Ejaculated Spermatozoa Research Articles

Superior Live Birth Rates, Reducing Sperm DNA Fragmentation (SDF), and Lowering Miscarriage Rates by Using Testicular Sperm Versus Ejaculates in Intracytoplasmic Sperm Injection (ICSI) Cycles from Couples with High SDF: A Systematic Review and Meta-Analysis

This study aimed to compare sperm DNA fragmentation (SDF) levels between ejaculate and testicular sperm and evaluate clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles using testicular sperm (T-ICSI) versus ejaculate sperm (E-ICSI) in males with high ejaculate SDF, prior ICSI failures, or severe male infertility. A systematic review of major databases and a subsequent meta-analysis were performed to compare clinical outcomes in men with high SDF, oligozoospermia, or prior ICSI failures undergoing T-ICSI or E-ICSI. Thirteen studies met the inclusion criteria. Outcomes analyzed included SDF levels, fertilization rate (FR), clinical pregnancy rate (CPR), live birth rate (LBR) per embryo transfer (ET), and miscarriage rate (MR) per pregnancy. The mean difference (MD) and odds ratio (OR) were calculated for each outcome. Paired assessments of SDF showed significantly lower levels in testicular sperm compared to ejaculated sperm (MD = −25.42 [−31.47, −17.30], p < 0.00001). While no significant difference in FR was observed in T-ICSI cycles overall (OR = 0.94 [0.74, 1.20]), a subgroup analysis revealed significantly higher FR with E-ICSI in men with oligozoospermia and no prior ICSI failures (OR = 0.61 [0.52, 0.71], p < 0.00001). CPR was significantly higher in T-ICSI cycles (OR = 2.13 [1.35, 3.36], p < 0.001; n = 540 ET), along with a significantly lower MR (OR = 0.31 [0.14, 0.70], p = 0.004; n = 35) and increased LBR (OR = 2.40 [1.32, 4.36], p = 0.004; n = 446 ET). In conclusion, using testicular sperm in cases of elevated ejaculate SDF, oligozoospermia, or prior failed ICSI cycles enhances the selection of sperm with lower DNA damage, leading to improved pregnancy rates, reduced miscarriage rates, and higher live birth rates. However, the studies included were rated as having a moderate to serious risk of bias. Further well-designed randomized controlled trials are necessary to confirm these findings with stronger evidence.

Gonadotropin-releasing hormone II and its receptor regulate motility, morphology, and kinematics of porcine spermatozoa in vitro.

The second form of gonadotropin-releasing hormone (GnRH-II) and its receptor (GnRHR-II) are abundantly produced within the porcine testis and immunolocalize within the seminiferous tubules, suggesting a role in spermatogenesis and/or sperm function. The objective of this study was to quantify GnRH-II and GnRHR-II abundance within boar reproductive tract tissues and examine their role in porcine sperm function. Immunoblotting revealed GnRHR-II abundance was 12-fold greater (P < 0.0001) within the testis compared with other reproductive organs. Within seminiferous tubules, GnRHR-II prominently immunolocalized to elongating spermatids. In ejaculated spermatozoa, GnRHR-II immunolocalized to the connecting piece. GnRH-II was also detected in seminal plasma, likely originating from the testis as GnRH-II concentrations were greatest in testicular homogenates (P < 0.0001) compared with other reproductive tissues. To assess the effects of GnRH-II/GnRHR-II on sperm function, extended semen samples were treated with GnRHR-II analogues and evaluated via computer-assisted sperm analysis (CASA). In Experiment 1, semen treatment with increasing concentrations of GnRHR-II agonist (D-ala6 GnRH-II) revealed that two concentrations (0.1 and 100µM) tended to decrease the percentage of bent sperm tails versus vehicle-treated semen (P < 0.10). In Experiment 2, semen treatment with increasing concentrations of GnRHR antagonist (SB-75/Cetrorelix) indicated that only 10µM SB-75 impaired CASA metrics compared with vehicle-treated samples (P < 0.05). In Experiment 3, semen treatment with both 100µM D-ala6 GnRH-II and 10µM SB-75 partially rescued sperm motility and morphology measures. These data suggest that GnRH-II and its receptor regulate porcine sperm function in an autocrine/paracrine manner.

Post-testicular spermatozoa of a marine teleost can conduct de novo cytoplasmic and mitochondrial translation.

