Conventional Semen Research Articles

Sperm DNA integrity and male infertility: a narrative review and guide for the reproductive physicians.

Background and ObjectiveConventional semen analysis (SA) remains an essential tool in the initial male fertility evaluation and subsequent follow-up. However, it neither provides information about the functional status of spermatozoa nor addresses disorders such as idiopathic or unexplained infertility (UI). Recently, assessment of sperm DNA fragmentation (SDF) has been proposed as an extended sperm test that may help overcome these inherent limitations of basic SA. In this review, we aim to: (I) discuss the pathophysiological aspects of SDF, including natural repair mechanisms, causes, and impact on reproductive outcomes; (II) explain different assessment tools of SDF, and describe potential therapeutic options to manage infertile men with high SDF; and (III) analyse the strengths, weaknesses, opportunities and threats (SWOT) of current research on the topic.MethodsThis review was constructed from original studies, systematic reviews and meta-analyses that were published over the years up until August 2021, related to the various aspects of SDF.Key Content and FindingsDifferent mechanisms lead to high SDF, including defective chromatin packaging, apoptosis, and seminal oxidative stress. The relevance of sperm DNA integrity to male fertility/infertility has been supported by the frequent observation of high levels of SDF in infertile men, and in association with risk factors for infertility. Additionally, high SDF levels have been inversely correlated with the outcomes of natural pregnancy and assisted reproduction. Terminal deoxynucleotidyl transferase dUTP nick end labelling, sperm chromatin structure assay, sperm chromatin dispersion, and Comet assay are four commonly used assays for measurement of SDF. Addressing lifestyle risks and underlying conditions, antioxidants, hormonal therapy, and advanced sperm selection techniques have all been proposed as potential therapeutic options to lower SDF.ConclusionsThe sum of literature provides evidence of detrimental effects of high SDF on both natural and assisted fertility outcomes. Standardization of the techniques used for assessment of SDF and their incorporation into the work up of infertile couples may have significant implications on the future management of a selected category of infertile men with high SDF.

P-032 Sperm parameters can predict the success of assisted reproductive technology. Single-center and retrospective analysis of assisted reproductive technology cycles from 1992 to 2020

Abstract Study question Male parameters are largely underestimated in assisted reproductive technology (ART) success, so it was evaluated whether conventional semen parameters could predict assisted reproductive technology success Summary answer Sperm motility plays a role in predicting in vitro fertilization (IVF) success, while sperm morphology is the relevant parameter in intracytoplasmic sperm injection (ICSI) cycles. What is known already The detection of predictive markers would be very useful to choose the ART type and usually the male counterpart was relegated to a secondary role. Obviously, no convincing evidence that ART outcomes may be dependent on sperm parameters exists. The variation over the years of the reference values in semen analysis, together with the poor inter-laboratory standardization of sperm assessment, contributes to this very complex scenario. It remains undeniable that identifying thresholds of seminal parameters with prognostic significance in terms of pregnancy rate would have a considerable clinical impact in the assisted reproduction field. Study design, size, duration A single-center, retrospective, observational study was carried out including couples attending the Santa Maria Nuova Hospital-IRCCS of Reggio Emilia for infertility between January 1992 and December 2020. Only fresh ART cycles performed in a single third-level fertility center were considered for this study with a total of 22,013 cycles. Fertilization rate was the primary end-point, representing a parameter immediately dependent on male contribution. Pregnancy and live birth rates were considered in relation to semen variables. Participants/materials, setting, methods Both partners were aged over 18 years and satisfied national criteria to access ART procedures. The studies considering common sperm parameters obtained by conventional semen analysis, that is, sperm number, motility, and morphology. Semen analyses were performed according to the World Health Organization (WHO) manual available at the time of the ART cycle. Ovarian stimulation was performed applying different hormone stimulating protocols and ART approach used are IVF or the ICSI. Main results and the role of chance Considering the entire ART cohort, 5819 cycles were IVF (26.4%) and 16,194 were ICSI (73.6%). Considering strong ART outcomes, the overall pregnancy rate was 20.4% (4,368 cycles) for biochemical and 20.2% (4,314 cycles) for clinical pregnancies, respectively. Among the latter, the overall live birth rate was 63.3%. Interestingly, both biochemical (20.8% vs. 19.1%, p < 0.001) and clinical (20.7% vs. 18.5%, p < 0.001) pregnancy rates were significantly higher in ICSI than IVF cycles. On the contrary, the live birth rate was not significantly different between ART methodologies (64.0% in ICSI vs. 60.9% in IVF, p = 0.074). Only 610 cycles (2.8%) were interrupted since no oocytes were retrieved after gonadotropins stimulation. Considering only cycles with oocytes retrieved, the fertilization rate was significantly higher in ICSI (42.4±39.7%) compared to IVF cycles (38.4±49.2%) (F = 27.343, p < 0.001). In IVF setting, both progressive motility (p = 0.012) and motility after capacitation (p = 0.002) significantly predicted the fertilization rate (statistical accuracy = 71.1%). Sperm motilities also predicted pregnancy (p < 0.001) and live birth (p = 0.001) rates. In ICSI, sperm morphology predicted fertilization rate (p = 0.001, statistical accuracy = 90.3%). Limitations, reasons for caution These results, although statistically significant, show confidence intervals at the limits of significance. Therefore, this data must be carefully considered. From entering the ART path to its outcome, the variables encountered are numerous. The identification of a single parameter that influences, slightly, the final outcome, indicates a considerable clinical weight. Wider implications of the findings Interestingly, sperm motility plays a role in predicting in vitro fertilization success, while sperm morphology is the relevant parameter in ICSI. These parameters may be considered reliable tools to measure the male role on ART outcomes, potentially impacting the clinical management of infertile couples. Trial registration number not applicable

