Comparative Analysis of the Effects of Magnesium Oxide Nanoparticles on Sperm Parameters in Fresh and Frozen Samples.

Background: Freezing is a crucial technique in reproductive science utilized for the preservation of sperm samples. However, the process of freezing and thawing sperm can result in detrimental effects on sperm quality. One of the major mechanisms underlying this decline in sperm quality is the generation of reactive oxygen species during the freeze process. The purpose of the current study was to investigate the effects of magnesium oxide nanoparticles on frozen sperm parameters. Methods: Semen samples were collected from 8 fertile men, aged 30 to 42 years, with normozoospermia, following 3 to 5 days of abstinence. The samples were divided into fresh (n=3), freeze (n=3), and control (n=2) groups. Three fresh experimental groups were only exposed to MgO NPs with concentrations of 5, 25, and 50 µg/ml and three freezing experimental groups were frozen after being treated with MgO NPs, thawed, and analyzed after 30 min. Results: Our findings revealed that the progressive movement and vitality of sperm experienced a significant decline, while non-progressive and immotile sperm showed a notable increase in both fresh and frozen experimental groups exposed to MgO NPs. However, the application of MgO NPs during fresh and freezing processes demonstrated an effective preservation of pH, morphology, and DNA fragmentation in sperm cells. Conclusion: The analysis revealed that MgO NPs negatively impact sperm motility and viability in both fresh and freeze analysis. Also, the use of MgO NPs in fresh and frozen processes effectively maintains the pH, morphology, and fragmentation of DNA in sperm cells.

Sperm Quality in 1252 Adolescents and Young Adults (AYAs) Undergoing Fertility Preservation Due to Cancer or Nonmalignant Diseases.

Purpose: To investigate the quality of emergency-collected semen samples aimed at sperm cryopreservation provided by adolescents and young adults (AYAs) presenting with cancer or nonmalignant diseases. Methods: This is a prospective cohort study of postpubertal males referred for sperm cryopreservation who provided at least one semen sample for fertility preservation at the Reproductive Medicine Clinic of Karolinska University Hospital, Stockholm, Sweden, between January 2009 and January 2020. Sperm quality was assessed by total sperm count, concentration, and motility. Sperm quality by disease groups was compared with the reference population data of fertile men defined by the World Health Organization (WHO). Results: Among the 1252 patients who provided samples for cryopreservation, 1063 had cancer and 189 had nonmalignant diseases. The most common malignant indications included testicular cancers (n = 501) and Hodgkin lymphoma (n = 102). Among those with nonmalignant disease, 35% (n = 66) had testicular disease. Sperm quality was significantly lower in all groups of patients with cancer compared with the reference population. In total, azoospermia was found in 8% of the patients with cancer, in 9% of those with nonmalignant testicular disease, and in 3% of the remaining men with nonmalignant disease. Conclusion: Sperm quality in adult patients with cancer was significantly impaired compared with the WHO reference population standards for fertile men. For adolescent patients, standard reference values are lacking. AYAs wishing to preserve fertility should receive individualized counseling regarding sperm quality at the time of cryopreservation, and in selected cases, banking of additional samples should be recommended depending on the sperm quality parameters.

Cryopreserved testicular spermatozoa among patients with azoospermia.

PURPOSE: To investigate cryopreserved testicular spermatozoa among patients with azoospermia. METHODS: In this retrospective study spanning from October 1993 to December 2021, we examined men diagnosed with azoospermia who underwent testicular spermatozoa cryopreservation. Data from medical records included utilization and disposal of sperm samples, age at initial cryopreservation. We analyzed the data over 20 years using Kaplan-Meier curves, compared age with the log-rank test, and assessed hazard ratios (HR) with 95% confidence intervals (CI) using Cox regression analysis. RESULTS: A total of 356 patients with a mean age of 32.1 ± 6 were included. Of these, 225 patients utilized thawed testicular sperm for fertility treatments, with 118 patients using all their frozen straws and 107 patients partially using their stored straws. Additionally, 29 patients opted for disposal (six patients partially used their testicular spermatozoa before disposal), resulting in 108 patients who neither used nor disposed of their straws. From a laboratory standpoint, nearly 90% of patients contributed a single testicular sample, which was subsequently divided and cryopreserved as straws, with a median of 4 straws per sample. Notably, in the older age group (> 35 years old), there were a significantly lower usage rate and a higher disposal rate compared to the younger age groups (p < 0.05 for both), corroborated by univariable Cox analysis. CONCLUSIONS: This extensive study unveils unique patterns in the preservation and disposal of testicular spermatozoa among azoospermic patients. Most patients utilize a significant portion of their stored samples, while older patients tend to use their testicular spermatozoa less frequently.