Translational silence of spermatozoa has long been considered the norm in animals. However, studies in mammals have shown that the mitochondrial ribosomal machinery is selectively activated during capacitation in the female reproductive tract, while cytosolic ribosomes remain inactive. Here, using quantitative proteomics in a piscine model species, we show that proteins involved in mRNA processing and cytoplasmic translation are predominantly accumulated in immature spermatozoa within the extratesticular excurrent ducts, while those related to flagellar motility are enriched in ejaculated (mature) sperm. Based upon invitro incubation of isolated spermatozoa, motility assays and polysome profiling, we further show that 80S cytoplasmic and 55S mitochondrial ribosomes are actively involved in the translation of motility- and osmoadaptation-related proteins. These findings thus reveal that post-testicular piscine spermatozoa can maintain de novo protein synthesis through both mitochondrial and cytoplasmic ribosomal activity, which is necessary for the acquisition of full sperm function.

Should testicular sperm retrieval be implemented for intracytoplasmic sperm injection in all patients with severe oligozoospermia or cryptozoospermia?

The choice between utilizing testicular or ejaculatory sperm for intracytoplasmic sperm injection (ICSI) in men with severe oligozoospermia or cryptozoospermia is a crucial aspect of managing male infertility. This study aimed to identify a predictive factor that could guide this decision-making process. Seventy-five infertile men with severe oligozoospermia or cryptozoospermia were included in the analysis. On the day of ovum pick-up, these participants were divided into three groups (n=25 each) based on their sperm concentrations: cryptozoospermia, 0.1-1 million/mL and 1-5 million/mL. Patients in each group underwent ICSI, which involved the insemination of sibling oocytes using either ejaculated spermatozoa or testicular spermatozoa obtained via fine-needle aspiration. We evaluated the rates of fertilization, cleavage, high-quality embryo production, and blastocyst formation. In patients with sperm concentrations below 1 million/mL, testicular sperm demonstrated higher rates of fertilization (p<0.001), cleavage (p=0.01), high-quality embryo formation (p=0.003), and blastocyst development (p=0.04) compared to ejaculated sperm. In cases of cryptozoospermia, testicular sperm was associated with a higher fertilization rate (p<0.001) and a marginally higher rate of high-quality embryos (p=0.06). Conversely, in patients with sperm concentrations exceeding 1 million/mL, ejaculated sperm yielded superior outcomes. This study underscores the significance of considering sperm concentration when advising on sperm retrieval techniques to improve ICSI outcomes in men diagnosed with severe oligozoospermia or cryptozoospermia. Further research is necessary to confirm predictive factors that assist in decision-making regarding the source of sperm, whether from ejaculate, testicular aspiration, or biopsies.

Evaluation of specific fractions of Large White Yorkshire boar semen

The current study was conducted to evaluate the quality of specific fractions of three Large White Yorkshire (LWY) boar semen samples. Fifteen semen samples were collected by gloved hand method as an initial 10 mL of sperm rich fraction (SRF- F1), remaining SRF (F2) and whole semen. Samples were evaluated for colour, volume, pH, sperm concentration and progressive motility. The first 10 mL of SRF of boar semen contained 25 per cent of the total ejaculate spermatozoa and was able to withstand cooling, freezing and thawing better than the spermatozoa of the bulk ejaculate. A significant difference between the fractions was observed in the volume of semen ejaculated, pH and sperm concentration across different fractions of the same ejaculate. The differences in semen fractions were attributed to the differences in the composition of seminal plasma of the initial fraction of SRF Keywords: Boar semen, sperm rich fraction, semen evaluation

SEMEN CHARACTERISTICS AND SCROTAL BIOMETRY OF WEST AFRICAN DWARF RAMS FED AMMONIUM SULPHATE FORTIFIED DIET

Ammonium sulphate (AS) is non-protein nitrogen sources widely used as a feed additive for ruminants to provide sulphur (S) to the diet. This study assessed the semen characteristics and scrotal biometry of West African dwarf rams fed AS fortified diets at 0. 2.5, 5.0 and 7.5g/kg to the concentrate diet as T1, T2, T3 and T4 respectively. Sixteen WAD rams with average body weight of 12.20+0.12 kg were used and fed for 105 days. Each treatment had four replicates while semen was collected once from all replicates in the treatments. There was significant difference in the sperm concentration (x 109/mL) with T4 (160.06) having the highest while T1 (136.00) had the lowest. The total sperm ejaculate followed the same trend as sperm concentration with the lowest value recorded for rams fed T1 (98.24) while those on T4 (150.66) had the highest value. There were significant differences in the values recorded for the scrotal circumference and length. however, scrotal width, testicular weight, scrotal length and scrotal circumference values in T4 was numerically higher than other treatments. It can be concluded that AS fortified diets at 7.5g/kg improved the semen quality positively.

Cryopreservation of rare ejaculate and testicular spermatozoa, a novel simple technique; the culture dish slice freezing: A pilot study.

Cryopreservation of rare ejaculate and testicular spermatozoa, a novel simple technique; the culture dish slice freezing: A pilot study.

