Sperm Preservation Research Articles

Fertility Preservation in Postpubertal Males Undergoing Cancer Treatment in a Middle-Income Country: Is it Possible Despite the Barriers?

Increased survival rates in childhood cancer have led to an emphasis on the importance of treatment-related infertility. Fertility preservation methods should be explained to every patient and their families (PaFs) before treatment. Establishing good communication with PaFs is crucial in this regard despite many barriers such as cultural and financial barriers. Routine feasibility of sperm preservation (SP) in adolescent males newly diagnosed with cancer was evaluated after the implementation of reimbursement for the procedure and storage by the national healthcare system. Males <18 years of age planned to undergo cancer treatment between 2021 and 2023 were included. Patient and their families were informed by the treating physician about the disease, treatment modality, side effects, the importance of SP, and the method. Information about the purpose and technique of SP was provided to the patient alone in a comfortable environment. Questions from PaFs were answered, and consent was obtained. The procedure and storage cost was covered by the social security institution. Seventeen patients (median age 15) (15 with bone/soft tissue sarcoma, 1 brain tumor, and 1 Hodgkin lymphoma) were included. There were no refusals for SP. Eleven patients were able to provide sperm samples. Of the 6 patients who could not provide sperm, 3 stated embarrassment, 2 anxiety, and 1 cancer-related pain. Increasing awareness among physicians about the importance of treatment-related infertility, allocating sufficient time to inform PaFs about fertility preservation, and providing information taking into account societal or socio-cultural factors will contribute to preventing treatment-related infertility. Establishing close communication with the Reproductive Health Center (RHC), along with the rapid and cost-free nature of the procedure, enhances success.

Damage to Mitochondria During the Cryopreservation, Causing ROS Leakage, Leading to Oxidative Stress and Decreased Quality of Ram Sperm.

Semen cryopreservation can achieve long-term preservation of sperm. Ice crystal damage, as well as oxidative stress, result in mitochondrial dysfunction and a reduction in sperm motility after thawing. However, limited information exists regarding the impact of reactive oxygen species (ROS) and mitochondria on the cryopreservation of ram sperm. The primary objective of this study was to investigate the relationship between ROS and mitochondria concerning sperm quality during the cryopreservation of ram sperm. This investigation assessed sperm motility, kinematic characteristics, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, expression of mitochondrial respiratory genes (NDUFV2, SDHA, CYC1, and COXIV), ROS levels, malondialdehyde (MDA) content, phosphatidylserine externalisation rate, sperm ultrastructure, mtDNA copy number, expression of apoptosis-related genes (Bax, Caspase-3, and Caspase-8), Cytochrome C, and Caspase-3 content. The results showed the cryopreservation significantly (p < 0.05) decreased motility, kinetic parameters, membrane integrity, acrosome integrity, MMP, ATP, mRNA expression levels of mitochondrial respiratory-related genes, and significantly (p < 0.05) increased ROS levels, MDA content, phosphatidylserine externalisation rate, damage of sperm ultrastructure, mtDNA copy number, mRNA expression levels of apoptosis-related genes, Cytochrome C and Caspase-3 content compared to the fresh semen group. In conclusion, the cryopreservation causes damage to mitochondria, leading to increased ROS and subsequent oxidative stress. This process also initiates mitochondrial dysfunction and interferes with the electron transport chain, ultimately resulting in decreased MMP and ATP production. Furthermore, the liberation of Cytochrome C prompted the increase in Caspase-3 expression and subsequent sperm apoptosis occurred, ultimately leading to a deterioration in sperm quality after thawing.

