05). CMA3 presented differences between fertile donors and all other groups (p < .001). Interestingly, patients with RPL, who were able to achieve a pregnancy, and infertile patients showed similar values of CMA3 (p > .05). Receiver operating characteristic curves and the profiles obtained by the combination of Comet assays and CMA3 indicate that the CMA3 test may be an interesting approach to distinguish those subjects with higher pregnancy loss risk from fertile donors (CMA3 area under the curve 0.928, with a confidence interval of 0.849-1.000). The present work shows that DNA condensation is related to oxidative damage, which affects mainly protamine-rich regions. The profiles observed in different clinical groups showed that CMA3 might be useful for the diagnosis of RPL risk when combined with Comet assays.

Proteomic Analysis in Seminal Plasma of Fertile Donors and Infertile Patients with Sperm DNA Fragmentation.

Seminal plasma proteomics studies could represent a new approach for the determination of molecular elements driving male infertility, resulting in a better male infertility characterization. The aim of this study is to investigate proteomic differences in seminal plasma samples from fertile and infertile individuals. For that, semen samples were selected according to semen analysis, clinical pathology, and values of sperm DNA fragmentation (alkaline and neutral Comet assay and Sperm Chromatin Dispersion test). A total of 24 seminal plasma samples classified in four groups were processed: fertile donors (FD), recurrent miscarriage patients (RM), asthenoteratozoospermic patients (ATZ), and asthenoteratozoospermic patients with varicocele (ATZ-VAR). Results obtained by 2D-differential gel electrophoresis (2D-DIGE) revealed 26 spots significantly increased in fertile donors when compared to patient groups. Also, eight spots in the ATZ group and two in the ATZ-VAR group were decreased compared to the other groups. Twenty-eight proteins were identified by mass spectrometry (MS), most of them involved in metabolic and cellular processes and with a catalytic or binding function. Protein-protein interactions through Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) tool suggest that a large part of them were associated with each other. Furthermore, most of them were associated with ubiquitin C, indicating that it could play an important regulation role, resulting in a potential male infertility biomarker.

Characterization of Nuclease Activity in Human Seminal Plasma and its Relationship to Semen Parameters, Sperm DNA Fragmentation and Male Infertility.

PURPOSE: Some studies have shown that complementary biomarkers are needed in semen analysis to provide a more accurate diagnosis for couples with infertility problems. To our knowledge no study has been done to determine the relationships among nuclease activity in seminal plasma, semen parameters, sperm DNA fragmentation and male infertility. MATERIALS AND METHODS: A total of 94 semen samples were collected according to WHO 2010 semen analysis parameters. Samples were analyzed using the single radial enzyme diffusion method for nuclease activity in seminal plasma, and alkaline and neutral Comet assay for sperm DNA fragmentation. Samples were obtained from 11 fertile donors with proven fertility, 17 patients with normozoospermia in an infertile couple, and 16 patients with asthenozoospermia, 19 with teratozoospermia, 21 with asthenoteratozoospermia and 10 with azoospermia. RESULTS: Nuclease activity analyzed in seminal plasma was higher in patients than in controls. It correlated with sperm motility and morphology, and sperm DNA fragmentation measured by the alkaline Comet assay. No correlation with sperm DNA fragmentation was measured by the neutral Comet assay. ROC curves to determine male infertility revealed 0.658 sensitivity, 0.727 specificity and 0.705 cm(2) AUC for the single radial enzyme diffusion method, 0.918, 1 and 0.994 cm(2) for the alkaline Comet assay, and 0.917, 0.250 and 0.373 cm(2), respectively, for the neutral Comet assay. CONCLUSIONS: Nuclease activity in seminal plasma corrected by sperm count is a good variable to predict male infertility. Results indicate that it could be a useful complementary parameter for male infertility diagnosis.

Impact of Seminal Plasma Antioxidants on DNA Fragmentation and Lipid Peroxidation of Frozen-Thawed Horse Sperm.

Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen-thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolox-equivalent antioxidant capacity (TEAC), plasma ferric reducing antioxidant capacity (FRAP) and cupric reducing antioxidant capacity (CUPRAC)); and the oxidative stress index (OSI) of their seminal plasma (SP). Based on total motility and plasma membrane integrity (SYBR14+/PI-) after thawing, ejaculates were hierarchically (p < 0.001) clustered into two groups of good- (GFEs) and poor-(PFEs) freezability ejaculates. LPO and DNA fragmentation (global DNA breaks) were higher (p < 0.05) in the PFE group than in the GFE group, with LPO and DNA fragmentation (global DNA breaks) after thawing showing a positive relationship (p < 0.05) with SP OSI levels and ROS production. In addition, sperm motility and membrane integrity after thawing were negatively (p < 0.05) correlated with the activity levels of SP antioxidants (PON1 and TEAC). The present results indicate that LPO and DNA fragmentation in frozen-thawed horse sperm vary between ejaculates. These differences could result from variations in the activity of antioxidants (PON1 and TEAC) and the balance between the oxidant and antioxidant components present in the SP.