In Vitro Gene Conservation Status and the Quality of the Genetic Resources of Native Hungarian Sheep Breeds.

Studies revealed a global loss of genetic resources for local sheep breeds. Therefore, the current study aimed to introduce and highlight the progress made on Hungary's existing gene conservation program (small Gene Bank). Furthermore, we evaluated breed (Tsigai, Cikta, and Racka), season, and individual variabilities (n = 24) of the pre-freeze and post-thaw semen stored in the Gene Bank to enhance the gene conservation of the breeds. The samples were cryopreserved manually, and post-thaw spermatozoa were analyzed for motility (CASA), viability, chromatin structure, and morphometry of the sperm nuclei. Ejaculate volume, spermatozoa concentration, subjective motility and standard motility, kinematic parameters, and spermatozoa's head area standard deviation of the post-thaw samples differed significantly among breeds (p < 0.05). Season affected ejaculate volume, total spermatozoa number/ejaculate, STR, BCF, and ALH. We observed a significant (p < 0.001; 0.05) breed and season interaction on concentration, total spermatozoa number/ejaculate, VCL, LIN, WOB, spermatozoa's head average perimeter and nucleus length (Tsigai and Cikta differed but were statistically the same as Racka). Similarly, season significantly (p < 0.05) affected the proportion of ejaculate suitable for freezing. There was a significant (p < 0.05) difference in kinematic parameters and viability among the rams across the breeds. The spermatozoa's head morphometry of the Tsigai and Cikta breeds differed significantly (p < 0.05) among the rams. There were individual and breed differences in many spermatozoa quality parameters. The stored samples are of good quality, with more than 40% having intact membranes and low abnormal chromatin condensation.

P-271 Deep sequencing of spermatozoal accessible chromatin reveals complex arrangements underlying spermatozoal chromatin packaging and fertilization

Abstract Study question Could structural aspects of sperm DNA packaging contribute to fertilization and early embryo development? Summary answer Structural aspects of sperm DNA include complex patterns of repetitive-DNA and developmental gene packaging that could delineate a paternal impact on fertilization and early development. What is known already Genetics, epigenetics and transcriptomics of ejaculate human spermatozoa could have important effects on the establishment of embryonic chromatin architecture that may impact early fetal development. Spermatozoa are transcriptionally silent; however, a small fraction (∼5%-15%) of their chromatin escapes repackaging by protamines and remains in a more open nucleosomal conformation that may be poised for transcription. The sensitivity of this fraction to progressive salt/detergent disruption is likely assayed in sperm chromatin dispersion (SCD) or halo tests that are commonly used in assessments of sperm DNA fragmentation. Study design, size, duration Eleven healthy male donors and 9 women undergoing IVF treatment agreed to participate in this study. Eleven normozoospermic semen samples were donated by male donors, aged 25-39 years, who abstained from sexual activity for 3 days. Seven MII oocytes and 5 pronuclear zygotes were donated from 5 and 4 women, respectively, undergoing IVF treatment. The samples were collected within a time frame of 6 months. Female donors provided either MII oocytes or pronuclear zygotes. Participants/materials, setting, methods Sperm chromatin was processed by differential salt extraction tailored with endonuclease or micrococcal nuclease treatment and RNA isolated using established protocols. Oocytes and pronuclear zygotes were analyzed at the single cell level. DNA library construction employed the NEBNext Ultra approach (NEB) and RNA libraries the Ovation Single Cell (NuGEN) for Illumina. Bioinformatics analysis included FastQC, HISAT2, StringTie, GREAT, edgeR and other source-software. Public datasets were employed to further increase the statistical power. Main results and the role of chance The aim of our approach was to dissect the accessible sperm chromatin fraction, identifying regions of great importance that could contribute towards the formation of the zygote and/or early embryonic development. Differing extraction methods revealed complementary accessible and resistant fractions in sperm, with accessible regions being significantly enriched for chromatin important in very early embryonic development. These chromatin fractions contained various genomic features including promoter sequences, CpG-islands and CTCF-binding sites that could attract transcription binding factors in the paternally-derived chromatin. Accessible sperm chromatin also contains high levels of specific DNA repeat classes of retroviral origin, known to impact early gene expression and gene expression programs. Accessible sperm chromatin was strongly associated with developmental and regulatory signatures with Bonferroni P values ranging between 2.07x10-4 and 1.03x10-20, potentially revealing a role for paternal chromatin in the initial start-up of early zygotic development. We further investigated paternally- and maternally- derived transcripts and identified mono-allelically transcribed genes present in the zygotes, that were entirely absent from all seven oocyte transcriptomes, suggesting a paternal origin, and potentially important roles following oocyte fertilization. Limitations, reasons for caution Due to the restraints of obtaining human material, the study relied on a relatively small sample size. However, sequencing results showed quite homogeneous clustering in both DNA/RNA samples, with at least 5 biological replicates from at least 4 donors per group, potentially reducing the impact of low sample size. Wider implications of the findings A better understanding of differentially accessible sperm chromatin and residual RNA, could help support the discovery of biomarkers that can be used to select more competent spermatozoa for IVF cycles, resulting in higher success rates in couples undergoing ART. Trial registration number not applicable