Dexamethasone and azithromycin enhance goat sperm preservation quality by regulating lipid metabolism

Phospholipase A (PLA) in goat semen aggregates with egg yolk in semen diluent, leading to sperm death. The aim of this study is to address the issue of sperm death caused by the interaction between PLA and egg yolk, and to explore the protective effect and metabolic regulation mechanism of the combination of dexamethasone (DXMS) and azithromycin (AZM) on goat sperm under low temperature conditions. At a low temperature of 4°C, different concentrations of DXMS were added to semen diluents containing 30μg/mL AZM to detect the quality of goat sperm. The optimal concentration of DXMS was determined to be 20μg/mL. On the 5th day of storage, antioxidant capacity, total cholesterol (TC) levels, energy metabolism, and metabolomics analysis were performed on the sperm of the 20μg/mL DXMS group. The results showed that there was no aggregation caused by the interaction between PLA and egg yolk in the group containing 30μg/mL AZM at 4°C. 20 μg/mL DXMS significantly improved sperm motility, plasma membrane integrity, acrosome integrity, glutathione peroxidase (GPX) (P<0.05), catalase (CAT) (P<0.01), and superoxide dismutase (SOD) activity (P<0.01). The content of reactive oxygen species (ROS) and Fe2+ significantly decreased (P<0.01), while the content of ATP (P<0.01) and TC (P<0.05) significantly increased. Through metabolomics analysis, a total of 56 differential metabolites (P<0.05) were screened, including 5a, 6-Anhydrotetracycline, Betamethasone, and 11-Dehydrocorticosterone, mainly enriched in 8 metabolic pathways (P<0.05), including steroid hormone biosynthesis, glycerophospholipid metabolism, and choline metabolism in cancer. Among them, 5 metabolic pathways are related to lipid metabolism. The results indicate that AZM effectively inhibits the aggregation of PLA and yolk, and the combination of AZM and DXMS enhances the preservation quality of goat sperm during low-temperature preservation by regulating lipid metabolism.

Protective Effects of Betaine on Boar Sperm Quality during Liquid Storage and Transport.

Boar semen is commonly used in artificial insemination (AI) for pig breeding, but its quality can be negatively affected by liquid preservation and transportation, leading to reduced fertility rates. Vibration and temperature fluctuations are critical factors that significantly impact semen quality during storage and transportation, influencing the success rate of AI procedures. Betaine, a naturally occurring compound known for its role in maintaining male fertility, demonstrates potential for improving the preservation and transportation of liquid-preserved boar sperm. The present study demonstrated that betaine supplementation in the semen extenders at 0.5 mg/mL had a significant protective effect on boar sperm motility during storage at 17 °C for 3 to 5 days. During road transportation, 2.5 mg/mL betaine showed significant protective effects on boar sperm progressive motility, while 0.4 mg/mL betaine notably improved boar sperm mitochondrial activity and antioxidant capacity, and reduced lipid peroxidation damage. Simulation models also demonstrated that betaine supplementation increased the proportion of sperm displaying progressive motility and possessing intact acrosomes, regardless of the storage temperature (17 °C or 25 °C), and effectively mitigated the damage caused by vibration at a speed of 200 r/min. Overall, supplementing liquid-preserved boar semen extenders with betaine shows promise in mitigating damage to sperm quality during storage and transportation.

Comparison between a new device for the semen quality analysis and the manual microscopic evaluation in a not specialistic clinical laboratory

Abstract Objectives Semen analysis investigates different parameters of human semen with a high relevance in fertility workup, confirmation of sterility by post vasectomy, in pathologies follow-up such as varicocele and in all cases where sperm preservation is required. Manually seminal fluid examination is characterized by poor reproducibility. Aim of this study was to evaluate the performance of an automatic device in semen analysis by comparing its results with those obtained with the manual microscopy. Materials Fifty samples (age 18–59 years) were analyzed simultaneously by the manual and automated method. Manual analysis was performed by at least two experienced operators. Concentration and motility were determined by means of standard manual analysis and by the automated LensHooke™ analyzer following the last WHO guidelines. Results We compared the concentration (million/mL) of spermatozoa obtained from manual and instrumental count and different classifications obtained: normal, oligospermic, cryptospermic and azoospermic samples. The Wilcoxon test does not show a statistically significant difference. The Bland-Altman plot showed a slightly higher value for the manual count. Second, we compared the morphology and the samples classification in morphological normal and abnormal. Third, spermatozoa motility obtained from the manual and instrumental count was compared with a different classification in normal total motility and asthenozoospermia. Statistical tests showed respectively for morphology and motility a moderate and a very good agreement. Conclusions Our study demonstrates that the LensHooke™ shows an acceptable agreement with the manual microscopic seminal fluid evaluation. The use of this simple device could help to standardize reports in non specialistic laboratories.

