Sperm Quality Parameters Research Articles

O-020 What is the recovery time for sperm parameters in men who have suffered a mild Covid-19 infection?

Abstract Study question What is the time required in men who have suffered a mild Covid-19 infection, for the seminal parameters to be restored to the pre-Covid values? Summary answer The effect of Covid-19 infection on the concentration and sperm motility is clearly confirmed and is maintained over time, at least beyond 100 days. What is known already Although several studies have been performed on the effect of SARS-CoV-2 on reproductive health in the short term vs. healthy controls, and the negative effect on sperm parameters has been demonstrated, there is a scarcity of data showing the degree to which the virus affects the semen parameters in one group of patients after coronavirus infection compared to before infection. Moreover, there are no conclusive data regarding the long-term effects of SARS-CoV-2 on semen parameters. Study design, size, duration This prospective study was carried out in 6 reproductive centers between February 2020 and October 2022 including 45 male patients with a confirmed diagnosis of mild COVID-19. For each of the semen parameters (volume, sperm count, active and total motility, normal morphology, and vitality) a standardized index was calculated by dividing the value of the parameter from the subsequent semen analysis by that from the semen analysis of the same individual prior to infection. Participants/materials, setting, methods The median age of the participants was 31 years, proving the absence of a relationship between age and any of the parameters analyzed. The post-Covid test was performed between 17 and 516 days after infection, with a median of 100 days. The time elapsed between both samples was a median of 238 days. The effect of time on the affectation of semen parameters was studied, setting 100 days as the cut-off point. Main results and the role of chance There were significant differences in semen parameters before and after COVID-19 in terms of semen volume, sperm concentration, progressive motility, total motility, and vitality which decreased significantly (p < 0,05) after SARS-CoV-2 infection compared to the pre-infection values. Morphology was not significantly affected. The total sperm count is the most clearly affected seminal parameter. Half of the individuals (median) obtained a total count in the second sample less than 57% of the initial one. Considering the time of spermatogenesis, the subjects were separated into two groups: those who performed the semen analysis in the first 100 days after infection and those who performed it later. No differences in any parameter were found between the two groups with U Mann-Withney test. Using the Wilcoxon test for paired samples, it was demonstrated that the deleterious effect of infection on sperm concentration and motility not only does not improve with time, but it becomes more significant in late sperm analysis, performed more than one hundred days after the infection. Limitations, reasons for caution It should be mentioned that impairment of semen parameters may not be due to a direct effect of the SARS-CoV-2 virus. There are likely to be additional factors that contribute to long-term sperm parameters decrease, but whose identity is currently unknown. Wider implications of the findings The present study revealed the long-term impacts of SARS-CoV-2 on various parameters of sperm quality even with a mild infection. Further efforts should be made to study the long-time effects of COVID-19 infection on male reproduction. Trial registration number Not Applicable

P-045 Effects of seminal BPA levels on human sperm parameters and gene expression

Abstract Study question Are high BPA levels in human seminal fluid associated with low sperm metrics and changes in gene expression? Summary answer A trend towards an inverse relationship between seminal BPA and sperm count was observed. Expression of androgen receptor mRNA was down-regulated with high BPA exposure. What is known already Bisphenol A (BPA) is a widespread industrial chemical, used as the key monomer of polycarbonate plastics and epoxy resins. While some countries have introduced bans/stringent regulation for BPA, it continues to be widely used especially in North America, where its use is only limited in baby products. BPA has been detected in human seminal fluid and has been correlated with decreased sperm counts and motility - crucial factors in determining male fertility, which continue to decrease. We have demonstrated that in vitro BPA exposure can reduce motility, mitochondrial membrane potential, and capacitation in mature bovine spermatozoa. Study design, size, duration In collaboration with CCRM, Toronto, seminal fluid samples were collected from 90 patients undergoing fertility assessment between 2021 and 2023. Typical evaluations such as sperm counts, motility, and morphology were recorded by the clinic (following WHO guidelines). Samples were then shipped to the University of Guelph for further investigations. Information on outcomes for patients who underwent subsequent IVF cycles like fertilization rates and pregnancy rates were also recorded at the clinic. Participants/materials, setting, methods At the University of Guelph, seminal BPA levels were measured through an enzyme-linked immunosorbent assay (ELISA; My BioSource), then compared to sperm metrics obtained by the clinic. Next, a GeNorm analysis was performed to determine the most stable reference genes for subsequent qPCR analysis. The relative mRNA expression of three key genes related to male reproduction (androgen receptor (AR), cyp17a1 and cyp11a1) were quantified and compared between groups with high and low BPA content. Main results and the role of chance BPA concentrations in the seminal fluid samples ranged between 0-3000 pg/mL, with most samples containing 35-1000 pg/mL. When samples were graphed according to increasing BPA levels, no relationships were observed between BPA and motility or morphology rates. However, a negative trend between BPA and sperm count was detected, although this was not significant. Samples were then divided into high- (total 15 samples with >700 pg/mL) and low- (total 15 samples with <100 pg/mL) BPA content groups. The GeNorm M value indicated that HPRT1 and HMBS were the most stable reference genes across the groups. qPCR analysis on three biological replicates in technical triplicates showed that androgen receptor mRNA expression was significantly higher in sperm exposed to low seminal BPA compared to the high BPA exposure group (p = 0.03913). This finding is particularly interesting as AR expression has previously been positively correlated with sperm count, motility and morphology. cyp17a and cyp11a mRNA expression was undetectable in human spermatozoa under our experimental conditions. Future steps include increasing the sample size to increase the power of the correlation studies between sperm quality parameters and ART success rates. Furthermore, other fertility-related genes will be investigated at both the mRNA (qPCR) and protein levels (flow cytometry). Limitations, reasons for caution Relatively small sample size; duration of study must be extended to gain information on outcomes of ART cycles; many samples in the mid-range of BPA are excluded from gene expression studies. Wider implications of the findings These results can ultimately display a relationship between BPA exposure and male fertility and provide further insights into the molecular mechanisms through which BPA exerts its effects. Ultimately, this research can drive changes to guidelines/exposure limits for BPA, especially in North America. Trial registration number not applicable