P-077 The role of glycosylation in the male reproduction revealed by omics technologies

Abstract Study question What is the current status of art and future perspective of glycosylation in the male reproduction using omics-based technology? Summary answer The precise site-specific glycoproteome of fertile human semen was generally established while the alteration in the pathogenic processes needed to be further investigated. What is known already The entire lifespan of human sperm concomitant with the glycosylation, which have been considered to be profoundly implicated in the male reproduction. In the past, only a few glycan patterns or glycoproteins in the male reproductive system were characterized due to technical limitations. Traditional strategies based on either glycomics or deglycosylated proteomics also are far from sufficient for fully understanding glycosylation in male reproduction. The development of the precise site-specific glycosylation of human semen was profiled, which laid a foundation on subsequently unveiling its role, while many challenging questions remain and require the assistance of advanced glycoproteomics. Study design, size, duration The original research and reviews in the PubMed and Web of Science databases were searched using terms regarding glycoproteome and male reproduction until September 2023. Participants/materials, setting, methods PubMed and Web of Science were used to search articles and reviews with the following main keywords pertaining to the glycoproteomics: “glycosylation”, “glycan”, “glycoprotein”, “glycome”, “glycoproteome”, “glycomics” and “glycoproteomics”, combined with “semen”, “spermatozoa”, “seminal plasma”, “epididymis”, “fertilization” and other key terms related to male reproduction. The search period included all publications until now (September 2023). The language was limited to English, and the species in most studies was limited to humans. Main results and the role of chance The precise site-specific glycoproteome of fertile human ejaculate spermatozoa and seminal plasma was generally established. Both are highly abundant in glycans and glycoproteins and share heavy fucosylation as the most significant feature. Their functions are mainly involved in immunomodulation, capacitation, and sperm-oocyte binding. Spatiotemporal regulation was found in glycosylation during male reproduction. Intracellular glycan synthesis occurs in testicular sperm, and a variety of glycan-modifying enzymes and extracellular vesicles are responsible for sperm maturation. Alterations in the semen glycoproteome were observed in male reproduction disorders and are associated with oligoasthenozoospermia, decreased sperm function, male accessory gland disease and immune dysregulation. Impaired glycosylation may be the result of genetic, endocrine, and oxidative stress and infection. Some idiopathic male infertility may be attributed to glycosylation abnormalities. Hence, glycoproteins have the potential to be biomarkers in male reproductive diseases and assisted reproductive technology. We point out that the mapping of the normal semen N-glycoproteome was the first step and there are many challenges such as the identification of the O-linked glycoproteome. Advanced glycoproteomic techniques developed recently have the potential to address them and promote the glycoproteome research in male reproduction. Limitations, reasons for caution There are no more than 20 articles regarding the glycomics or glycoproteomics of the semen and the samples processing is not ideal. Wider implications of the findings Our work hope to enlighten the biologists to depict the overall landscape of the male reproductive glycoproteome including spermatogenesis and maturation. Moreover, we provide adequate information for reproductive clinicians and technicians for the clinical translation of glyco-related biomarkers and therapeutic targets. Trial registration number not applicable

P-028 Pregnancy and perinatal outcomes in male patients with temporary ejaculation failure undergoing assisted reproductive technology