Advancements in Understanding and Enhancing Antioxidant-Mediated Sperm Cryopreservation in Small Ruminants: Challenges and Perspectives.

Cryopreservation poses significant challenges to the preservation of sperm integrity and function, particularly in small ruminants where cryodamage is pronounced. This review explores the molecular mechanisms underlying sperm cryodamage and strategies for improving cryopreservation outcomes, with a focus on the role of antioxidants. Cryopreservation-induced alterations in proteins and RNA transcripts critical for sperm function, including motility, capacitation, fertilization, and embryo development, are discussed. Proteomic, transcriptomic, and epigenomic advancements have provided valuable insights into these mechanisms, offering potential biomarkers for predicting sperm freezability and enhancing cryopreservation strategies. Combining technologies such as mass spectrometry and flow cytometry allows for a comprehensive understanding of molecular and cellular changes induced by the freezing-thawing process. However, challenges remain in optimizing cryoprotectant formulations and antioxidant supplementation to improve post-thaw sperm fertility. Further research is needed to explore a wider range of novel cryoprotectants, antioxidants, and proteins for cryopreservation media, as well as to validate their efficacy in enhancing sperm viability and function. Additionally, investigations into the effects of cryopreservation on RNA transcripts and epigenetic factors in small ruminant species are warranted to advance our understanding of sperm preservation. Overall, this review highlights the importance of antioxidants in mitigating cryodamage and underscores the need for continued research to refine cryopreservation protocols and improve reproductive outcomes in small ruminants.

Testicular Cancer Survivorship and Fertility Preservation.

Testicular cancer disproportionally affects men of reproductive age making fertility an important aspect of testicular cancer survivorship. Men with testicular cancer have more semen parameter abnormalities and a higher incidence of infertility compared to the general population. All treatment options for testicular cancer negatively affect fertility with recovery rates varying by treatment. For these reasons, clinicians should offer sperm cryopreservation, ideally before orchiectomy to maximize the possibility of biologic paternity, if desired. Several innovations have positively impacted this space including direct-to-consumer cryopreservation and bench research demonstrating the feasibility of reintroducing testicular cells post-therapy.

Semen cryopreservation for an oncological reason: a retrospective study.

RESEARCH QUESTION: How do cancer type and treatment affect semen quality before and after treatment, and what effect does it have in their clinical management of infertility? Also, what is the rate of patients using cryopreserved semen samples after treatment? DESIGN: Patients who cryopreserved spermatozoa for oncological reasons between 2000 and 2022 in IVI clinics in Spain were retrospectively reviewed. Semen parameters were analysed before and after treatment, and utilization and destruction rates were calculated. Total motile sperm count (TMSC) was used for assisted reproductive technology (ART) counselling. RESULTS: A total of 724 patients cryopreserved their semen during the study period. The semen parameters of the cancer patients' semen before and after treatment were heterogeneous, with significant differences between cancer type and semen parameters. The utilization rate was relatively low (0.4%), whereas the destruction rate was 23.1%. CONCLUSION: Cancer and antineoplastic treatment affect everyone differently. Therefore, sperm cryopreservation should be offered to all patients before starting treatment to ensure their reproductive future. Furthermore, in addition to considering the semen parameters defined by the World Health Organization, it is important to use TMSC in the diagnosis of men to choose appropriate ART according to type of cancer.

Assessment of reproductive outcomes of fresh versus cryopreserved ejaculated sperm samples-a retrospective analysis of 44 423 oocyte donation ICSI cycles.

STUDY QUESTION: 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? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: A retrospective multicenter observational cohort study, including data from 37 041 couples and 44 423 ICSI procedures from January 2008 to June 2022, was carried out. The group using frozen sperm included 23 852 transferred embryos and 108 661 inseminated oocytes, whereas the fresh sample group comprised 73 953 embryos replaced and 381 509 injected oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): None declared. TRIAL REGISTRATION NUMBER: Not applicable.