O-132 Is there an association between sDF index and serum testosterone in human follicle stimulating hormone alfa (r-hFSH alfa) treated males with idiopathic infertility?

Abstract Study question Is there correlation between testosterone serum levels and sperm DNA fragmentation (sDF) index before and after follicle-stimulating hormone (FSH) administration in male idiopathic infertility? Summary answer Correlation between testosterone and sDF was highlighted for the first time in FSH-treated men. Significant amelioration of conventional semen parameters and sDF index were confirmed. What is known already Testicular overstimulation is the yearned therapeutic goal when exogenous FSH is empirically administered in male idiopathic infertility. Although a robust physiological rationale theoretically supports the FSH use in male idiopathic infertility, useful markers to evaluate its efficacy are still far to be identified. Whether pregnancy outcomes remain the expected valuable endpoint for couple infertility treatment, the identification of reliable, and possibly early, markers of therapeutic response to FSH in males is mandatory. Randomized controlled clinical trials (RCTs) on this topic focused, as main outcomes of FSH treatment effectiveness, on testicular seminiferous component, not considering involvement of testosterone-secreting compartment. Study design, size, duration A retrospective post-hoc analysis of raw data extracted from two RCTs in which 148 idiopathic infertile men were treated with FSH. Participants/materials, setting, methods A retrospective post-hoc re-analysis was performed on raw data of RCTs in which idiopathic infertile men were treated with FSH and both testosterone serum levels and sDF were reported among primary and/or secondary endpoints. Additional data regarding couple infertility history, age, anthropometric variables, FSH treatment scheme and semen variables were included in a single dataset. Data were evaluated by logistic regression analyses. Main results and the role of chance Two RCTs were included accounting for 148 patients (median age 37, 25-52 years). After three months of FSH administration, a significant increase was observed in FSH levels (p < 0.001), inhibin B (p = 0.012), sperm concentration (p = 0.003), total sperm number (p = 0.021), progressive motility (p < 0.001) and normal sperm morphology (p < 0.001). Moreover, an overall sDF index reduction was confirmed after treatment (19.3±9.4 versus 16.4±6.8, p = 0.002). SDF was found significantly inversely related to sperm concentration both at baseline and after FSH treatment (Rho -0.325, p < 0.001 and Rho -0.316, p = 0.001, respectively). Interestingly, sDF index after treatment showed a significant inverse correlation with testosterone serum levels (Rho -0.327, p = 0.002). Multivariate stepwise linear regression analyses using sDF index as dependent variable identified testosterone as a predictor for sDF index change (p = 0.005). Similarly, logistic regression analysis highlighted testosterone and SHBG levels as predictive of sDF reduction after FSH administration (p = 0.043 and p = 0.005, respectively). Combining raw data of published RCTs investigating FSH administration to idiopathic infertile men, a significant amelioration of conventional semen parameters together with a reduction in sDF were confirmed. Intriguingly, a potential correlation between serum testosterone and sDF was highlighted for the first time. Limitations, reasons for caution Since the analysis was performed on raw data obtained in previous RCTs, heterogeneity among patients enrolled and evaluated should be considered interpreting these results. Moreover, a real and accurate evaluation of intratesticular testosterone levels, rather than serum levels should be more informative to the objective of the study. Wider implications of the findings The highlighted significant correlation between testosterone serum levels and sDF is opening a completely unexplored field in the possibility to identify early predictors of FSH therapy response in male idiopathic infertility. Trial registration number Not applicable

O-251 Defining the exclusive role of male genome integrity on conceptus development