Abstract Study question When temporary ejaculation failure occurs on the oocyte retrieval day, choose surgical sperm retrieval or oocyte cryopreservation? Summary answer TESA-ICSI and oocyte cryopreservation are both relatively efficient and safe way. Embryos have better developmental potential through TESA-ICSI. What is known already Nearly 9% of men among couples who use assisted reproductive technology have temporary ejaculation failure (TEF) on the oocyte retrieval day. If they fail to provide semen sample, the couples may face three choices (1. surgical sperm retrieval; 2. oocyte cryopreservation; 3. canceling the oocyte retrieval). The aim of the study was to compare the impact of testicular sperm aspiration (TESA) or oocyte cryopreservation on the clinical outcomes of intracytoplasmic sperm injection (ICSI) patients with TEF. Study design, size, duration The retrospective study included 345 male patients with TEF in our University-affiliated IVF center from 2015 to 2021. Participants/materials, setting, methods Three hundred and forty-five male patients were divided into two groups. Two hundred patients chose TESA-ICSI (the TESA group), while another 145 patients chose oocyte cryopreservation, waiting for the man’s next successful sperm ejaculation and subsequent ICSI (the frozen oocyte group). We investigated the embryo development, pregnancy and perinatal outcome, and included the data from fresh embryo transfer (ET) cycle. Herein, all patients in the frozen oocyte group received hormone replacement and fresh ET. Main results and the role of chance Firstly, there were no significant difference in the female BMI, FSH, antral follicle count (AFC), AMH, and male semen quality between the two groups. For embryo development, both two groups have the similar normal fertilization rate (71.5% vs 69.7%, P = 0.242). However, the frozen oocyte group has lower rate of Day3 high quality embryo (31.1% vs 49.3%, P &amp;lt; 0.001), available embryo rate (49.8% vs 55.8%, P = 0.043), and available blastocyst rate (18.6% vs 33.7%, P &amp;lt; 0.001). For pregnancy outcome, the clinical pregnancy rate (44.2% vs 50.0%), pregnancy loss rate (4.8% vs 2.9%) and live birth rate (38.5% vs 46.1%) were similar. And there were no significant differences in the rates of Gestational diabetes mellitus, hypertensive disorders of pregnancy, placenta previa and fetal malformation between the two groups. Limitations, reasons for caution This study only included data from fresh embryo cycles, not yet from thawed embryo cycles. Although we presented complete embryo development data for each couple, there may be some bias in pregnancy and perinatal outcomes due to the lack of data from thawed embryo cycles. Wider implications of the findings The present study suggested that TESA-ICSI and oocyte cryopreservation are both relatively efficient and safe way to help the patients encountering TEF. Due to the embryos with better developmental potential through TESA-ICSI, choosing the surgical sperm retrieval might have more have more advantages, and this has to be further verified. Trial registration number REC No. (2023(S142))

Concentration of fatty acid binding protein as a new indicator of ejaculate fertility

Objective. To determine the marker function of fatty acid binding protein (FABP) concentration in seminal plasma (SP) in order to assess ejaculate fertility used in in vitro fertilization (IVF) treatment of infertility in couples. Materials and methods. The study involved semen samples of 96 men of reproductive age: the study group (n=63) – patients with a decrease in concentration and/or total content of spermatozoa in ejaculate, the comparison group (n=33) – men with normal concentration and number of spermatozoa in ejaculate. The content of FABP in SP was determined by enzyme-linked immunosorbent assay using the test system “FABP – ELISA – BEST” (A-9102, Vector-Best, Russia). In order to determine the informative value of using the concentration of FABP in SP as a criterion of ejaculate fertility of men of the study group, the predictive value of positive and negative IVF results was determined by calculating the diagnostic sensitivity, specificity and efficiency. Results. The content of FABP in SP accounted for 1.29 ± 0.24 ng/mL, the median and interquartile range comprised 1.23 [1.13–1.35] ng/mL, ranging from 1.08 to 2.79 ng/mL. The study revealed statistically significant intergroup differences (Mann-Whitney test U=79.00; p=0.000016), a weak correlation between the level of FABP and the concentration of spermatozoa (R=0.578008) and their number in ejaculate (R=0.583599). The diagnostic sensitivity of the test for FABP in SP of the men of the study group accounted for 81.82%, specificity – 78.95%, efficiency – 80.95%. Conclusion. Seminal plasma FABP can act as a marker of spermatogenesis disorders. The study of this protein in ejaculate provides the accuracy of predicting the outcome of in vitro fertilization.

Assessment of reproductive outcomes of fresh versus cryopreserved ejaculated sperm samples-a retrospective analysis of 44423 oocyte donation ICSI cycles.