P-053 Sperm aneuploidy and DNA integrity pre- and post-chemotherapy in men with testicular cancer

Abstract Study question Are fragmentation levels (single-double stranded DNA breaks) or aneuploidy frequencies in sperm increased in patients with testicular cancer who recovered spermatogenesis after chemotherapy (CT)? Summary answer Similar levels of sperm DNA fragmentation and sperm aneuploidy frequency are found in men with testicular cancer pre- and post-chemotherapy. What is known already Testicular cancer is the most frequent solid tumor among young men. Therefore, the restoration of fertility and achievement of fatherhood in survivors have become important concerns. The integrity of genetic material in sperm is crucial since some paternal genes are essential for early embryo development. Although previous studies analyzed the effect of CT on sperm DNA integrity, they did not differentiate single vs. double-stranded DNA breaks. Few data are available on aneuploidy in sperm from testicular cancer survivors, so it remains to be elucidated whether cancer treatments may lead to an increased risk of aneuploidy syndromes in offspring. Study design, size, duration Prospective multicenter study including 31 males with a history of testicular cancer who requested semen cryopreservation between 2007-2021. All patients included underwent CT and recovered spermatogenesis once treatment ended. Post-CT samples were collected and seminal parameters, single-double stranded DNA breaks and the proportion of sperm aneuploidies were compared with one aliquot of the initial ejaculate. The project was approved by an Institutional Review Board and written informed consent for participation was obtained from each subject. Participants/materials, setting, methods Patient´s age prior CT was 31.23yrs (IC 95%: 29.29-33.17). Mean interval time between 1st and 2nd samples was 4.53yrs (IC 95%: 3.42-5.64). TUNEL quantified overall sperm DNA fragmentation by flow cytometry analyzing at least 20,000 cells, while γH2AX staining was applied for double DNA fragmentation assessment. Sperm aneuploidies were calculated by using fluorescence in situ hybridization (FISH) probes for chromosomes 13, 18, 21, X, Y. A paired t-test was used to compare variables pre- and post-CT. Main results and the role of chance Similar results were found when sperm quality parameters were compared. Mean sperm concentration pre-CT was 33.03M/mL (IC 95%: 23.98-42.07) and 36.41 M/mL (IC 95%: 27.85-44.96) in post-CT ejaculates (N.S.). A comparable rate of motile spermatozoa was observed in pre-CT vs. post-CT samples [(43.81% (IC 95%: 38.42-49.19) vs. 44.73% (IC 95%: 38.95-50.51), N.S.]. Regarding TUNEL assay results, the sperm DNA fragmentation rate was 21.65% (IC 95%: 17.66-25.63) in pre-CT samples and 23.00% (IC 95%: 19.90-26.61) in post-CT samples (N.S.). Furthermore, similar results were obtained when double-stranded fragmentation levels (H2AX) were compared between pre and post-CT [10.2% (IC 95%: 6.45-13.96) vs. 12.44% (IC 95%: 8.75-16.13) N.S.]. Comparable sperm disomic proportions were found in PRE and POST-CT samples: [(Cr.13 =0.02%, Cr.18 =0.01%, Cr.21=0.30%, Cr.XY=0.84%) vs. (Cr.13 =0%, Cr.18 =0%, Cr.21=0%, Cr.XY=0.33%), N.S]. Results were also evaluated according to the time elapsed between the PRE and POST -CT samples. No significant differences were found for seminal values (sperm concentration, p = 0.26; motility, p = 0.68), overall sperm DNA fragmentation (p = 0.68), double-strand DNA fragmentation (p = 0.90) and the frequency of sperm disomies (Cr. 13 p = 0.79, Cr.18 p = 0.46, Cr. 21 p = 0.85, Cr.XY p = 0.05). Limitations, reasons for caution The main limitations of the present study are that we only recruited patients who recovered spermatogenesis and that our data evaluated a long-term effect of CT. Wider implications of the findings All men wishing to have their own children after gonadotoxic treatment should receive adequate counseling about the side effects of the therapy. Based on our results on sperm DNA integrity and euploidy, patients who recovered spermatogenesis should not be advised to use samples that were preserved before undergoing CT. Trial registration number na

P-418 Medical sperm cryo-preservation in adolescents: what proportion needs long term storage?