Abstract Study question To determine the effect of sperm DNA fragmentation on embryo development by ruling out a female factor component. Summary answer By utilizing healthy donor oocytes, it is possible to quantify the sole deleterious effect of sperm DNA fragmentation and explore the ooplasmic repair mechanism. What is known already In approximately 50% of couples with unexplained ART failure, a subtle male factor is present that is missed in a conventional semen analysis assessing concentration, motility, and morphology. Additional information on male gamete competence can be acquired by assessing Sperm Chromatin Fragmentation (SCF). Indeed, a fragmented male genome can give rise to poorly developing embryos, leading to impaired implantation, lower pregnancy, and higher miscarriage rates. It has been previously seen that an oocyte, according to its age, can repair the damaged DNA contributed by the male gamete. Study design, size, duration In last decade, 316 couples, who have an elevated SCF, underwent ICSI cycles and resulted in disappointing clinical outcomes. To exclude an eventual confounding female factor, couples who utilized donor oocytes were identified and clinical outcome was compared to a control. To measure the role of an impaired sperm genome and a concurrent oocyte repair mechanism, we compared clinical outcomes between cycles in the same couples who used their own and subsequently donor oocytes. Participants/materials, setting, methods We included 381 couples screened for SCF; 65 underwent ICSI cycles with donor oocytes; of these, 46 underwent a previous ICSI cycle with their own oocytes. Fertilization, implantation, clinical pregnancy, and delivery rates were compared between above-mentioned groups. A TUNEL assay was used to measure SCF (≥500 spermatozoa were assessed/sample at a 15% threshold). Paired t and Chi-square tests were used to compare ages and clinical outcomes, respectively. Main results and the role of chance We included 381 couples (maternal age, 37.8 ± 4.2; paternal age, 41.8 ± 8.2), whose male partners had the following semen parameters: average volume of 2.5 mL, concentration of 25.4x106/mL, 33% motility, and normal morphology of 1.7. Of these, 316 had elevated average DNA fragmentation of 25.5% and utilized their own oocytes in 683 ICSI cycles, resulting in a 67.5% fertilization, 12.2% implantation, 27.9% clinical pregnancy, and a 22.5% ongoing/delivery. Of the 381 couples, 65 underwent ICSI utilizing donor oocytes (SCF of 24.3%) resulting in a significantly higher fertilization (78.4%) (P < 0.00001) and embryo implantation rates (34.2%) (P < 0.0001). Similarly, albeit not statistically significant, the clinical pregnancy and ongoing/delivery rates increased from 27.9% to 37.1% and from 22.5% to 30.0%, respectively. To further explore the exclusive role of the male gamete in embryo development, we identified a cohort of patients (n = 46), with an elevated average DNA fragmentation of 23.6%, who underwent an ICSI cycle using their own oocytes and a subsequent cycle using donor oocytes. Compared to cycles where couples used their own oocytes, fertilization, embryo implantation, clinical pregnancy, and ongoing/delivery rates rose from 67.5% to 76.6% (P < 0.0001), 2.8% to 24.7% (P < 0.00001), 8.4% to 38.8% (P < 0.0001), and 1.2% to 28.6% (P < 0.00001), respectively. Limitations, reasons for caution Although this study attempts to control for a concurrent confounding female factor, it cannot be completely excluded. It is difficult to assess the extent and contribution of the ooplasmic repair mechanisms on the male genome. Wider implications of the findings Ooplasmic repair mechanisms of healthy female gametes appear to repair or improve the deleterious impact of sperm DNA fragmentation on ART outcomes. Trial registration number not applicable

P-074 Sperm deoxyribonucleic acid integrity decreases with age and exhibits a rapid decline beyond the age of 35: a retrospective evaluation of 3446 semen samples

Abstract Study question Does advancing paternal age correlate with sperm DNA fragmentation index (DFI) and is there a cut-off age beyond which sperm DFI increases significantly? Summary answer In infertile men, DFI correlate with advancing paternal age and should be routinely screened starting 35 years of age. What is known already In recent decades, birth rates have substantially increased for men older than 30 years because of advanced age of marriage, rising life expectancy at birth, modern societal norms, and accessibility to assisted reproductive technology (ART). Advanced paternal age has been associated with a decline in conventional semen parameters (volume, concentration, motility, DFI), as well as, reduced fertility, increased risk of miscarriage, structural chromosomal aberrations and complex epigenetic disorders. Several studies have recommended testing sperm DFI in infertile men with advanced age (≥40 years) as it may provide prognostic information for couple attempting natural and assisted reproduction. Study design, size, duration This is a retrospective study of 3446 semen samples from patients under investigation for infertility between April 2016 and January 2022. Semen samples were obtained after 2-3 days of sexual abstinence. Patients were stratified into seven groups based on their age: patients ≤ 29 years (n = 127; 3.7%), 30-35 years (n = 868; 25.2%), 36-39 years (n = 863; 25.0%), 40-45 years (n = 1017, 29.5%), 46-49 years (n = 321; 9.3%), 50-55 years (n = 179, 5.2%) and ≥ 56 years (n = 71, 2.1%). Participants/materials, setting, methods Conventional semen parameters were assessed according to the WHO criteria and DFI was evaluated by TUNEL assay using the APODirect Kit run on BDAccuriC6 flow cytometer. Pearson’s r was used for correlation analysis between sperm concentration, DFI and paternal age. DFI results for each stratified patient group were evaluated by one-way ANOVA, followed by Tukey pos-hoc multiple comparison test. Results are presented as the mean±standard error and a P-value of < 0.05 was considered statistically significant. Main results and the role of chance In this cohort of men with a mean age of 39.5 years ± 0.1 (range 23-76 years), sperm deoxyribonucleic acid (DNA) fragmentation (21.1% ± 0.2) was positively correlated with age (r = 0.23, p˂0.001). In contrast, the correlation between sperm concentration and age was non-significant (r = 0.03, p = 0.07). Mean DFI in patients segregated into seven age groups were: ≤29 years (15.7% ± 0.8), 30-35 years (17.7% ± 0.4), 36-39 years (19.7% ± 0.4), 40-45 years (22.6% ± 0.4), 46-49 years (26.2% ± 0.9), 50-55 years (26.7% ± 1.2) and ≥ 56 years (31.1% ± 2.0). Mean %DFI level in the 26-29 and 30-35 age groups were non-significantly different (p = 0.65). However, mean %DFI level in the 36-39 age group was significantly higher than in the 26-29 and 30-35 age groups (p = 0.02 and p = 0.03, respectively). Mean %DFI level in the older age groups (40-45, 46-49, 50-55 and ≥ 56 years) were all significantly higher than in the 26-29 or 30-35 age groups (p˂0.001). Using a %DFI threshold level of 16.9%, 46.0% of patients ˂36, 52.2% of men aged 36-39, 60.2% of men aged 40-45, 67.3% of men aged 46-49, 72.6% of men aged 50-55 and 74.7% of men aged ≥56 years had an elevated DFI. Limitations, reasons for caution This is a retrospective analysis that did not account for confounding variables (e.g., clinical diagnosis, gonadotoxin exposure, febrile illness) that may affect conventional sperm parameters and DFI. Wider implications of the findings Our results underline the relationship between paternal age and sperm DFI and demonstrate a significant decline in sperm DNA fragmentation in men over the age of 35 years. The data suggest that we may want to reconsider the age cut-off we traditionally use to define advanced paternal age. Trial registration number No trial registration