Does the use of frozen sperm affect live birth rate (LBR) and cumulative LBR (CLBR) compared to fresh sperm samples in oocyte donation ICSI cycles? Although there were slight decreases in pregnancy rates (PRs) and LBR, as well as CLBR per embryo replaced and per embryo transfer (ET), when frozen sperm samples were used compared to fresh ejaculates, their clinical impact was limited. Sperm cryopreservation is part of the daily routine in reproduction clinics worldwide because of its many advantages in cycle planning. Nonetheless, there is a lack of agreement in terms of its impact on the outcomes of ICSI cycles. Previous studies showed conflicting conclusions and focused on different populations, which makes reaching consensus on the impact of sperm freezing-thawing complicated. Moreover, classical parameters are used to assess cycle success: pregnancy, live birth and miscarriage rates per ET. This study reports those measurements plus CLBR, which more accurately reflects the impact of the technique on the likelihood of achieving a newborn. A retrospective multicenter observational cohort study, including data from 37041 couples and 44423 ICSI procedures from January 2008 to June 2022, was carried out. The group using frozen sperm included 23852 transferred embryos and 108661 inseminated oocytes, whereas the fresh sample group comprised 73953 embryos replaced and 381509 injected oocytes. Outcomes measured per first ET and per ET were compared between groups using Fisher's exact test and Chi-squared test, as appropriate. Binary-logistics regression models were used to adjust the analyses according to clinically relevant co-variables. Kaplan-Meier curves plotted the CLBR per oocyte inseminated, per embryo replaced and per ET, and compared between groups using the Mantel-Cox test. Cox regressions were employed for the multivariate analyses of CLBR. The frozen sperm group showed a slightly lower biochemical (3.55% and 2.56%), clinical (3.68% and 3.54%) and ongoing (3.63% and 3.15%) PR compared to the cycles using fresh sperm, respectively, both per first ET and per ET. LBR was 4.57% lower per first ET and 3.95% lower per ET in the frozen sperm group than the fresh sperm group. There was also a subtle increase of 2.66% in biochemical miscarriage rate per ET when using frozen versus fresh sperm. All these differences remained statistically significant after the multivariate analysis (adjusted P ≤ 0.001). There were statistically significant differences in CLBR per embryo replaced and per ET but not per oocyte used (adjusted P = 0.071). Despite the statistical significance of the differences between the groups, those using frozen sperm required only 0.54 more oocytes injected, 0.45 more embryos transferred and 0.41 more ET procedures, on average, to achieve a live birth compared to the fresh samples. The retrospective nature of the study subjects the data to biases or potential errors during annotation on the source clinical and cycle records. This study uses multivariate analyses to control biases as much as possible. Using the oocyte donation model also contributes to reducing heterogeneity in the oocyte quality factor. The large sample sizes included in this study allowed for the detection of small changes in cycle success rates between groups. Although statistically significant, the decrease in PRs, LBR, and CLBR when using frozen sperm can be clinically overlooked in favor of the many benefits of sperm cryopreservation. None declared. Not applicable.

What is the maximal timeframe between sperm acquisition to sperm cryopreservation, in different "culture" conditions?

Most of the literature about postmortem sperm retrieval (PMSR) deals with the controversies surrounding ethical and legal aspects, while the optimal time interval between the death and viable sperm acquisition is indefinite. In an attempt to aid fertility specialists, while counseling whether to pursue and adopt PMSR, we aim to explore the maximal time frame from ejaculated sperm acquisition to sperm cryopreservation in different "culture" conditions, observations that might be extrapolated to PMSR requests. Five healthy men with normal semen analysis were enrolled. The sperm specimen from each man was diluted to 6.5 mL. After extracting 0.5 mL for cryopreservation, the remaining 6 mL were divided into three tubes: one was maintained in room temperature (23-25 °C), the second in an incubator (37 °C), and the third in a refrigerator (4 °C). Thereafter, every day, a 0.5 mL of each sample was extracted, examined, and cryopreserved. A week later, all the cryopreserved samples were thawed and tested for sperm motility and viability. While at room temperature, frozen/thawed sperm were still motile (6.5%) and viable (9.9%) up to 96 h; those maintained in the refrigerator, following freezing/thawing were immotile already at 48 h in culture, but still viable (6.0%) up to 72 h in culture. Those maintained in the incubator demonstrated the worse results with negligible motility (1.5%) and viability (3.7%) following freezing/thawing, already after 48 h in culture. The timeframe cut-off between ejaculated sperm acquisition and cryopreservation should be 72 h, unless sperm was maintained at room temperature, where it might be longer. It would be prudent to check for sperm vitality prior to freezing in cases where only immotile sperms are present.

Global Practice Patterns in the Evaluation of Non-Obstructive Azoospermia: Results of a World-Wide Survey and Expert Recommendations.

Non-obstructive azoospermia (NOA) represents the persistent absence of sperm in ejaculate without obstruction, stemming from diverse disease processes. This survey explores global practices in NOA diagnosis, comparing them with guidelines and offering expert recommendations. A 56-item questionnaire survey on NOA diagnosis and management was conducted globally from July to September 2022. This paper focuses on part 1, evaluating NOA diagnosis. Data from 367 participants across 49 countries were analyzed descriptively, with a Delphi process used for expert recommendations. Of 336 eligible responses, most participants were experienced attending physicians (70.93%). To diagnose azoospermia definitively, 81.7% requested two semen samples. Commonly ordered hormone tests included serum follicle-stimulating hormone (FSH) (97.0%), total testosterone (92.9%), and luteinizing hormone (86.9%). Genetic testing was requested by 66.6%, with karyotype analysis (86.2%) and Y chromosome microdeletions (88.3%) prevalent. Diagnostic testicular biopsy, distinguishing obstructive azoospermia (OA) from NOA, was not performed by 45.1%, while 34.6% did it selectively. Differentiation relied on physical examination (76.1%), serum hormone profiles (69.6%), and semen tests (68.1%). Expectations of finding sperm surgically were higher in men with normal FSH, larger testes, and a history of sperm in ejaculate. This expert survey, encompassing 367 participants from 49 countries, unveils congruence with recommended guidelines in NOA diagnosis. However, noteworthy disparities in practices suggest a need for evidence-based, international consensus guidelines to standardize NOA evaluation, addressing existing gaps in professional recommendations.