Applying a heat transfer mathematical model for the cryopreservation of rainbow trout (Oncorhynchus mykiss) sperm: How straw location over liquid nitrogen level affects freezing rate and fertilization yield.

Cryopreservation of rainbow trout semen under field conditions was analyzed. Straw location over liquid nitrogen level is a crucial variable that affects freezing rate and fertilization yield due to changes in nitrogen vapor external temperature. The objectives were: to analyze cryopreservation protocols by experimentally measuring the cooling rates and fertilization yield of 0.5 ml plastic straws located in nitrogen vapor at different heights corresponding to different external temperatures; to numerically simulate the freezing process, by solving the heat transfer partial differential equations with the corresponding thermo-physical properties of the biological system and the plastic straw; to evaluate and analyze the surface heat transfer coefficient (h) during the freezing process of the straws; to introduce a new variable, the characteristic freezing time (tc), that enables comparison between protocols; this variable was defined as the elapsed period between the initial freezing temperature and a final reference temperature of -40 °C (temperature in which more than 80 % of the water is in a frozen state). The mathematical model predicted the temperature distribution inside the straw, showing a low effect of straw plastic materials (polyethylene-terephthalate glycol, polyvinyl-chloride, and polypropylene) on freezing rates. The average h value obtained from numerical simulations was 25.5 W/m2 K, close to that obtained from the analytical Nusselt correlation for natural convection. An improvement on fertilization trials was observed when the average external nitrogen temperature was -129.6 °C (temperature range: -94 to -171 °C) with an average tc of 56.8 s (ranging between 47 and 72 s). These results corresponded to a height above the level of liquid nitrogen of 2 cm. Comparison with literature reported data showed satisfactory results. Applying mathematical models in the cryobiology field achieved results that are relevant for cryopreservation activities.

Effects of different extenders on epigenetic patterns and functional parameters of bull sperm during cryopreservation process.

The cryopreservation process induces alterations in cellular parameters and epigenetic patterns in bull sperm, which can be prevented by adding cryoprotectants in the freezing extenders. The purpose of this study was to compare the protective effects of two extenders based on soybean lecithin (SLE) and egg yolk (EYE) on epigenetic patterns and quality parameters of sperm such as motility parameters, mitochondrial membrane integrity, DNA fragmentation, viability, and apoptotic-like changes of bull sperm after cryopreservation. Results demonstrated that cryopreservation significantly (p < .05) reduced the level of DNA global methylation, H3K9 histone acetylation, and H3K4 histone methylation in both frozen groups compared to the fresh sperm. Also, the level of H3K9 acetylation was lower in the frozen SLE group (21.2 ± 1.86) compared to EYE group (15.2 ± 1.86). In addition, the SLE frozen group had a higher percentage of viability, progressive motility, and linearity (LIN) in SLE frozen group compared to EYE frozen group. However, no difference was observed in mitochondrial membrane integrity and DNA fragmentation between SLE and EYE frozen groups. While soybean-lecithin-based extender showed some initial positive impacts of epigenetics and semen parameters, further investigations can provide useful information for better freezing.

Effects of meteorology and lunar cycle on the post-thawing quality of avian sperm.

Introduction: Various climatological and lunar cycle parameters have a direct impact on animal reproduction, and in the case of the avian species, spermatozoa are extremely sensitive to heat stress. These parameters could influence sperm freezability, which will ultimately affect post-thawing semen quality, being sperm motility in roosters a relevant indicator of this quality as it is highly related to fertility. Therefore, the objective of the present study is to determine which are the climatological and lunar cycle parameters that have a greater effect on sperm freezability in roosters. Methods: Sperm was obtained from 16 Utrerana breed roosters and a total of 27 replicates were performed. A pool was made with those ejaculates that met the minimum quality criteria for each replicate, and four freezing-thawing samples per replicate were analyzed. The straws were thawed, and sperm motility was evaluated, classifying the results obtained into four seminal quality groups according to the guidelines of the Food and Agriculture Organization of the United Nations (Group 1: Good, Group 2: Satisfactory, Group 3: Acceptable but undesirable and Group 4: Unsatisfactory). The following traits were recorded for each day of semen collection: maximum temperature, minimum temperature, maximum barometric pressure, minimum barometric pressure, maximum gust, wind direction, mean wind speed, sunshine hours, rainfall, moon phase, and percentage of illuminated lunar surface over the total area. Results: A discriminant canonical analysis was performed to determine which of these parameters offered the most information when classifying an ejaculate in each quality group, with minimum temperature, the new moon as moon phase, minimum barometric pressure, and rainfall being the most significant variables. Discussion: According to the results obtained, semen quality decreases when temperature and precipitation are lower, pressure is higher, and when there is a new moon phase. Therefore, these environmental conditions should be avoided for sperm collection and processing.