P-064 Clinical outcomes of 127 patients with recurrent implantation failure treated with testicular sperm aspiration (TESA)

Abstract Study question Are the embryological, clinical and newborn outcomes using aspirated testicular sperm improved in cases with recurrent implantation failure previously treated with ejaculated sperm? Summary answer Aspirated testicular sperm enabled to obtain significant higher embryological, clinical and newborn outcomes in cases with recurrent implantation failure previously treated with ejaculated sperm. What is known already High levels of sperm DNA fragmentation (SDF) were associated to poor clinical outcomes (1-Simon et al., 2017). Testicular sperm display lower SDF than ejaculated sperm (2-Sakas and Alvarez, 2010), improving clinical outcomes in cases with abnormal semen parameters (3-Awaga et al., 2018; 4-Kang et al., 2018), recurrent implantation failure (RIF) and pregnancy loss (RPL) (5-Esteves et al., 2017), and elevated SDF (6-Ambar et al., 2021). As only a few studies are specifically dedicated to RIF, we expanded the number of cases and first provided full demographic, stimulation, embryological, clinical and newborn outcomes. References 1-(https://doi.org/10.4103/1008-682X.182822); 2-(https://doi.org/10.1016/j.fertnstert.2009.10.046); 3-(https://doi.org/10.1016/j.rbmo.2018.08.017); 4-(https://doi.org/10.1038/s41598-018-26280-0); 5-(https://doi.org/10.1016/j.fertnstert.2017.06.018); 6-(https://doi.org/10.5534/wjmh.200084 Study design, size, duration We retrospectively evaluated during consecutive years (2010-2020) 63 patients with recurrent implantation failure, which accepted to perform testicular sperm aspiration (TESA) as an alternative treatment. These patients presented a long history of failed treatments (153 cycles) using ejaculated sperm. From these cycles, no pregnancy ensued. The present study compares 127 treatment cycles, 80 with testicular sperm (17 cases repeated TESA) and 47 with ejaculated sperm from the same patients performed at the present IVF clinic. Participants/materials, setting, methods Patients were screened for karyotype abnormalities, for Y-chromosome microdeletions (7-Gonçalves et al., 2016), and for SDF with the TUNEL assay (8-Sá et al., 2015). Conventional semen analysis was performed according to World Health Organization guidelines (9-WHO, 2010). Male evaluation and TESA was performed by the same experienced urologist (LF) according to established protocols (10-Madureira et al 2014). The procedure was performed entirely on an outpatient basis, with no complications reported. References 7-(https://doi.org/10.4103/1008-682X.172827); 8-(https://doi.org/10.1016/j.rbmo.2015.06.019); 9-(https://apps.who.int/iris/handle/10665/44261); 10-(https://doi.org/10.1111/j.2047-2927.2014.00231.x). Main results and the role of chance The mean ages were 35.5±3.4 (26-42)-female and 38.1±5.7 (29-59)-male. There were 4 abnormal karyotypes (3-female, 1-male), all without known relevance. Most cases had asthenozoospermia and teratozoospermia (65.1%), or oligoasthenoteratozoospermia (41.8%). Of the 19 cases with <5M/ml, none presented Y-chromosome microdeletions. Although we do not routinely perform SDF testing, 15 patients had previous SDF values (12, >20%; 8, >36%). Female basal characteristics and testicular evaluation were under normal values. The TESA procedure took about 15-20 min, and the time of laboratorial search around 30-60 min. Cases using testicular sperm showed significant higher rates of fertilization (64% vs 73%-p=0.005), blastocyst development (47% vs 62%-p=0.010), implantation (6% vs 27%-p=0.000), clinical pregnancy (10% vs 39%-p=0.001), live birth delivery (5% vs 28%-p=0.005) and newborn (5% vs 32%-p=0.000) than ejaculated sperm. No significant differences were observed regarding the rates of embryo cleavage (95% vs 94.8%) and high quality embryos (89.4% vs 94%), in the mean number of transferred embryos (1.8±0.4 vs 1.9±0.4), or in the abortion rate (2 cases-50% vs 7 cases-25.9%). Cases using testicular sperm had 22 frozen-thawed embryo transfer cycles, enabling per initiated cycle a cumulative pregnancy rate of 45%, live birth delivery rate of 31.3% and newborn rate of 37.5% (32 newborn). Limitations, reasons for caution Although presenting the higher number of cycles using TESA in the treatment of RIF, this number needs to be increased for drawing more definitive conclusions, as these women present a diversity of conditions, rendering subgrouping difficult. In the future, it would also be important to evaluate SDF in all cases. Wider implications of the findings In conclusion, the present results gave further evidence for the superiority of using testicular sperm instead of ejaculated sperm in cases with recurrent implantation failure. Data also evidences the security of using testicular sperm aspiration, as there were no pregnancy or delivery complications, or congenital anomalies among the 32 newborn. Trial registration number Not Applicable