ICSI using testicular spermatozoa after failure of ICSI with ejaculated spermatozoa could be a good choice: A propensity score-matched cohort study.

Ejaculated spermatozoa are considered to possess a higher fertilisation potential than testicular spermatozoa. In selected cases, the use of testicular spermatozoa from non-azoospermic infertile men resulted in a higher implantation and pregnancy rate than the use of ejaculated spermatozoa. The primary objective was to compare the live birth rate and cumulative live birth rate between couples with failed intracytoplasmic sperm injection procedure using ejaculated spermatozoa who subsequently had an intracytoplasmic sperm injection cycle with testicular spermatozoa and those who subsequently had an intracytoplasmic sperm injection cycle with ejaculated spermatozoa. The secondary objective was to determine the indications for the use of testicular spermatozoa after intracytoplasmic sperm injection failure with ejaculated spermatozoa. A retrospective study of matched couples using propensity score matching analysis was performed. After an intracytoplasmic sperm injection failure (cycle_1), intracytoplasmic sperm injection with either ejaculated spermatozoa (ejaculated sperm group), or testicular spermatozoa (testicular sperm group), was performed (cycle_2). The matching was on intracytoplasmic sperm injection performed in cycle_1 according to spermatozoa used (testicular or ejaculated) in cycle_2. Logistic regression was used to evaluate the influence of sperm origin on cumulative live birth rate. Univariate analysis on parameters of cycle_1 was used to identify the prognostic factors to propose an intracytoplasmic sperm injection with testicular spermatozoa in case of cycle_1 failure. The study outcomes were live birth rate and cumulative live birth rate. Among the 6034 couples available, 63 were selected to constitute the testicular sperm group and 63 were selected by propensity score matching to constitute the ejaculated sperm group. After matching, the DNA fragmentation index was higher in the testicular sperm group (13.43%±9.65% vs. 8.93%±4.47%, p=0.013); no significant difference was observed for the fertilisation rate, the number of obtained embryos, blastulation rate and frozen embryo rate. In cycle_2, the live birth rate was higher in the testicular group (22.2% vs. 0.0%, p<0.001), as was the cumulative live birth rate (25.4% vs. 6.3%, p=0.065). The prognostic factors identified for the proposal of intracytoplasmic sperm injection procedure with testicular spermatozoa after intracytoplasmic sperm injection failure with ejaculated spermatozoa were: teratozoospermia, cryptozoospermia and high DNA fragmentation index. According to the present study and current knowledge, the use of testicular spermatozoa after failed intracytoplasmic sperm injection procedure in non-azoospermic men could be proposed instead of sperm donation in case of high sperm DNA fragmentation index, cryptozoospermia and teratozoospermia. A good oocyte response to ovarian stimulation during the previous assisted reproductive technology attempt will increase the chance of success. Although the main limitation of the current study is its retrospective nature, the use of the propensity score matching to perform causal inference study increases its reliability. The present study supports that the use of testicular spermatozoa outside the classical indication of azoospermia is a good option when the indication is well established. However, before proposing a testicular biopsy, an improvement in sperm characteristics should be considered by treating the causes of sperm alteration.

A Possible Role for Nerve Growth Factor and Its Receptors in Human Sperm Pathology.

Nerve growth factor (NGF) signalling affects spermatogenesis and mature sperm traits. In this paper, we aimed to evaluate the distribution and the role of NGF and its receptors (p75NTR and TrKA) on the reproductive apparatus (testis and epididymis) and sperm of fertile men (F) and men with different pathologies, namely varicocele (V) and urogenital infections (UGIs). We collected semen samples from 21 individuals (31-40 years old) subdivided as follows: V (n = 7), UGIs (n = 7), and F (n = 7). We submitted the semen samples to bacteriological analysis, leucocyte identification, and analysis of sperm parameters (concentration, motility, morphology, and viability). We determined the seminal plasma levels of NGF, interleukin 1β (IL-1β), and F2-isoprostanes (F2-IsoPs), and the gene and protein expression of NGF receptors on sperm. We also used immunofluorescence to examine NGF receptors on ejaculated sperm, testis, and epididymis. As expected, fertile men showed better sperm parameters as well as lower levels of NGF, F2-IsoPs, and IL-1β compared with men with infertility. Notably, in normal sperm, p75NTR and TrKA were localised throughout the entire tail. TrKA was also found in the post-acrosomal sheath. This localisation appeared different in patients with infertility: in particular, there was a strong p75NTR signal in the midpiece and the cytoplasmic residue or coiled tails of altered ejaculated sperm. In line with these findings, NGF receptors were intensely expressed in the epididymis and interstitial tissue of the testis. These data suggest the distinctive involvement of NGF and its receptors in the physiology of sperm from fertile men and men with infertility, indicating a possible role for new targeted treatment strategies.