Abstract Study question What proportion of adolescents who have cryo-preserved sperm for medical indications will have normal semen analysis after treatment and no longer require stored sperm samples? Summary answer More than half of adolescent patients with cryo-preserved sperm will not need continued storage as sperm parameters return to normal after completion of treatment. What is known already Worldwide, about 12,400 young adults are diagnosed with cancer every year. With modern treatment 80% of them will achieve a long-term cure1. Lymphomas, brain tumours, sarcomas, and carcinomas account for more than 90% of cases, for which treatment tends to be predominantly gonadotoxic. Sperm cryo-preservation prior to starting treatment is recommended by all clinical guidelines. Although there is evidence that treatment could cause prolonged azoospermia, especially alkylating or platinum-based agents, several therapy regimes seem to induce only a temporary reduction in sperm count. In some cases spermatagonial stem cells can repopulate the seminiferous tubules after chemotherapy-induced damage2. Study design, size, duration This was a retrospective cohort study on male adolescent who underwent medical sperm cryo-preservation between 1985 and 2014. The data was retrieved from a prospective held secure electronic database. Within 10 years of storage a review was carried out to ascertain if they met criteria for continued storage. A total of 323 teenagers were included in the study. Participants/materials, setting, methods In a tertiary fertility preservation centre, 323 adolescents had sperm cryopreservation before undergoing potentially gonadotoxic treatment. All patients were offered repeat semen analysis within 10 years from storage. The indication for discarding sperm within this period were analysed. The primary outcome was the proportion of patients who did not have adverse impact on sperm. Their diagnosis, treatments and outcomes including death were included in the analysis. Statistical analysis was performed using IBM SPSS Statistics. Main results and the role of chance According to WHO criteria, at time of cryo-preservation 168/323 (52%) adolescents had a normal semen analysis, 120/323 (37,1%) had oligospermia and 35/323 (10,8%) had azoospermia. For the 198 adolescents included in the final analysis, median age was 16 (IQR 15-16). Haematological cancer was the predominant group [94/198 (47%)] followed by sarcoma/osteosarcoma [58/198 (29%)]. Outcome analysis revealed that 99/198 (50%) patients had repeated semen analyses. Median time between the pre- and post-treatment analysis was 10 years (IQR 7-10). Of those who had a subsequent semen analysis, 59/99 (58,4%) showed improvement, 23/99 (22,8%) had no change and 17/99 (16.8%) revealed decline in sperm quality parameters compared to the stored sample. Post treatment, spontaneous pregnancy was reported by 16 (8%) patients. Sperm was discarded for patient demise in 35.8% (71/198) of cases and for inability to contact the patient since storage in 13.1% (26/198). Limitations, reasons for caution One limitation is the retrospective design and the relatively small number of cases included in the study. A second limitation is that although we have information of semen analysis, we did not have information on pregnancies achieved for patients with a normal semen analysis. Wider implications of the findings This is one of the largest case series of outcomes in adolescents. We can anticipate retention of fertility potential in half of the adolescents freezing sperm. A repeat semen analysis facilitates reassurance to the patients and allows discarding sperm when in remission. Patients can refreeze sperm if disease relapse occurs. Trial registration number Non applicable

P-048 Vitrification of human semen impairs the mobility of spermatozoa when compared to the conventional slow-freezing

Abstract Study question Does vitrification provide a better method for human sperm cryopreservation than slow freezing in terms of survival, mobility, maturity, and DNA fragmentation? Summary answer Vitrification of human semen yields similar outcome as slow-freezing considering survival, mobility, DNA fragmentation, and maturity but the mobility after thawing is lower. What is known already Slow freezing is the most common technique for cryopreservation of human sperm. Unfortunately, many sperm cells die during the process or lose their mobility and are thereafter unable of fertilization. Meanwhile, vitrification of animal sperm showed good outcomes in terms of sperm survival and motility while being easier to perform than slow freezing. So far, many studies focused on survival and mobility of human sperm after vitrification compared to slow freezing, with results still not unified. They also neglected the importance of sperm maturity and DNA fragmentation caused by the process. DNA damage can affect embryo development or cause abortion. Study design, size, duration The study was performed from March January to July 2022. It included 69 male participants who underwent a diagnostic spermiogram at the Division of gynaecology and obstetrics of University Medical Centre Ljubljana. All participants gave written consent of using the remaining of sample, after performing the spermiogram, for our study. Sperm samples were divided in three aliquots, one saved as native sample, one intended for vitrification, and one intended for slow freezing. Participants/materials, setting, methods Participants in the study had normal sperm quality parameters according to 5th edition of WHO manual. Slow freezing was performed using Sperm Freezing medium (Origio). For vitrification sperm was diluted in solution consisting of Sperm preparation medium (Origio), sucrose, and HSA in 1:1 (v/v) ratio, incubated for five minutes and then vitrified by adding 30 microliter droplets directly into liquid nitrogen. After thawing sperm survival, mobility, maturity, and DNA fragmentation were assessed. Main results and the role of chance The data are presented as mean values (± standard deviation) when normally distributed and as medians (IQR) when not normally distributed. The patient’s age was 35.0±5.1 years. In native sperm sample, sperm concentration was 69.6±59.0x106 cells per millilitre and total motility was assessed to be 60.0% (50.0%-70.0%). After thawing we noticed significantly lower mobility (p < 0.001), while also the mobility was significantly lower after vitrification when compared to slow-freezing (17.6% (12.4%-25.3%) and 14.5% (9.0%-22.6%), p = 0.007). Interestingly, there was no difference in survival after thawing the samples (20.5±10.7 % after slow freezing and 23.7±13.7 % after vitrification). Also there was no difference in maturity of sperm when checked with protamine staining (native vs. slow-freezing vs. vitrification; 53.6±15.2 % vs. 53.9±16.0 % vs. 52.4±15.2 %, p = 0.905) and also when checked with hyaluronan binding assay (78.0% (67.5%-82.4%) vs. 79.4% (71.7%-82.95%) vs. 75.3% (67.5%-81.1%), p = 0.607) . While sperm maturity was not affected with cryopreservation, DNA fragmentation was significantly higher after cryopreservation (native vs. slow-freezing vs. vitrification; 19.7±9.5%, vs. 24.7±11.6 vs. 27.1±15. %, p = 0.049). Limitations, reasons for caution We believe our study would be better by obtaining higher number of participants and by being performed over longer time period. We would also like to further optimise vitrification protocol. More knowledge is also needed in poor sperm samples, since we only included sperm samples of good quality. Wider implications of the findings We believe our finding are of great importance for the field of andrology. Our results show vitrification of human sperm can be comparable to slow freezing in most of parameters analysed, but lower mobility after vitrification is reason for caution. Trial registration number 20210024