O-250 Strong differences in surface molecule expression profile in both microvesicles and exosomes between patient and semen donor groups: new fertility biomarker?

Abstract Study question Are the extracellular vesicles (exosomes and microvesicles) presented in seminal plasma a potential novel biomarkers of male infertility? Summary answer There are significant differences in surface molecules (CD9, CD29, CD40, CD49d, CD73, CD282 and CD284) expressed in extracellular vesicles of patient and control groups What is known already Male infertility has been progressively increasing in recent decades. Therefore, it is essential to find new tools that help us to diagnose the male factor. One of the potential markers could be the study of the extracellular vesicles (EV), mainly exosomes (EXO) and microvesicles (MV), present in the seminal plasma. Recent works have shown that these extracellular vesicles would have an important role in the modulation of the maternal immune system, as well as in regulating the sperm activation. Therefore, the study of the extracellular vesicles can be a useful model to increase the diagnosis of the infertile male Study design, size, duration This prospective study, performed between March 2019 to January 2022, included 44 semen samples from healthy semen donors (control group) and 44 semen samples from infertile males seeking infertility treatment with their partners (study group). Men under drug treatment and/or with infectious or chronic diseases were excluded from the study Participants/materials, setting, methods After conventional semen analysis, semen samples were ultra-centrifuged to obtain EVs enriched plasma seminal. The samples were then stained with fluorescent antibodies against the following surface molecules: CD81, CD63, CD9, CD40, CD44, CD49d, CD73, CD284 and CD29. After incubation, samples were analyzed by flow cytometry (Cytoflex cytometer) The percentage and mean expression of each molecule in EVs populations (EXOs and MVs) were determined. Statistical analysis was performed by t-Student test; significance established under 0.05 Main results and the role of chance Conventional semen analysis showed significant differences between control and patient group in sperm concentration (77.2 · 106 vs 40.2 · 106 cells / ml, respectively, p < 0.01), percentage of progressive sperm (60 vs 40.3 %, respectively, p < 0.01) and total progressive sperm cells (170 · 106 vs 69 · 106 spermatozoa, respectively, p < 0.01). Flow cytometry analysis demonstrated that the MVs population is larger than the EXOs population in both control and patient group. With respect to the surface molecule expression in EXOs population, patient group showed significant higher expression of CD9 (31.18 vs 20.1 % control group, p < 0.01), CD40 (8.94 vs 3.84 % control group, p < 0.001), CD49d (69.07 vs 46.73 %, control group, p < 0.001), CD73 (68.17 vs 51.41 % control group, p < 0.001) and CD284 (61.48 vs 53.45 % control group, p < 0.01), and significant lower expression of CD29 (19.49 vs 29.54 % control group, p < 0.01) molecules compared to control group. When MVs populations were analyzed, significant differences between patient and control group were found in the expression of CD63 (13.01 vs 1.65 %, respectively p < 0.001), CD40 (9.23 vs 1.34 %, respectively p < 0.001), CD282 (17 vs 10.22 %, respectively p < 0.01) and CD29 (17.11 vs 7.8 %, respectively p < 0.001) molecules Limitations, reasons for caution This study enrolled male partners of couples seeking infertility treatment with dissimilar disorders and seminal diagnosis (astheno, oligo, and teratozoospermia). Therefore, further studies are needed lowering the heterogeneity of the study group. No data about reproductive outcome of these patients are available at this moment Wider implications of the findings These results suggest that the differences found in the expression profiles between men with proven fertility and infertile men may be related to molecular mechanisms implicated in sperm function, and/or female immune system modulation and, for that reason, may be used as a new potential marker of male fertility Trial registration number 1904-MAD-045-AP