Seminal plasma removal for medium-term preservation of ram sperm at 5 °C

This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

Prostate radiotherapy may cause fertility issues: a retrospective analysis of testicular dose following modern radiotherapy techniques

BackgroundProstate cancer in younger men is rare but not exceptional. Radiotherapy is a cornerstone of prostate cancer treatment and yet, its impact on fertility is scarcely reported in literature. Given the radiosensitivity of testicular tissue, this study aimed to determine the testicular dose using modern radiotherapy techniques for definitive prostate irradiation.MethodsOne hundred radiotherapy plans were reviewed. Testicles were contoured retrospectively without dosimetric optimization on testicles.ResultsThe median testicular dose was 0.58 Gy: 0.18 Gy in stereotactic plans, 0.62 Gy in Volumetric Modulated Arc Therapy plans and 1.50 Gy in Tomotherapy plans (p < 0.001). Pelvic nodal irradiation increased the median testicular dose to 1.18 Gy versus 0.26 Gy without nodal irradiation (p < 0.001). Weight and BMI were inversely associated with testicular dose (p < 0.005). 65% of patients reached the theoretical dose threshold for transient azoospermia, and 10% received more than 2 Gy, likely causing definitive azoospermia.ConclusionDespite being probably lower than doses from older techniques, the testicular dose delivered with modern prostate radiotherapy is not negligible and is often underestimated because the contribution of daily repositioning imaging is not taken into account and most Treatment Planning Systems underestimate the out of field dose. Radiation oncologists should consider the impact on fertility and gonadal endocrine function, counseling men on sperm preservation if they wish to maintain fertility.Trial registration: retrospectively registered.

Poor Sperm Chromatin Condensation Is Associated with Cryopreservation-Induced DNA Fragmentation and Cell Death in Human Spermatozoa.

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze-thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze-thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze-thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3-8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at -196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all rs ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial rs ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (rs = 0.366, p = 0.014) and necrosis (rs = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial rs ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (rs = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze-thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood.

Carboxyfullerene C60 preserves porcine sperm by enhancing antioxidant capacity and inhibiting apoptosis and harmful bacteria.

This study used a porcine model to systematically investigate whether carboxyfullerene C60(CF-C60) can be used for sperm preservation. The results indicated that CF-C60 supplementation can preserve porcine sperm quality during storage at 17°C. This effect was attributable to an improvement in the antioxidant capacity of sperm through a decrease in the reactive oxygen species (ROS) level. Additionally, CF-C60 can maintain mitochondrial function, inhibit sperm apoptosis through the ROS/Cytochrome C (Cyt C)/Caspase 3 signaling pathway, and mediate suppression of bacterial growth through the effects of ROS. Finally, the results of artificial insemination experiments indicated that insemination with CF-C60-treated sperm can increase the total number of offspring born and reduce the number of deformed piglets. Thus, CF-C60 is safe for use as a component of semen diluent for sperm storage.