Supplementation of Extender with Melatonin Improves the Motility, Mitochondrial Membrane Potential, and Fertilization Ability of Cryopreserved Brown-Marbled Grouper Sperm.

Sperm cryopreservation is a valuable tool for breeding, conservation, and genetic improvement in aquatic resources, while oxidative damage will cause a decline in sperm quality during this progress. Melatonin (MT), a natural antioxidant hormone, is used as an additive in sperm cryopreservation to reduce cellular damage from oxidative stress. Here, we aimed to investigate the effect of adding MT to the freezing medium in sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus). Different concentrations of MT (0, 0.1, 0.25, and 0.5 mg/mL) were tested. We evaluated sperm motility, viability, apoptosis, mitochondrial membrane potential (MMP), and fertilization ability to assess the effects of MT supplementation. Our results demonstrated that the addition of MT to the extender improved the post-thaw motility, MMP, and fertilization ability of brown-marbled grouper sperm. The total motility, curvilinear velocity, straight linear velocity, and average path velocity in MT-treated groups (0.1 and 0.25 mg/mL) exhibited significantly higher values than that of the control group. A higher MMP (p < 0.05) was observed in the group treated with 0.25 mg/mL MT, suggesting that supplementation of MT in the extender might be able to protect mitochondrial membrane integrity effectively. Regarding fertilizing ability, 0.25 mg/mL MT yielded a significantly higher hatching rate than the control. An adverse effect was found with the concentration of MT up to 0.5 mg/mL, suggesting the possible toxicity of a high-dose addition. In this study, we optimized the sperm cryopreservation protocol of brown-marbled grouper, which might be valuable for sperm cryopreservation and sample commercialization of groupers and other fish.

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.

Paternal Age Amplifies Cryopreservation-Induced Stress in Human Spermatozoa.

The global fall in male fertility is a complicated process driven by a variety of factors, including environmental exposure, lifestyle, obesity, stress, and aging. The availability of assisted reproductive technology (ART) has allowed older couples to conceive, increasing the average paternal age at first childbirth. Advanced paternal age (APA), most often considered male age ≥40, has been described to impact several aspects of male reproductive physiology. In this prospective cohort study including 200 normozoospermic patients, 105 of whom were ≤35 years (non-APA), and 95 of whom were ≥42 years (APA), we assessed the impact of paternal age on different endpoints representative of sperm quality and cryopreservation tolerance. Non-APA patients had superior fresh semen quality; DNA fragmentation was notably increased in APA as compared to non-APA individuals (21.7% vs. 15.4%). Cryopreservation further increased the DNA fragmentation index in APA (26.7%) but not in non-APA patients. Additionally, APA was associated with increased mtDNAcn in both fresh and frozen/thawed sperm, which is indicative of poorer mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, as indicated by reduced incidences of unreacted acrosome in relation to fresh counterparts in non-APA (from 71.5% to 57.7%) and APA patients (from 75% to 63%). Finally, cryopreservation significantly reduced the phosphorylation status of proteins containing tyrosine residues in sperm from young males. Therefore, the present findings shed light on the effects of paternal age and cryopreservation on sperm quality and serve as valuable new parameters to improve our understanding of the mechanisms underlying sperm developmental competence that are under threat in current ART practice.