ANALYSIS OF THE QUALITY OF SEXED BULL SEMEN BY COMPUTER-ASSISTED SEMEN ANALYSIS-CASA

The quality of the ejaculate is determined by examination of the macroscopic (volume, density, color, smell) and microscopic properties of the sperm (concentration of spermatozoa in 1 ml and the total number of spermatozoa in the ejaculate, morphology, vitality, mobility of spermatozoa and integrity of the acrosomal membrane). Computer assisted semen analysis (CASA) is an automated system that measures sperm motility, kinetics and concentration. Some systems have modifications for evaluating the morphological characteristics of spermatozoa. The assessment of morphological characteristics of spermatozoa can be performed by microscopic examination (presence of normal and pathological spermatozoa, integrity of sperm cell membrane and acrosome abnormalities). The Blom method, as the method of the staining of the sperm smear with eosin and nigrosine, determines the vitality of the semen. In modern cattle breeding, sexed bull semen (semen from which desired gender of calf will be delivered) is used more and more. The analysis of sexed semen, and especially the comparison of its quality with conventional semen, is an increasingly common requirement on the cattle production market. The aim of the work was to examine the quality of 15 samples of sexed bull semen, originating from imports. The tests included determination of concentration and kinetics using the CASA system, as well as assessment of vitality and morphological characteristics by Blom staining. The average sperm concentration in 15 samples was 19.61x106/ml of ejaculate. The total motility was 39.42%, while the number of immobile spermatozoa in the samples was 60.58%. The number of morphologically normal spermatozoa varied from 2.91x106 to 6.11x106, and the percentage of pathological spermatozoa from 8% to 28%. Taking into account the results obtained and the absence of standards related to sexed semen in the currently valid Regulation on the manner of marking of sperm, the manner of keeping records on sperm production, as well as on the conditions that must be met by animal sperm in terms of quality (Official Gazette of the Republic of Serbia, 38/14), it is necessary to carry out more frequent analyzes of sexed semen using the CASA method in order to obtain values that would become an integral part of the Regulation and thereby facilitate the manipulation of sexed semen on the market.

Effect of Sexed and Conventional Semen on Pregnancy Rate in Murrah Buffaloes under Field Condition

The study was conducted on twenty Murrah buffaloes selected at random from the field to evaluate the effect of sexed and conventional semen on pregnancy rate. The buffaloes selected were initially treated with pre-synchronization medicines. The buffaloes in spontaneous estrus with vaginal discharge and frequent micturition were divided into two groups containing ten animals. The buffaloes from group I and II were inseminated at mid-estrus with sexed and conventional semen, respectively. The average pregnancy rate and female calves born for AI with sex-sorted semen were 40% and 100% and for AI with conventional semen 50% and 40%, respectively. The pregnancy rate was lower, whereas the rate of female calves born was greater after AI with sex-sorted semen than conventional semen.

The female to male calf sex ratio is associated with the number of services to achieve a calf and parity of lactating dairy cows.

Commercial dairy producers may get frustrated by the lower ratio of female to male calves born because female calves are more valuable than bull calves. Our objective was to determine if parity or stage of lactation at the time of breeding, using conventional semen, influenced the sex of the calf. Data from the University of Illinois and the University of New Hampshire dairy herds were collected and summarized for calf sex, the number of services to achieve a calf and the lactation number when conception of that calf occurred. Logistical regression procedures were used to analyze the dataset via version 9.4 of SAS. The final dataset contained 2,987 calvings, which consisted of 1,406 females and 1,581 males (47.1% and 52.9% for females and males, respectively). The frequency distribution of the number of services to achieve a calf was highest for the first service and progressively declined with increasing services (52.06%, 21.66%, 10.75%, 6.66%, 4.22%, and 4.65% for 1 to 6 services, respectively). The frequency distribution of calvings by lactation number was greatest for first lactation cows becoming pregnant with their second calf and declined with increasing parity (35.49%, 28.22%, 17.01%, 9.61%, 5.02%, 2.51%, 1.14%, 0.70%, and 0.30% for lactation numbers 1 to 9, respectively). Logistic stepwise regression indicated that the number of services to achieve a calf was significant in predicting the ratio of female to male calves. Calculation of odds ratios indicated that as the lactation number increased the likelihood of getting a bull calf decreased. Parity, services, and parity by services interaction were significant for cows having a greater number of parities and cows with a greater number of services yielding more heifer calves. However, an interaction occurred where cows with greater number of services along with greater parities more likely to have a bull calf. These data provide evidence that increasing the number of services to achieve a calf and increasing age of the cow increased the probability of a heifer calf being born. These data indicate that cows with greater parties (lesser cull rate) are more likely to produce heifer calves.