Sperm Quality and Fertility of Livestock Animals.

Recent research has focused on the understanding of the causes of subfertility observed in livestock species, evidencing that different factors could underlie this condition [...].

Animal board invited review: An update on the methods for semen quality evaluation in swine - from farm to the lab.

Pig breeding is mainly conducted through artificial insemination with liquid-stored semen. It is, therefore, crucial to ensure that sperm quality is over the standard thresholds, as reduced sperm motility, morphology or plasma membrane integrity are associated with reduced farrowing rates and litter sizes. This work aims to summarise the methods utilised in farms and research laboratories to evaluate sperm quality in pigs. The conventional spermiogram consists in the assessment of sperm concentration, motility and morphology, which are the most estimated variables in farms. Yet, while the determination of these sperm parameters is enough for farms to prepare seminal doses, other tests, usually carried out in specialised laboratories, may be required when boar studs exhibit a decreased reproductive performance. These methods include the evaluation of functional sperm parameters, such as plasma membrane integrity and fluidity, intracellular levels of calcium and reactive oxygen species, mitochondrial activity, and acrosome integrity, using fluorescent probes and flow cytometry. Furthermore, sperm chromatin condensation and DNA integrity, despite not being routinely assessed, may also help determine the causes of reduced fertilising capacity. Sperm DNA integrity can be evaluated through direct (Comet, transferase deoxynucleotide nick end labelling (TUNEL) and its in situ nick variant) or indirect tests (Sperm Chromatin Structure Assay, Sperm Chromatin Dispersion Test), whereas chromatin condensation can be determined with Chromomycin A3. Considering the high degree of chromatin packaging in pig sperm, which only have protamine 1, growing evidence suggests that complete decondensation of that chromatin is needed before DNA fragmentation through TUNEL or Comet can be examined.

Expression of miR-138 in cryopreserved bovine sperm is related to their fertility potential.

BACKGROUND: MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules of 22-24 nucleotides that regulate gene expression. In the last decade, miRNAs have been described in sperm of several mammals, including cattle. It is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans; however, little is known about the content, expression, and function of sperm-borne miRNAs in early bovine embryo. In this study, total sperm RNA was isolated from 29 cryopreserved sperm samples (each coming from a separate bull) using a RNeasy kit and treatment with DNase I. RNA concentration and purity were determined through an Epoch spectrophotometer and an Agilent Bioanalyzer. The expression of 10 candidate miRNAs in bovine sperm (bta-miR-10a, bta-miR-10b, bta-miR-138, bta-miR-146b, bta-miR-19b, bta-miR-26a, bta-miR-34a, bta-miR-449a, bta-miR-495 and bta-miR-7), previously identified in testis and/or epididymis, was evaluated with RT-qPCR. The cel-miR-39-3p was used as a spike-in exogenous control. Nonparametric Mann-Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility [HF; non-return rates (NRR) ranging from 39.5 to 43.5] and those with subfertility (SF; NRR ranging from 33.3 to 39.3). Several sperm functionality parameters (e.g., viability, membrane stability or oxygen consumption, among others) were measured by multiplexing flow cytometry and oxygen sensing technologies. RESULTS: RNA concentration and purity (260/280 nm ratio) (mean ± SD) from the 29 samples were 99.3 ± 84.6 ng/µL and 1.97 ± 0.72, respectively. Bioanalyzer results confirmed the lack of RNA from somatic cells. In terms of the presence or absence of miRNAs, and after applying the Livak method, 8 out of 10 miRNAs (bta-miR-10b, -138, -146b, -19b, -26a, -449a, -495, -7) were consistently detected in bovine sperm, whereas the other two (bta-miR-10a, and -34a) were absent. Interestingly, the relative expression of one miRNA (bta-miR-138) in sperm was significantly lower in the SF than in the HF group (P = 0.038). In addition to being associated to fertility potential, the presence of this miRNA was found to be negatively correlated with sperm oxygen consumption. The expression of three other miRNAs (bta-miR-19b, bta-miR-26a and bta-miR-7) was also correlated with sperm function variables. CONCLUSIONS: In conclusion, although functional validation studies are required to confirm these results, this study suggests that sperm bta-miR-138 is involved in fertilization events and beyond, and supports its use as a fertility biomarker in cattle.