Safety evaluation of single-sperm cryopreservation technique applied in intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is a technique that directly injects a single sperm into the cytoplasm of mature oocytes. Here, we explored the safety of single-sperm cryopreservation applied in ICSI. This retrospective study enrolled 186 couples undergoing ICSI-assisted pregnancy. Subjects were allocated to the fresh sperm (group A)/single-sperm cryopreservation (group B) groups based on sperm type, with their clinical baseline/pathological data documented. We used ICSI-compliant sperm for subsequent in vitro fertilization and followed up on all subjects. The recovery rate/cryosurvival rate/sperm motility of both groups, the pregnancy/outcome of women receiving embryo transfer, and the delivery mode/neonatal-related information of women with successful deliveries were recorded. The clinical pregnancy rate, cumulative clinical pregnancy rate, abortion rate, ectopic pregnancy rate, premature delivery rate, live birth delivery rate, neonatal birth defect rate, and average birth weight were analyzed. The two groups showed no significant differences in age, body mass index, ovulation induction regimen, sex hormone [anti-Müllerian hormone (AMH)/follicle-stimulating hormone (FSH)/luteinizing hormone (LH)] levels, or oocyte retrieval cycles. The sperm recovery rate (51.72%-100.00%) and resuscitation rate (62.09% ± 16.67%) in group B were higher; the sperm motility in the two groups demonstrated no significant difference and met the ICSI requirements. Group B exhibited an increased fertilization rate, decreased abortion rate, and increased safety versus group A. Compared with fresh sperm, the application of single-sperm cryopreservation in ICSI sensibly improved the fertilization rate and reduced the abortion rate, showing higher safety.

Fertility preservation in males with cancer of trends, region development, and efficacy in mainland China from 16 regions Chinese sperm banks.

PURPOSE: Male cancer survivors experience confusion about fertility following cancer treatment. The aims of this study were to evaluate survivors' semen quality in different tumor type groups in China and to analyze the current situation and challenges of male cancer patients with sperm cryopreservation. METHODS: This was a multicenter retrospective study of male patients with cancer who underwent sperm cryopreservation in 16 regions of the national sperm banks over an 11-year period from 2010 to 2020. RESULTS: The number of male cancer patients with sperm cryopreservation showed an overall upward trend. The development of male cancer fertility preservation (FP) in the eastern, central, and western regions of Chinese displayed imbalance. There are seven tumor types for sperm preservation in the top incidence ten tumor types, including lymphoma, leukemia, nasopharyngeal carcinoma, sarcoma, thyroid cancer, and brain tumor. Moreover, nasopharyngeal carcinoma is a high incidence rate in China, which is related to high sperm preservation rate, different from other countries. The most percentage of males receiving sperm cryopreservation in the testicular cancers (15-39 years old) of China in 2020 was 5.55%, 1.29% in the lymphoma, and 0.39% in the leukemia. According to the type of cancer, a statistically significant lower pre-sperm density, total sperm output, and post-sperm density was observed in testicular cancers. It is worth noting that the prevalence of azoospermia 22.2% in leukemia patients attribute to urgent treatment before sperm cryopreservation. Disposition of cryopreserved sperm categories included continued storage (47.2%), discarded (9%), death (0.9%), and use (3.7%). CONCLUSION: This study provides the first comprehensive national statistical census and review of fertility preservation in male cancer patients with respect to trends, prevalence, and cancer types. The development of male cancer fertility preservation in China is imbalanced and percentage of males receiving sperm cryopreservation in the adolescent and young adult cancers was low. Sixteen human sperm banks from China analyze current problems and challenges, and then prioritize steps toward the achievement of the FP strategy framework for Healthy China 2030.

A Pilot Analysis of Whole Transcriptome of Human Cryopreserved Sperm.

Sperm cryopreservation is a procedure widely used to store gametes for later use, to preserve fertility in patients prior to gonadotoxic treatments or surgery, and for sperm donation programs. The purpose of the study was to assess the impact of cryopreservation on human sperm transcriptome. Semen samples were collected from 13 normospermic men. Each sample was divided into two aliquots. The total RNA was immediately extracted from one aliquot. The second aliquot was frozen and total RNA was extracted after a week of storage in liquid nitrogen. The RNA samples were randomized in four pools, each of six donors, and analyzed by microarrays. The paired Significance Analysis of Microarray was performed. We found 219 lower abundant transcripts and 28 higher abundant transcripts in cryopreserved sperm than fresh sperm. The gene ontology analysis disclosed that cryopreservation alters transcripts of pathways important for fertility (i.e., spermatogenesis, sperm motility, mitochondria function, fertilization, calcium homeostasis, cell differentiation, and early embryo development), although the increase of some transcripts involved in immune response can compensate for the harmful effects of freezing.

Development of sperm cryopreservation protocol for patin buah, Pangasius nasutus.