Effect of different sources of dietary zinc on sperm quality and oxidative parameters.

Zinc has a critical physiological role in sperm function. The purpose of this study was to investigate the effect of different sources of zinc on sperm quality. For this purpose, 18 Zandi lambs with an average weight of 32 ± 1.2 kg were subjected to three treatments in a completely randomized design. Experimental treatments include (1) control treatment of basal diet without zinc supplementation, (2) basal diet with 40 mg/kg of zinc supplementation from zinc sulfate source and (3) basal diet with 40 mg/kg of zinc supplementation with organic source. At the end of feeding period, lambs were slaughtered. To determine the effect of experimental treatments on sperm quality, the testes were transferred to the laboratory. After that, epididymal spermatozoa were evaluated for sperm motility parameters, abnormal morphology, viability, membrane functionality, malondialdehyde (MDA) and antioxidant activity [glutathione peroxidase (GPx), superoxide dismutase (SOD), total antioxidant capacity (TAC)], sperm concentration and testosterone level. Zinc sulfate administration decreased MDA levels compared to other treatments and increased GPx and TAC activity compared to the control (P < 0.05), although SOD activity was not affected by any supplementation. Also, the use of zinc sulfate supplementation increased the percentage of total and progressive motility compared to the control group (P < 0.05). Membrane integrity and sperm viability were also affected by zinc sulfate supplementation (P < 0.05). Therefore, the results of this study showed that the use of zinc sulfate, can improve sperm motility and survival indices and its antioxidant capacity.

Microbial composition of goat buck's ejaculates is modified by the process of preparing and storing refrigerated semen doses

Ejaculates present their own microbiota, and a link between ejaculates' microbiota and sperm quality and fertility exists. With the development of artificial insemination in animal breeding, ejaculates must be manipulated by diluting them with extenders and storing them at temperatures below body temperature. The effects that these processes have on the original semen microbiota have never been studied. This study explores the effects of the protocol for preparing refrigerated goat buck semen doses and storing on seminal microbiota. Semen from six adult goat bucks of the Murciano-Granadina breed (24 ejaculates) was used, cooled to 4 °C in a skimmed milk-based extender, and stored at this temperature for 24 h. Samples were taken in different steps: in the raw ejaculates (ejaculates), after dilution with the refrigeration extender (diluted), immediately after reaching 4 °C (chilled 0 h) and the samples refrigerated at 4 °C and stored at this temperature for 24 h (chilled 24 h). Sperm quality (motility and integrity of plasma and acrosomal membrane, and mitochondrial functionality) was also evaluated. Bacterial 16S rRNA sequencing was used to study the seminal microbiota. Our results indicated that both refrigeration and storage at 4 °C negatively affected sperm quality parameters. Preparing semen doses and their subsequent conservation caused a significant change in the bacterial community structure. Raw ejaculates showed a lower Pielou's evenness index than the other samples (diluted, chilled 0 h and chilled 24 h). Ejaculates also had a lower Shannon's diversity index (3.44) than the diluted semen (4.17) and the semen chilled for 24 h (4.43). Regarding beta diversity, significant differences were detected between ejaculates and the other treatments. Differences were also found in unweighted UniFrac distances between the semen chilled for 0 h and that chilled for 24 h. At the genus level, marked effects of preparing doses and their subsequent conservation were also evident: 199 genera that were absent in ejaculates were found in the semen chilled and stored for 24 h; 177 genera that were present in ejaculates disappeared after 24-h refrigeration. In conclusion, the extender and protocol for preparing refrigerated goat buck semen doses considerably modify microbial ejaculate composition.

FeTPPS, a Peroxynitrite Decomposition Catalyst, Ameliorates Nitrosative Stress in Human Spermatozoa.