The application of mean number of DNA breakpoints in sperm cryopreservation

Growing concerns over declining male semen quality and rising infertility have shifted attention to male fertility. Sperm cryopreservation emerges as a crucial tool in preserving male fertility, especially for patients who need proactive preservation, such as cancer patients before undergoing radiation or chemotherapy. Although cryopreservation does not directly address infertility, effective preservation can support future fertility. However, the process may compromise sperm DNA integrity. Despite their impairment, damaged sperm often retain vitality and may still have the potential to fertilize an egg. Nonetheless, if damaged sperm fertilize an egg, excessive DNA damage could impede embryo implantation and development, despite the egg's repair capabilities. Consequently, precise detection of sperm DNA damage is crucial and urgent. To better address the issue of sperm DNA damage detection, we have introduced a novel fluorescence biosensor technology known as the TDT/SD Probe. This technology utilizes terminal deoxynucleotidyl transferase (TdT) and strand displacement probes to accurately detect the number of sperm DNA breakage points during the cryopreservation process. Experimental results reveal that the number of sperm DNA breakpoints significantly increases after both sperm vitrification (8.17 × 105) and conventional slow freezing (10.80 × 105), compared to the DNA breakpoints of fresh semen samples (5.19 × 105). However, sperm vitrification has the least impact on sperm breakage points. This research provides innovative means for further optimizing sperm preservation techniques by offering a novel DNA damage detection method, enabling more precise assessment of sperm DNA damage during the freezing process.

O-015 Enabling laboratory-quality male fertility analysis with 72-hour post ejaculation sample viability

Abstract Study question Can SPX72-treated semen samples sustain diagnostic viability and integrity for up to 72 hours, enabling comparable analysis of mail-in samples to fresh samples? Summary answer SPX72 preserves sperm viability, motility, and integrity up to 72 hours post-collection, equalling the diagnostic precision of fresh samples and enhancing at-home testing capabilities. What is known already Challenges with laboratory-based fertility assessments stem from logistical burdens and the necessity for timely sample delivery, often deterring male patient participation. Semen samples typically need to be assessed within 60 minutes of collection to ensure accuracy. Pre-existing solutions have failed to sufficiently prolong sample viability while matching the diagnostic accuracy of immediate laboratory analyses, an issue Sapyen's SPX72 medium addresses effectively. Study design, size, duration An exhaustive controlled study with over 100 semen samples compared SPX72-treated samples at 72 hours against fresh controls. The study incorporated actual postal logistics to emulate mail-in scenarios. Participants/materials, setting, methods A predetermined quantity of SPX72 per millilitre was added to the collected semen samples, with key indicators, including progressive and total motility, morphology, and DNA fragmentation, measured at intervals of 0, 24, 48, and 72 hours, contrasted with untreated, fresh samples. Main results and the role of chance SPX72 application preserved notable semen motility, evidenced by a mean progressive motility of 37.12% after 72 hours, a slight decrease from the initial 41.14%. This minor 4.02% reduction underscores SPX72's proficiency in maintaining diagnostic-grade motility over an extended duration. In terms of DNA integrity, SPX72 maintained DNA fragmentation at 14.78%, juxtaposed with 12.44% in control samples, a statistically insignificant variance (p-value of 1.15037E-08). This consistency in DNA fragmentation levels is pivotal for the accuracy of fertility diagnostic metrics. Additionally, SPX72 demonstrated its efficacy in stabilising the pH level of semen samples throughout the 72-hour period, ensuring an optimal environment for sperm preservation. This maintenance of pH is crucial as deviations can significantly affect sperm functionality and overall sample integrity. These results substantiate SPX72's capacity to preserve diagnostic sperm quality for up to three days, potentially redefining the logistics of male fertility testing by enabling accurate, laboratory-level assessment through a mail-in format. Limitations, reasons for caution Despite the controlled study and real-world postal validation, ongoing surveillance of sample stability in diverse global postal environments will be crucial to ensure consistent results across all geographies. Wider implications of the findings The data indicate that SPX72 sets a new standard for at-home fertility testing, matching clinical quality without the immediacy constraint. For the first time, home-based sperm diagnostics do not compromise clinical quality, revolutionising how male fertility is approached and managed. Trial registration number not applicable

P-006 Fertility outcomes in men diagnosed with oncohematological diseases who stored their semen for fertility preservation