The development of sperm cryopreservation for Pangasius nasutus is necessary in order to serve the growing demand of this species through artificial fertilization and the preservation of valuable strains of male broodstocks. In the present study, the basic protocol of sperm cryopreservation for P. nasutus was established by identifying the optimal conditions for optimum cryoprotectant, toxicity of cryoprotectants, extenders, freezing condition and dilution ratio. Methanol (MeOH) at 10% concentration had the best post-thaw motility (26.3 ± 0.9%) and curvilinear velocity (VCL) compared to dimethyl acetamide and dimethyl sulfoxide. MeOH was the least toxic cryoprotectant; sperm suspended in 5 and 10% MeOH maintained motility up to 50 min. No significant differences were detected between the three types of extenders tested (0.9% sodium chloride, Calcium-free Hanks' Balance salt solution and ringer solution). P. nasutus sperm had a narrow range of optimal cooling rate. Significantly higher post-thaw motility was identified when cooling at 9.23 °C min-1, obtained by freezing at height of 14 cm above liquid nitrogen vapor for 7 min, showing lower cooling rate is suitable for this species. However, when cooling below and above the optimal cooling rate, post-thaw motility dropped drastically. There were no significant differences among the dilution ratios investigated, indicating the volume of cryodiluent at all tested ratios (1:9, 1:19 and 1:49) was sufficient for the protection of cells during the cryopreservation process. The development of the protocol for cryopreserved P. nasutus sperm will assist artificial seed production and provide an important tool for genetic and breeding research.

Fertility preservation in adult male patients with cancer: a systematic review and meta-analysis.

STUDY QUESTION: Does sperm cryopreservation serve as a feasible and effective method for preserving fertility in adult male patients with cancer? SUMMARY ANSWER: Sperm cryopreservation is an effective fertility preservation method and may benefit patients with cancer. WHAT IS KNOWN ALREADY: Sperm cryopreservation is the only way to efficiently preserve male fertility. It is an important procedure in ART. Recently, due to remarkable advances in cancer treatment, an increasing number of studies have reported the outcomes of sperm cryopreservation in patients with cancer. STUDY DESIGN SIZE DURATION: We conducted an extensive literature search for relevant studies published through to 31 December 2021, in the following databases: CENTRAL, CNKI, Cochrane Systematic Reviews, EMBASE, MEDLINE, PUBMED, and Web of Science. The search terms used were '(cryopreservation OR freeze OR freezing OR banking OR cryostorage OR storage) AND (sperm OR semen OR spermatozoon) AND (cancer OR tumor OR malignancy OR neoplasm)'. PARTICIPANTS/MATERIALS SETTING METHODS: We included all studies that reported offering or attempting to cryopreserve sperm before or during cancer treatment in male patients considered at risk of treatment-related fertility impairment. We evaluated the eligibility of all data in each study. The major exclusion criteria were as follows: non-cancer patients; pediatric and adolescent cancer patients; not reporting the use of cryopreserved sperm; use of fresh semen for ART; not reporting the number of patients with cancer offered sperm cryopreservation or attempting to do so before or during treatment; using an experimental fertility preservation technique such as preservation of testicular tissue or spermatogonial stem cells; duplicate data; abstracts, case report, comments, reviews, or editorials; insufficient data reported. The quality of the included studies was assessed using the Newcastle-Ottawa scale and the Methodological Index for Non-Randomized Studies. MAIN RESULTS AND THE ROLE OF CHANCE: This meta-analysis included 69 non-randomized studies, with 32 234 patients referred for sperm analysis and 23 178 patients cryopreserving at least one sperm sample. The pooled failed-to-cryopreserve rate was 10% (95% CI, 8-12%), and the sperm disposal and sperm use rates were 23% (95% CI, 16-30%) and 9% (95% CI, 8-10%), respectively. The pregnancy, miscarriage, and delivery rates were 28% (95% CI, 22-33%), 13% (95% CI, 10-17%), and 20% (95% CI, 15-25%), respectively. Subgroup analysis showed higher pregnancy and delivery rates, as well as a lower failed-to-cryopreserve rate, in recent studies compared to those released a decade ago. The studies from Asia reported higher sperm disposal and pregnancy rates than in other continents. Our analysis showed clinical pregnancy rates per cycle of 34% (27-41%), 24% (14-35%), and 9% (5-15%) and delivery rates per cycle of 23% (17-30%), 18% (11-26%), and 5% (1-9%) for ICSI, IVF, and IUI, respectively. LIMITATIONS REASONS FOR CAUTION: As with all meta-analyses, some limitations should be considered. The first limitation of our study is that the data span 36 years. During this time, the World Health Organization has revised its sperm analysis standards, and other important changes have been made. There is also a limitation in that the outcome does not analyze the correlation between the type of cancer and sperm quality. Many of the earlier studies were limited by small sample sizes and a lack of control groups. Furthermore, almost all studies did not consider the severity of the disease, which could potentially have a substantial impact on the results. Consequently, further research should evaluate the effect of the type of cancer and, in particular, the severity of the condition on sperm quality in order to draw more precise conclusions. Similarly, it is inappropriate that most studies failed to differentiate between patients with different types of tumors and instead drew generalized conclusions that are presumed to apply to all patients with cancer. In the present analysis, we did not have in-depth information on patients' disease, and although extensive efforts were made to conduct a thorough systematic review and meta-analysis of the outcomes for patients with various types of tumors, the results must be acknowledged as being subject to bias. However, the use of average results obtained in each study, without the patient-level data, might also represent a source of bias. WIDER IMPLICATIONS OF THE FINDINGS: Sperm cryopreservation is an effective fertility preservation method and may benefit patients with cancer. The observed utilization rate of frozen sperm at 9% may underestimate the actual usage, as the short follow-up period is inadequate for obtaining comprehensive data on the use of frozen sperm in young cancer survivors. ART plays an important role in fertility preservation and the achievement of pregnancy, with this meta-analysis showing that ICSI delivers better clinical outcomes than IVF or IUI in patients with cancer undergoing fertility preservation. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (grant no. 82001634, 81960550), and the China Postdoctoral Science Foundation (2019M661521). There are no competing interests to declare. REGISTRATION NUMBER: CRID 42022314460.