Excessive levels of reactive nitrogen species (RNS), such as peroxynitrite, promote nitrosative stress, which is an important cause of impaired sperm function. The metalloporphyrin FeTPPS is highly effective in catalyzing the decomposition of peroxynitrite, reducing its toxic effects in vivo and in vitro. FeTPPS has significant therapeutic potential in peroxynitrite-related diseases; however, its effects on human spermatozoa under nitrosative stress have not been described. This work aimed to evaluate the in vitro effect of FeTPPS against peroxynitrite-mediated nitrosative stress in human spermatozoa. For this purpose, spermatozoa from normozoospermic donors were exposed to 3-morpholinosydnonimine, a molecule that generates peroxynitrite. First, the FeTPPS-mediated peroxynitrite decomposition catalysis was analyzed. Then, its individual effect on sperm quality parameters was evaluated. Finally, the effect of FeTPPS on ATP levels, motility, mitochondrial membrane potential, thiol oxidation, viability, and DNA fragmentation was analyzed in spermatozoa under nitrosative stress conditions. The results showed that FeTPPS effectively catalyzes the decomposition of peroxynitrite without affecting sperm viability at concentrations up to 50 μmol/L. Furthermore, FeTPPS mitigates the deleterious effects of nitrosative stress on all sperm parameters analyzed. These results highlight the therapeutic potential of FeTPPS in reducing the negative impact of nitrosative stress in semen samples with high RNS levels.

Evaluation of long-term cryostorage on sperm function and fertility of brown-marbled grouper (Epinephelus fuscoguttatus)

Cryopreservation of fish semen is a valuable technique for reproductive management in aquaculture. The aim of this study was to evaluate the effect of long-term storage in liquid nitrogen on post-thaw sperm functional parameters of brown-marbled grouper (Epinephelus fuscoguttatus). Samples were cryopreserved in a simple freezing medium (10 % DMSO + 0.3 M Glucose), sperm quality parameters were assessed at different storage periods (one month, one year, 2 years and 3 years). Our results indicated that cryostorage periods had a significant effect on the quality of frozen-thawed spermatozoa. Compared with fresh semen, cryopreserved sample was characterized by an overall decline in motility, viability, mitochondrial membrane potential (MMP), ATP content and increased level of lipid peroxidation with the storage time going on. For the analyzed quality parameters, there were no significant differences in storage time cryopreserved over one month. After 3 years of storage, frozen sperm still showed relatively high total motility, viability and MMP (all over 80 %), yielding acceptable fertilizing capacity close to that of fresh sperm. Therefore, it can be concluded that brown-marbled grouper sperm could be effectively cryopreserved for at least 3 years. The results of this study would be helpful to optimize gamete management in routine hatchery practices.

The relationship between sperm quality parameters and stallion fertility

There are stallions with apparently good sperm quality that achieve low pregnancy rates when their semen is used for artificial insemination (AI). Thus, additional methods of evaluating sperm quality that result in better methods of predicting stallion fertility, are essential. Semen samples were obtained in April-June from 8 stallions, 4 trotters and 4 warmblood sport horses, which were sampled Monday, Wednesday and Friday each week. From each stallion, 1-4 samples were obtained, and evaluated after cold storage for 24 hours. The samples were evaluated for kinematic variables using CASA, and for membrane integrity, mitochondrial membrane potential (MMP), production of reactive oxygen species, acrosome integrity and chromatin integrity using flow cytometry. Relationships with fertility, calculated as number of pregnant mares/number of inseminated mares for samples used for artificial insemination after transport in cold storage, were evaluated by linear regression, following log-transformation of variables found not to be normally distributed. The following sperm variables were found to have a positive relationship with fertility: Straightness (r2=0.86, p= 0.0009), Viable with High mitosoxred/Low MMP (r2=0.70, p= 0.01), Viable non acrosome reacted (r2=0.68, p= 0.01), Viable by calceinviolet (r2=0.67, p= 0.01), Viable non H2O2-producing (r2=0.66, p= 0.01), Viable with High mitosoxred/High MMP (r2=0.65, p= 0.02), Viable non superoxide-producing (r2=0.60, p= 0.02), Linearity (r2=0.56, p= 0.03). The only variable found to have a negative relationship with fertility was Amplitude of lateral head displacement (r2=0.63, p= 0.02). The other 26 measured variables did not show a significant relationship with fertility. The results of the study shows that both kinematic variables as well as flow cytometric analysis can be valuable for relating fertility with sperm quality in vitro. The flow cytometry results indicate that analysis of the production of different reactive oxygen species can be valuable when predicting stallion fertility. Acknowledgments: Special thanks to Menhammar, Västerbo, Markebäck and Stockholm Seminstation for providing stallion semen. This work was supported by funding from FORMAS (2018-01083; awarded to AJ). The work was also supported by the Swedish University of Agricultural Sciences via the Cells for Life Platform.

CpG DNA methylation changes during epididymal sperm maturation in bulls

BackgroundDuring epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions.ResultsCaput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda.ConclusionsSpecific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

Melatonin Ameliorates Cyclophosphamide-Induced Spermatogenesis Disorder by Reducing Pyroptosis