#110 : Diagnostic Approach and Clinical Recommendations for Non-obstructive Azoospermia: Global Andrology Forum Survey Results

Background and Aims: Non-obstructive azoospermia (NOA) is the most common form of male infertility, characterized by an absence of sperm in ejaculate and is caused by testicular failure. While NOA afflicts 10-38.3% of infertile males, there is no specific recommendations in EAA/EAU guidelines on its clinical diagnosis or WHO guidelines on semen analysis. Therefore, we conducted a global survey to assess the standard approach of practitioners in the diagnosis and management of NOA. Method: The Global Andrology Forum (GAF) conducted a survey on 436 independent practicing entities from 55 countries. We compiled data from 336 respondents (andrologists, urologists, fertility specialists). Information collected included frequency of NOA, varicocele, frequency of obtaining semen, interval between obtaining semen samples, hormone evaluation (FSH, LH, prolactin, total/free testosterone, SHBG, estradiol, inhibin), genetic testing (karyotype, Y-chromosome deletion, CFTR, Kal1) and referral to genetic counsellors. Results: The majority of participants (80%) either reported &lt;10% or 10-25% NOA frequency in their population (Fig. 1A). 40.6% of participants stated that 10-25% of men with NOA cases have a clinically significant varicocele (Fig. 1B). For NOA diagnosis, majority of participants cited the requirement of at least two semen samples, with a period of 14-29 days as the interval between two semen analyses. To achieve high-quality semen, consideration must be given to an abstinence period (2-7days) and the complete collection of ejaculates. The survey reported FSH (97%), LH (86.9%) and total testosterone (92.9%) tests as most frequently used for NOA patients (Fig. 2A). The initial hormonal evaluation of a patient with suspected NOA should include total testosterone levels and LH, as these hormones are the primary regulators of spermatogenesis. Karyotype analysis (86.2%), Y-microdeletion (88.3%), CFTR (21.9%) and Kal1 (2.7%) were most routinely performed genetic tests (Fig. 2B). Referrals to genetic counsellors is recommended as these defects have the potential to be passed to future generations. Conclusion: This global study evaluates NOA frequency, clinical diagnosis and management by specialists, in an effort to fill the gap in NOA research. Concomitantly, we provide GAF expert recommendations to enhance our collective understanding of NOA, optimize patient care and improve the management of NOA.

Comparative RNA isolation methods from fresh ejaculated spermatozoa in Sahiwal cattle (Bos indicus) and Murrah buffalo (Bubalus bubalis) bulls for high quality and enhanced RNA yield

Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. The aim of the study was to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bulls. Ten fresh ejaculates from each Sahiwal (n = 10 bulls × 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) resulted significantly (p < .05) higher total RNA quantity (300–340 ng/µL) and better purity in different concentrations of spermatozoa viz., 30–40 million, 70–80 million and 300–400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm.

Factors influencing timing of puberty in Mexican gray wolves (Canis lupus baileyi)

Abstract The reintroduced population of Mexican gray wolves (Canis lupus baileyi) continues to rely on a carefully managed captive breeding program. Although success of that program depends on detailed knowledge of reproductive processes, age of puberty has not been determined. This study assessed male puberty status (presence of sperm in ejaculates), maintained in nine US facilities, during their first breeding season. Variables possibly associated with puberty were also evaluated. There was a significant effect of body weight, testis size, facility latitude, and date of collection, whereas a statistical trend was found for age and inbreeding coefficient. Social factors, including being housed with sire, had no effect. Over half the males in their first breeding season were producing sperm, although in some cases sperm quality was poor, suggesting possible infertility. Although there was no minimum body weight associated with presence of sperm, likelihood increased with increasing body weight, highlighting a possible critical role for nutrition. The trend for sperm production in males collected later in the breeding season and at lower latitudes suggests that later collections, especially at higher latitudes, might reveal a higher percentage of pubertal males. These results have potential implications for breeding program management and introduce the possibility that more wild gray wolves than previously thought might produce sperm during their first year, even if they do not sire young.