Women's Adherence to Healthy Dietary Patterns and Outcomes of Infertility Treatment.

Importance: Increasing evidence suggests that specific foods and nutrients may improve infertility treatment outcomes in women. However, less is known about the role of dietary patterns. Objective: To investigate whether women's adherence to a priori-defined dietary patterns promoted for the prevention of chronic conditions is associated with outcomes of infertility treatment. Design, Setting, and Participants: This prospective cohort study was conducted at a fertility center at an academic medical center in Boston, Massachusetts. Women undergoing infertility treatment cycles, including intrauterine insemination cycles and in vitro fertilization with or without intracytoplasmic sperm injection were included. Data were collected from January 2007 to October 2019, and data were analyzed from February to December 2022. Exposures: Women's pretreatment diet was assessed with a validated food frequency questionnaire from which 8 a priori-defined scores were calculated (higher score indicates greater adherence): (1) Trichopoulou Mediterranean diet, (2) alternate Mediterranean diet, (3) Panagiotakos Mediterranean diet, (4) Healthy Eating Index, (5) Alternate Healthy Eating Index, (6) American Heart Association (AHA) index, (7) Dietary Approaches to Stop Hypertension index, and (8) plant-based diet. Main Outcomes and Measures: The adjusted probability of clinically relevant outcomes (live birth as a primary outcome and clinical pregnancy and pregnancy loss as secondary outcomes) was evaluated across quartiles of adherence to each dietary pattern using multivariable generalized linear mixed models to account for repeated cycles. Results: This analysis included 612 women with a median (IQR) age of 35.0 (32.0-38.0) years. There was no association between women's adherence to the 8 a priori dietary patterns and probability of clinical pregnancy or live birth following in vitro fertilization or intrauterine insemination. However, an inverse association was found between adherence to AHA dietary pattern and risks of total and clinical pregnancy loss. Among women who became pregnant during the course of infertility treatment, the adjusted probabilities of pregnancy loss in the lowest and highest quartile of the AHA dietary pattern were 0.41 (95% CI, 0.33-0.50) and 0.28 (95% CI, 0.21-0.36), respectively (P for trend = .02). The corresponding adjusted probabilities of clinical pregnancy loss were 0.30 (95% CI, 0.22-0.39) and 0.15 (95% CI, 0.10-0.23) (P for trend = .007). A similar pattern was observed for all other dietary patterns, with the exception of the plant-based diet pattern. Conclusions and Relevance: Findings of this cohort study suggest that preconception adherence to the AHA diet may be associated with a lower likelihood of pregnancy loss during the course of infertility treatment.

An Algorithm to Predict the Lack of Pregnancy after Intrauterine Insemination in Infertile Patients.

Increasing intrauterine insemination (IUI) success rates is essential to improve the quality of care for infertile couples. Additionally, straight referral of couples with less probability of achieving a pregnancy through IUI to more complex methods such as in vitro fertilization is important to reduce costs and the time to pregnancy. The aim of the present study is to prospectively evaluate the threshold values for different parameters related to success in intrauterine insemination in order to provide better reproductive counseling to infertile couples, moreover, to generate an algorithm based on male and female parameters to predict whether the couple is suitable for achieving pregnancy using IUI. For that, one hundred ninety-seven infertile couples undergoing 409 consecutive cycles of intrauterine insemination during a two-year period were included. The first year served as a definition of the parameters and thresholds related to pregnancy achievement, while the second year was used to validate the consistency of these parameters. Subsequently, those parameters that remained consistent throughout two years were included in a generalized estimating equation model (GEE) to determine their significance in predicting pregnancy achievement. Parameters significantly associated with the lack of pregnancy through IUI and included in the GEE were (p < 0.05): (i) male age > 41 years; (ii) ejaculate sperm count < 51.79 x 106 sperm; (iii) swim-up alkaline Comet > 59%; (iv) female body mass index > 45 kg/m2; (v) duration of infertility (>84 months), and (vi) basal LH levels > 27.28 mUI/mL. The application of these limits could provide a pregnancy prognosis to couples before undergoing intrauterine insemination, therefore avoiding it in couples with low chances of success. The retrospective application of these parameters to the same cohort of patients would have increased the pregnancy rate by up to 30%.

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.