As a chemotherapeutic drug, cyclophosphamide (CP) has a negative impact on male fertility due to its reproductive toxicity. Melatonin (Mel) promotes the male reproductive system and increases testosterone synthesis. This study is aimed at exploring the molecular mechanism of Mel as a protector of male fertility against CP-induced cytotoxicity. A CP toxicity model was established in adult ICR male mice by intraperitoneal injection of 100 mg/kg CP every other day for a week. Protective effects of Mel on the testis from CP-induced damage were evaluated using four groups of ICR male mice that received intraperitoneal injections of normal saline, 100 mg/kg CP, 10 mg/kg Mel, or the same dosage of CP and Mel, respectively. Testis morphology was observed by hematoxylin and eosin (HE) staining. Sperm quality parameters were evaluated, and sperm function was studied by in vitro fertilization (IVF). Proliferation, meiosis, and pyroptosis markers were examined by western blot. Results showed that CP treatment induced testis toxicity in a time-dependent manner with the most severe damage to the testis at two weeks post CP treatment. CP-treated mice showed reduced testicular weight and impaired spermatogenesis by downregulating PCNA and SYCP3, reduced serum testosterone levels, decreased sperm counts and motility, increased seminiferous tubule vacuolization, and oxidative damage to spermatogenic cells. All these effects, apart from testicular weight, could be ameliorated by Mel administration. The IVF experiment revealed that CP treatment reduced the rates of sperm fertilization and blastocyst development, which were also enhanced by Mel. Mel-treated mice also showed increased expression of proliferation-associated protein PCNA and meiosis-associated proteins REC8, STRA8, and SYCP3, which were all reduced by CP. Furthermore, Mel inhibited the pyroptosis of spermatogenic cells by reducing GSDMD and IL18 expression. In conclusion, this study indicated that Mel might protect the testis from CP-induced DNA damage to germ cells through the alleviation of pyroptosis.

Antioxidant effects of zinc-oxide nanoparticles on post-thaw quality and in vivo fertility of Beetal buck spermatozoa

Metallic nanoparticles are generally considered potential cryoprotectants during semen cryopreservation owing to their ability to improve the spermatozoa quality following cryopreservation when added in cryo-diluents. The present study aimed at investigating the effects of incorporating zinc oxide nanoparticles (ZnO-NPs) in the cryo-diluent on in vitro post-thaw sperm quality attributes, oxidant-antioxidant profile, and in vivo fertility rate in Beetal buck. After characterization, ZnO-NPs were added to the Tris-citric acid-fructose yolk extender. Semen samples, collected from five breeding bucks (5 ejaculates per buck) using an artificial vagina, were pooled. The pooled semen samples, distributed into five equal aliquots, were diluted in the Tris-citric acid cryo-diluent containing five different concentrations of ZnO-NPs (0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, and 0.0 mg/ml called control group), and were cryopreserved for 24 h. At post-thaw, various in vitro sperm quality parameters, and antioxidant enzyme profiles like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and reduced glutathione (GSH) levels were determined. For the in vivo fertility experiment, breeding does (n = 152), aging from 1.5 to 4 years, were artificially inseminated with spermatozoa with intact acrosome containing the same five concentrations of ZnO-NPs. Results demonstrated that the 1.0 mg/ml ZnO-NPs (crystallite size of 19.73 nm) improved (p < 0.01) the activities of antioxidant enzymes (SOD, CAT, and POD) with concomitantly decreased levels (p < 0.001) of lipid peroxidation (LPO) and reactive oxygen species (ROS) when compared with the other groups. Total spermatozoa motility, rapid velocity, and average path velocity were higher (p < 0.05) with the 1.0 mg/ml ZnO-NPs when compared with the control group. Progressive motility and straight-line velocity were higher (p < 0.05) with the 0.5, 1.0, and 1.5 mg/ml of ZnO-NPs addition compared with the 2.0 mg/ml and control groups. The sperm kinematics parameters; the amplitude of the lateral head displacement, the beat cross frequency, the straightness, and the linearity were not influenced by the ZnO-NPs inclusion. Supra-vital plasma membrane integrity was higher (p < 0.001) with the 0.5 mg/ml and 1.0 mg/ml of ZnO-NPs added groups when compared with either 2.0 mg/ml ZnO-NPs or the control group. The viable sperm with intact acrosome was higher (p < 0.05) in the 1.0 mg/ml ZnO-NPs group compared with the other groups. Low supplementation rates improved (p < 0.05) the DNA integrity when compared with the higher dose (2.0 mg/ml ZnO-NPs). The pregnancy per AI was higher (59.3 %; P < 0.05) in the does inseminated with semen added with the 0.5 mg/ml ZnO-NPs followed by 1.0 mg/ml (58.8 %), 1.5 mg/ml (58.6 %), and 2.0 mg/ml (46.6 %) when compared with the control group (33.3 %). We concluded that the 1.0 mg/ml ZnO-NPs added in the citric acid-fructose yolk extender improved the post-thaw quality of Beetal buck spermatozoa in terms of elevated activities of endogenous antioxidant enzymes, improved structural and functional integrity along with higher fertility.

The Effect of Adding Different Levels of Reduced Glutathione to Extender on the Quality of Cooled Ring-Necked Pheasant Semen.

Aim: Artificial propagation of ring-necked pheasant through semen preservation is of significance, as this species is facing enormous threats in its natural habitat. Semen preservation inevitably induces oxidative stress, and exogenous antioxidants need to be investigated for the preservation of ring-necked pheasant semen. Therefore, the current study was conducted to investigate the role of glutathione (GSH) in extender on the liquid storage of ring-necked pheasant semen. Materials and Methods: Semen was collected from 10 sexually mature males, evaluated for sperm motility, and pooled. Pooled semen was aliquoted for dilution with Beltsville poultry semen extender (1:5) at 37°C having GSH levels of 0.0 mM (Control), 0.2, 0.4, 0.6, and 0.8 mM. Extended semen was gradually cooled to 4°C and stored in a refrigerator (4°C) for 48 hours. Semen quality, that is, sperm motility, membrane integrity, viability, acrosomal integrity, and DNA integrity, was assessed at 0, 2, 6, 24, and 48 hours. Results: Sperm motility (%), plasma membrane integrity (%), viability (%), and acrosomal integrity (%) were recorded higher (p < 0.05), whereas DNA fragmentation (%) was recorded lower in extender supplemented with 0.4 mM GSH up to 48 hours of storage compared with 0.2, 0.6, and 0.8 mM GSH concentrations and control. Conclusion: It is concluded that 0.4 mM GSH in extender improves sperm quality parameters of ring-necked pheasant during liquid storage up to 48 hours at 4°C.