Produção de embriões in vitro com sêmen sexado de touros nelore

The objective was to analyze the effect of the type of semen, conventional and sexed, on the in vitro production of embryos. A total of 1,285 oocytes and 1,155 oocytes with sexed semen were matured and fertilized with conventional semen, obtaining 1,167 (90.55%) and 1,051 (90.68%) zygotes in fertilization with conventional and sexed semen, respectively (P=0.8411). In in vitro culture, embryo cleavage was evaluated in 769 (67.42%) of zygotes from conventional semen and 540 (55.12%) from zygotes from sexed semen (P=0.8411). There was no statistical difference in both analyzed variables. On day 7, the number of viable embryos produced was evaluated, being 428 (53.55%) conventional embryos and 138 (30.64%) viable embryos from sexed semen (P=0.0001). There was a significant difference, therefore, it obtained better embryonic conversion with conventional semen in relation to sexed semen of Nelore bulls.

Fertility Response of the Sex Sorted and Conventional Semen in Cattle

Fertility Response of the Sex Sorted and Conventional Semen in Cattle

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects.

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility. With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 °C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The role of impaired acrosomal exocytosis (IAE) in stallion subfertility: A retrospective analysis of the clinical condition, and an update on its diagnosis by high throughput technologies

Acrosomal dysfunction has been considered as a cause of subfertility in males of different species, including stallions. A subset of subfertile stallions with acrosomal dysfunction is unique because they have normal sperm quality (motility, morphology, viability, and DNA quality). The current work aims to describe the clinical characteristics of subfertile stallions that were diagnosed with Impaired Acrosomal Exocytosis (IAE) by using two high throughput methods: flow cytometry and molecular genetic analysis, and to identify the prevalence of subfertility due to IAE in stallions evaluated at Texas A&M University. Clinical data from 1,128 stallions evaluated during 17 years at a Veterinary Teaching Hospital was retrospectively analyzed. Only stallions with a history of subfertility not explained following a breeding soundness examination and/or conventional semen analysis, were included. For those stallions, the acrosomal exocytosis test (AE test), in which sperm is incubated at 37 °C for up to 2 h in the presence of the calcium ionophore A23187, was used to determine IAE. The difference in AE-Rate (AE-Diff) between each pair of fertile control stallion and subfertile stallion was categorized as: Normal: AE-Diff < 14%; Questionable: AE-Diff 15–29%; Abnormal: AE-Diff > 30%. In selected cases, blood or hair was procured for identification of the susceptibility genotype for IAE, A/A-A/A, in the FKBP6 gene, exon 5. Twenty-one (21) stallions (1.86% total population analyzed) had reduced fertility despite having acceptable sperm quality. Sperm from these stallions were subjected to the AE Test. Of these, 8 stallions had reduced sperm AE-rate, based on the AE Test (8/21; 38.1%). Subsequently, blood or hair samples from these 8 stallions which had either questionable (AE-Diff 15 – 29%; n = 5) or abnormal (AE-Diff > 30%; n = 3) responses to the AE Test were analyzed for the susceptibility genotype for IAE, A/A-A/A (FKBP6 gene, exon 5). Seven out of the eight (7/8) stallions carried this susceptibility genotype. All of these were Thoroughbreds. After 2 h of incubation, the viability in fertile stallion sperm was lower than in A/A-A/A stallions (4% vs. 25%, respectively; P < 0.05), while the AE-rate was higher for fertile than for A/A-A/A stallions (85% vs. 56%, respectively; P < 0.05). The use of two high throughput tests (i.e., flow cytometry and molecular genetic analysis) may complement each other in the diagnosis of IAE in breeding stallions. In this study, 5/7 subfertile stallions diagnosed with the IAE susceptibility genotype would have been diagnosed as normal with the AE Test. This study introduces a subset of stallions with the IAE genotype with fertility higher than has been previously reported (i.e., <15% per-cycle pregnancy rate), suggesting that IAE manifests as a broader range of subfertility.

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

BackgroundThe 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.ResultsThe 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 (β = − 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).ConclusionConsidering all these findings, this work sets a useful model to study how SDF negatively influences fertility.

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

Long-chain omega-3 fatty acids and their food sources have garnered interest as a potential nutrient with wide-range health benefits, including fertility. 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. 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. 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. 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.

Fertility analysis of bovine semen by in vitro fertilization.

The aim of the present study was to evaluate the efficiency of using in vitro fertilization to validate semen fertility for artificial insemination. Cryopreserved semen from ten bulls (five Nelore and five Brangus bulls) was evaluated using in vitro production of embryos (IVPE) and via fixed-time artificial insemination (FTAI). There was variation (p < 0.05) in the IVPE (20.9 to 53.7% of blastocyst production) and in the FTAI (42.0 to 56.0% of pregnant cows) results among the semen evaluated. According to the results, there was a positive correlation (rs = 0.8378; p = 0.0001) between the rate of blastocyst production (using IVPE) and the rate of pregnancy (using FTAI) using Nelore bull semen. Variation (p < 0.05) was also found using semen from Brangus bulls, in the rates of blastocyst production (36.5 to 47.0%) and pregnancy (45.6 to 52.2%) via FTAI. There was also a positive correlation (rs = 0.8786; p = 0.0001) between the rates of blastocyst production (IVPE) and pregnancy (FTAI) when using Brangus bull semen. According to the results, IVPE may be used in addition to conventional semen analysis to evaluate and validate the semen fertility of bulls for artificial insemination programs.