Cryopreservation of Limited Sperm Using A Combination of Sucrose and Taurine, Loaded on Two Different Devices, and Thawed at Two Different Temperatures.

BACKGROUND: Cryopreservation of sperm is essential for patients with low sperm counts and couples undergoing infertility treatment. The aim of this study was to compare the effects of Taurine (T) and Sucrose (S) in individual sperm cryopreservation utilizing cryotop and petri dish and thawing at 37 and 42°C. MATERIALS AND METHODS: In this experimental study, 17 normospermic semen samples were processed using the "Swim-up" procedure and progressively motile sperm were then isolated from these samples using an inverted microscope. Sperm were added to droplets of "sucrose medium" with 25 mM Taurine antioxidant (S+T) and the commercial cryoprotectant "Sperm Freeze" (CPA), loaded on a petri dish and cryotop. After rapid freezing of the samples, they were thawed at two different temperatures (37°C and 42°C), and the sperm classical parameters, viability, and DNA fragmentation were assessed. RESULTS: Statistical analysis displayed a significant increase in total and progressive motility in individual sperm freezing on cryotop with CPA and thawing at 42°C (P<0.05). Other parameters did not show any differences between the CPA and S+T groups and two thawing temperatures in either of the cryopreservation methods. CONCLUSION: Although, both cryoprotectants (CPA and S+T) may preserve individual sperm effectively using cryotop, the CPA and thawing at 42°C showed a better effect on the motility percentage of the small number of sperm.

Cold storage and cryopreservation methods for spermatozoa of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus.

BACKGROUND: Sea urchins have contributed greatly to knowledge of fertilization, embryogenesis, and cell biology. However, until now, they have not been genetic model organisms because of their long generation times and lack of tools for husbandry and gene manipulation. We recently established the sea urchin Lytechinus pictus, as a multigenerational model Echinoderm, because of its relatively short generation time of 4-6 months and ease of laboratory culture. To take full advantage of this new multigenerational species, methods are needed to biobank and share genetically modified L. pictus sperm. RESULTS: Here, we describe a method, based on sperm ion physiology that maintains L. pictus and Strongylocentrotus purpuratus sperm fertilizable for at least 5-10 weeks when stored at 0°C. We also describe a new method to cryopreserve sperm of both species. Sperm of both species can be frozen and thawed at least twice and still give rise to larvae that undergo metamorphosis. CONCLUSIONS: The simple methods we describe work well for both species, achieving >90% embryo development and producing larvae that undergo metamorphosis to juvenile adults. We hope that these methods will be useful to others working on marine invertebrate sperm.