(256) Sexual Function and Andrology Parameters in Men after Treatment of Anterior Urethral Strictures with the Optilume Drug Coated Balloon

Abstract Introduction The Optilume Urethral Drug Coated Balloon (DCB) is a novel endoscopic treatment for recurrent male anterior urethral strictures. An important topic of discussion with patients during counseling on treatment options includes the potential affect of treatment on sexual function and fertility. The ROBUST III study was a prospective, randomized study comparing standard of care endoscopic therapy against treatment with the Optilume DCB. Assessments included evaluation of sexual function and andrology parameters. Objective The objective was to study the potential affect of treatment with the Optilume DCB on sexual function and, for younger patients, fertility. Methods A total of 79 men were randomized to receive treatment with the Optilume DCB. Sexual function was assessed via the 15 item International Index of Erectile Function (IIEF) questionnaire. Erectile function, orgasmic function, and overall satisfaction sub-domains were assessed at 3, 6, and 12 months after treatment in patients that were sexually active at baseline. Fifteen additional subjects were enrolled in a non-randomized pharmacokinetic sub-study that included assessments of paclitaxel concentration in semen and sperm quality parameters through 6 months post-treatment. Results Average erectile function and overall satisfaction scores improved from baseline to 12 months post-treatment, while orgasmic function was not changed (Figure). The improvement in satisfaction was statistically significant at 12 months (p=0.003). No adverse events of de novo erectile or ejaculatory dysfunction were reported. Average ejaculate sperm concentration did not change from baseline to 6 months (49 million/mL vs 46.6 million/mL, p=0.88), nor did total spermatozoa per ejaculate (107.2 million vs 104.0 million, p=0.94). Paclitaxel concentration in semen after treatment was maximal at 30 days (2.99 ng/mL) and was reduced near the limit of quantitation of the analytical method by 6 months (0.12 ng/mL). Conclusions Treatment of male anterior urethral strictures with the Optilume DCB did not appear to have an impact on sexual function as measured by the IIEF and adverse event assessments. Average sperm quality parameters did not differ from baseline to 6 months post-treatment. Low levels of paclitaxel were detected in semen after treatment but were nearly undetectable by 6 months. Disclosure Yes, this is sponsored by industry/sponsor: Urotronic Inc. Clarification Industry funding only - investigator initiated and executed study. Any of the authors act as a consultant, employee or shareholder of an industry for: Boston Sci, Coloplast, Medtronic USA Inc., PROCEPT BioRobotic Corporation, NeoTract Inc., Janssen Biotech Inc., Endo Pharmaceuticals Inc., Tolmar Pharmaceuticals, Inc., 180 Medical Inc., AbbVie Inc., Pfizer Inc., Bluewind Medical, Astellas Pharma US Inc., Antares Pharma Inc., Axonic Modulation Technologies Inc., Allergan Inc., Terumo Medical Corporation, Dendreon Pharmaceuticals llc, Blue Earth Diagnostics Limited, Ferring Pharmaceuticals Inc., Olympus America Inc., Karl Storz Endoscopy-America, Clovis Oncology Inc., Dornier MedTech America Inc., Travere Therapeutics Inc., Valeant Pharmaceuticals North America LLC

Reaction time as a libido indicator and its relation to pre-freeze and post-thaw sperm quality in bulls.

Libido and sperm quality output relationship is already not clear in farm animals. The present study compared reaction time (RT) as a libido indicator and the pre-freeze and post-thaw sperm quality of AI bulls. Before the collection of ejaculates (n = 53, from 22 AI bulls [4.2 ± 1 years of age]), RTs were collected using a chronometer as the interval between the bull's arrival at the semen collection area and his first false mount (FM) on another male. The ejaculates were examined for their volume, concentration and motility. Subsequently, all aliquots were diluted with a commercial semen extender and equilibrated for 3 h before freezing. Frozen semen samples were thawed and examined for sperm kinematics using CASA, plasma membrane and acrosome integrity of sperm (PMAI) by flow cytometry. Additionally, the temperature humidity index (THI) values were assessed during the study. Multiple linear regression analysis was used to analyse the data. The results indicated that THI had a significant effect on libido (p < .001). However, libido had no effect on either pre- or post-thaw sperm quality parameters except for the velocity of the average pathway (VAP) (p < .05). Therefore, relying solely on RT -libido- as an indicator of bull sperm quality at AI stations may not be reliable, as it is a complex behavioural assessment.