PRM1 Gene Expression and Its Protein Abundance in Frozen-Thawed Spermatozoa as Potential Fertility Markers in Breeding Bulls.

Functional genes and proteins in sperm play an essential role in bulls’ reproductive processes. They are more accurate in determining bull fertility than conventional semen quality tests. Protamine-1 (PRM1) is a gene or protein crucial for packaging and protecting sperm DNA until fertilization affects normal sperm function. This study analyzes the genes and proteins potential from PRM1 as fertility markers for different breeds of bulls utilized in the artificial insemination programs, expected to be an accurate tool in interpreting bull fertility in Indonesia. This study used Limousin, Holstein, and Ongole Grade bulls divided into two groups based on fertility, high-fertility (HF) and low fertility (LF). The semen quality assessment included progressive motility (computer-assisted semen analysis), viability (eosin-nigrosine), and plasma membrane integrity (HOS test). Sperm DNA fragmentation (SDF) was assessed using the acridine orange staining and the Halomax test. Sperm PRM deficiency was evaluated with the chromomycin A3 method. Moreover, PRM1 gene expression was measured using qRT-PCR, and the PRM1 protein abundance was measured with the enzyme immunoassay method. Semen quality values, relative expression of PRM1 gene, and quantity of PRM1 protein were significantly higher (p < 0.05) in HF bulls than in LF bulls. The SDF and PRM deficiency values in LF bulls were significantly higher (p < 0.05) than HF bulls. Additionally, PRM1 at the gene and protein levels correlated significantly (p < 0.01) with fertility. Therefore, PRM1 is a potential candidate for fertility markers in bulls in Indonesia.

Correlations between abnormalities of morphological details and DNA fragmentation in human sperm.

ObjectiveAs the associations of sperm DNA fragmentation with morphology have not been examined in detail, this study aimed to investigate the relationship between abnormalities of morphological details and DNA integrity in human sperm.MethodsIn this cross-sectional study, men from infertile couples were enrolled at Hue Center for Reproductive Endocrinology and Infertility, Vietnam. Conventional semen parameters, including morphological details, were analyzed following the World Health Organization 2010 criteria. Sperm DNA fragmentation was evaluated using a sperm chromatin dispersion assay. The relationships and correlations between semen parameters, sperm morphology, and the type of halosperm and the DNA fragmentation index (DFI) were analyzed.ResultsAmong 130 men in infertile couples, statistically significant differences were not found in the sperm halo type between the normal and abnormal sperm morphology groups. The percentage of round-head spermatozoa was higher in the DFI >15% group (16.98%±12.50%) than in the DFI ≤15% group (13.13%±8.82%), higher values for amorphous heads were found in the DFI >15% group, and lower values for tapered heads were observed in the DFI ≤15% group; however, these differences were not statistically significant. Small-halo sperm and the DFI were positively correlated with round-head sperm (r=0.243, p=0.005 and r=0.197, p=0.025, respectively). ConclusionThe rate of general sperm morphological abnormalities in semen analysis was not related to sperm DNA integrity. However, round sperm heads were closely associated with sperm DNA fragmentation.

Economics of timed artificial insemination with unsorted or sexed semen in a high-producing, pasture-based dairy production system

This study used a stochastic simulation model to estimate the potential economic benefit of using timed artificial insemination (TAI) in combination with conventional unsorted (TCONV) and sexed (TSEX) semen in heifers only (TCONV-H, TSEX-H) and in both heifers and lactating cows (TCONV-HC, TSEX-HC) in a high-producing, pasture-based production system. The scenarios were compared with a conventional reproductive policy (CONV) in which heifers and cows were inseminated with conventional unsorted semen after estrus detection. Sensitivity analysis was also used to estimate the effect of hormone costs from TAI use on the profitability of each program relative to CONV. The mean annual (± standard deviation) profit advantage (ΔPROF) over CONV for TCONV-H, TCONV-HC, TSEX-H, and TSEX-HC scenarios were €3.90/cow ± 4.65, €34.11/cow ± 25.69, €13.96/cow ± 6.83, and €41.52/cow ± 42.86, respectively. Combined application of both technologies was shown to return a greater annual ΔPROF on average compared with that achievable from TAI alone. However, the risk of not returning a positive annual ΔPROF varied across the scenarios with higher risk in TCONV-H and TSEX-HC. Specifically, TCONV-H and TSEX-HC had a 24 and 18% chance, respectively, of not returning a positive annual ΔPROF. Sensitivity analysis showed that when hormone costs increased by €10/cow TCONV-H and TSEX-HC had a 38 and 23% chance, respectively, of not returning a positive annual ΔPROF. The range in ΔPROF for TCONV policies was most sensitive to the TAI pregnancy rate and TSEX policies were most sensitive to the relative fertility achieved with sexed compared with unsorted semen. This study has shown TAI and sexed semen are complementary technologies that can increase genetic gain and profitability in a pasture-based, dairy production system.