Microbiome structure of milt and ovarian fluid in farmed Arctic charr (Salvelinus alpinus)

Limited knowledge exists about the residing microbiome in gamete-related samples in fish. A potential effect between the seminal microbiome composition and sperm quality traits has been previously suggested in humans and livestock. Using a metabarcoding approach, we aimed to gain insights into the structure of the residing prokaryotes and microbial eukaryotes in ovarian fluid (n = 10) and milt (n = 84) from farmed Arctic charr - a species with highly variable reproductive success in captivity. In addition, sperm quality traits were recorded on the sampled males to investigate potential associations with the residing seminal microbiome. Higher microbial diversity was found in the ovarian fluid compared to the milt habitat. Even though the residing microbiome showed distinct differences between the two habitats, substantial overlap was observed, with >70% of the milt core microbiome being found in the ovarian fluid habitat. Statistically significant associations were found between the Shannon diversity index and sperm motility-related traits. Additionally, a fungal operational taxonomic unit (OTU) potentially belonging to the Leotiomycetes class was associated with sperm concentration and motility. Overall, our study documents the microbiome structure of gamete-related samples from Arctic charr. Even though some associations were obtained between sperm quality parameters and either microbiome diversity or with a fungal OTU, follow-up studies on a larger scale with more tank replicates are needed to confirm the robustness and causality of these relationships.

The Impact of Bacteriospermia on Semen Parameters Among Infertile Egyptian Men: A Case-Control Study.

This study aimed to detect the impact of bacteriospermia on semen parameters and sperm DNA fragmentation. This prospective case-control study was conducted over a period of 9 months. Samples were collected from andrology outpatient clinic attendants of Cairo University Hospitals. We enrolled 68 semen samples divided into two groups: a study group (34 semen samples with bacteriospermia) and a control group (34 semen samples without bacteriospermia). The characteristics of the semen, including morphology, motility, count, liquefaction, viscosity, pH, volume, and appearance, were evaluated according to the standard protocols. Patients with and without bacteriospermia had a comparable liquefaction time (p = .343), semen appearance and color (p = 1.00), semen pH (p = 1.00), velocity (p = .163), and total sperm count (p = .451). Patients with bacteriospermia were associated with lower progressive motility (p = .001), nonprogressive motility (p = .032), total motility (p = .001), and normal forms (p = .001). The prevalence of abnormal semen analysis was 64.71% in the study group compared with 35.29% in the control group. Staphylococcus aureus (67.6%) and Escherichia coli (14.7%) were the most commonly detected organisms. Samples from which Methicilin-resistant Staphylococcus aureus was isolated showed significant abnormalities in both progressive motility and normal morphology of sperm. Bacteriospermia has deteriorative effects on sperm quality parameters, such as semen volume, sperm motility, and sperm normal morphology.

Mitochondria-Stimulating and Antioxidant Effects of Slovak Propolis Varieties on Bovine Spermatozoa

Propolis is a natural honeybee product, which is known for its beneficial pharmacological effects and use in traditional medicine. This study aspired to investigate the chemical and antioxidant properties of five propolis specimens, followed by the analysis of their in vitro impact on bovine spermatozoa vitality and oxidative profile. Semen samples from 10 breeding bulls were incubated in the absence or presence of 50 µg/mL propolis extracts, and selected sperm quality parameters including motility, mitochondrial activity, cAMP concentration, NAD+/NADH ratio, reactive oxygen species (ROS) and superoxide production were assessed at 2 h and 24 h. Sperm motion behavior and mitochondrial function were stabilized particularly in the presence of propolis collected from Hrabské and Litpovský Hrádok, Slovakia. At the same time, all propolis specimens acted as significant ROS quenchers and stabilized the oxidative milieu of the sperm cultures. Our data suggest that propolis may stabilize the mitochondrial function of spermatozoa while preventing excessive oxidative insults, thereby showing the potential to sustain the sperm motility of extended semen.

Effects of extender type and storage time on sperm quality parameters of Kail ram semen stored at 5 °C

The study was designed to evaluate the effect of different extenders and storage times on sperm quality parameters of extended Kail ram semen. Semen was collected from five adult Kail rams using an artificial vagina. Semen samples with >70% total sperm motility were pooled, diluted with Tris (TR), sodium citrate (SC), and skim milk (SM)-based extenders, and stored at 5 °C. Sperm motility and kinematics, viability, and plasma and acrosomal membrane integrity were assessed every 24 hrs for 120 hrs. Sperm longevity was estimated by placing semen in a water bath at 37°C to assess sperm kinematics. Storage time as the main effect had a negative (P < 0.05) impact on sperm quality parameters. The percentages of total motile (TM), progressive motile (PM), rapid, and medium progressive (MP) motile sperm were similar at 24 hrs of storage for the three extenders. However, semen extended in TR had a higher (P < 0.05) percentage of TM, PM, rapid, and MP sperm compared to SM and SC extenders at 48 hrs of storage. The sperm kinematics (VCL, VSL, VAP, and ALH) of progressive sperm were similar for all three extenders up to 72 hrs of storage. In addition, semen extended in TR had a higher (P < 0.05) percentage of sperm with intact plasma and acrosomal membranes at a storage time of 48 hrs. At 37 °C, the percentage of TM and PM sperm was higher (P < 0.05) in the TR extender than in the SM and SC extenders at 60 minutes and beyond. In conclusion, the Kail ram sperm extended in TR and stored at 5 °C maintained better quality after 48 hrs storage than sperm extended in SM and SC extenders. At 37 °C, sperm extended in TR also retained better quality parameters at 60 min and afterward than sperm extended in SM and SC.