Abstract Study question What is the prevalence of conceptions among patients who underwent semen cryopreservation before receiving treatment for oncohematological disease? Summary answer Natural live birth rate after oncohematological treatments is around 38%(95%CI:31-45%), but can rise to 61%(95%CI:54-67%) with the use of frozen semen during ART cycles. What is known already Due to advancements in cancer treatments, there is a growing population of male adult cancer survivors. These individuals are at risk of experiencing treatment-induced infertility, and it is generally advised for them to preserve their semen before starting therapies. However, the details regarding the long-term utilization and effectiveness of frozen sperm samples in this particular population remain unclear. Study design, size, duration The study included males with oncohematological disease who stored semen (1987-2018) at Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico’s Assisted Reproductive Technology Unit in Milan. Data, gathered from records or proactive inquiry, assessed natural and ART conceptions post-treatment. The study also examined semen analysis, parenthood attempts, and the usage rates of stored spermatozoa as primary outcomes. Participants/materials, setting, methods The study is limited to men who meet the following eligibility criteria: i) semen cryopreserved for fertility preservation between 1987 and 2018; ii) diagnosis of oncohematological disease; iii) survival of at least 5 years from cryopreservation. Main results and the role of chance A total of 424 patients who underwent semen cryopreservation for oncohematological diseases were included in the study. The median [IQR] age at the time of sperm banking was 27 [23-33]. The most common indication was lymphoma (86%), and 102 subjects (24%) underwent hematopoietic stem cell transplantation. The median sperm concentration at the time of storage was 54 [20-98] million/ml. Among the participants, 242 (57%) men underwent post-treatment sperm analysis, revealing azoospermia in 87 of them (36%, 95%CI: 30-42%). Of the remaining, only 93 (38%) exhibited normal semen parameters. A total of 188 out of the 424 men included (44%) expressed a desire for childbearing and seventy-one achieved a natural live birth (38%, 95%CI: 31-45%). Eighty-nine individuals used their cryopreserved sperm samples, constituting 21% (95%CI: 17-25%) of the entire cohort. Among these 89 men, 45 experienced at least one live birth (51%, 95%CI: 40-61%). Notably, thirty-three men, despite actively seeking pregnancy, did not utilize their frozen semen. In summary, out of the 188 men interested in fatherhood, a total of 114 achieved a live birth (61%, 95%CI: 54-67%). Limitations, reasons for caution Our study is constrained by the lack of detailed information on the illness status, comorbidities, and specific cancer therapies administered. Furthermore, the data concern a single-center population, potentially limiting the generalizability of the results. Wider implications of the findings Oncohematological disease often require systemic therapies or even hematopoietic stem cell transplantation resulting in a very high percentage of infertile patients These findings support the need to prioritize sperm preservation before therapies in these patients, even in cases who are not candidates for stem cell transplantation in the first instance Trial registration number NA

Ultrasound-assisted deep eutectic solvents extraction of polysaccharides from Loquat leaf: Process optimization and bioactivity study

Loquat leaves are the by-product of loquat fruit production. Polysaccharides are one of the main active ingredients in loquat leaves. In this study, polysaccharides were extracted from loquat leaves by ultrasonic-assisted deep eutectic solvents (DESs) extraction method. Further, the extracted crude loquat leaf polysaccharides (CLLP) were purified and separated via S-8 resin and DEAE-52 cellulose column chromatography, respectively. Additionally, the effects of polysaccharides on activity of sperm in boar semen preserved in medium at 17 °C, were evaluated preliminarily. DES, composed of choline chloride/ethylene glycol (1:6, molar ratio), was proved to be the suitable solvent for LLP extraction. The optimized extraction conditions were water content 44 %, liquid-solid ratio 1:29 (g/g), extraction temperature 61 °C and extraction time 98 min. Under these conditions, the LLP yield was 57.82 ± 1.50 mg/g. A homogeneous polysaccharide (LLP1-2, Mw: 2.17 × 104 Da) was isolated from CLLP. Its total sugar, uronic acid and protein contents were 76.31 ± 1.25 %, 14.19 ± 0.67 % and 3.28 ± 0.42 %, respectively. Further, 800 μg/mL LLP1-2 could effectively enhance the antioxidant activity of sperm. This study laid a foundation for DESs and column chromatography in the field of polysaccharide extraction and separation, proving that LLP can be used as a natural antioxidant for sperm preservation.

Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation

BackgroundAntimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage.ResultsWe found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05).ConclusionsThe protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

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.

Positive In Vitro Effect of ROCK Pathway Inhibitor Y-27632 on Qualitative Characteristics of Goat Sperm Stored at Low Temperatures.

Y-27632, as a cytoskeleton protector, is commonly used for low-temperature preservation of cells. Goat sperm are prone to damage to the cytoskeleton under low-temperature conditions, leading to a loss of sperm vitality. However, the Y-27632 small molecule has not yet been used in research on low-temperature preservation of goat semen. This study aims to address the issue of low temperature-induced loss of sperm motility in goats by using Y-27632, and explore the regulation of Y-27632 on goat sperm metabolism. At a low temperature of 4 °C, different concentrations of Y-27632 were added to the sperm diluent. The regulation of Y-27632 on the quality of low temperature-preserved goat semen was evaluated by detecting goat sperm motility, antioxidant capacity, mitochondrial activity, cholesterol levels, and metabolomics analysis. The results indicated that 20 µM Y-27632 significantly increased plasma membrane integrity (p < 0.05), and acrosome integrity (p < 0.05) and sperm motility (p < 0.05), increased levels of superoxide dismutase (SOD) and catalase (CAT) (p < 0.01), increased total antioxidant capacity (T-AOC) (p < 0.05), decreased levels of malondialdehyde (MDA) and reactive oxygen species (ROS) (p < 0.01), and significantly increased mitochondrial membrane potential (MMP). The levels of ATP, Ca2+, and TC in sperm increased (p < 0.01). Twenty metabolites with significant differences were identified, with six metabolic pathways having a significant impact, among which the D-glutamic acid and D-glutamine metabolic pathways had the most significant impact. The artificial insemination effect of goat semen treated with 20 μM Y-27632 was not significantly different from that of fresh semen. This study indicates that Y-27632 improves the quality of low-temperature preservation of sperm by protecting the sperm plasma membrane, enhancing sperm antioxidant capacity, regulating D-glutamine and D-glutamate metabolism, and promoting the application of low-temperature preservation of semen in artificial insemination technology.

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

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. 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. 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 (15-39years 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%). This study provides the firstcomprehensive national statistical census and review of fertility preservation in male cancer patients with respect totrends, 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.

Legal Regulation of Embryo and Sperm Preservation Centers in Jordanian Law

A new category of banks called embryo and sperm preservation facilities has emerged as a result of the considerable improvements that have been made in this era across many centers, particularly the medical field. Yet, this development raised several legal issues because the Jordanian government did not adequately regulate it. The agreement reached between the centers for the preservation of sperm and embryos is considered to be special, deriving its specificity from the place and the purpose that the patient desired, and that delivery is not a requirement of the agreement, but rather an obligation that falls on the shoulders of the preservation centers. Accordingly, we hope that the Jordanian legislator will establish a legal system for these centers by adding a provision within the Jordanian Medical Liability Law No. (25) of 2018.

Resveratrol and male infertility: a systematic review of the literature.

The aim of this study is to provide an updated and comprehensive systematic review on the effects of resveratrol (RSV) in male infertility and sperm preservation. This systematic review followed the MOOSE guidelines for Meta-analyses and Systematic Reviews of Observational Studies and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols (PRISMA-P). Only original articles evaluating the effect of RSV on human spermatozoa in vivo or in vitro were included. Of the 1806 abstracts evaluated for eligibility, only 12 studies were included in the qualitative synthesis, of which three were prospective in-vivo studies and nine were in-vitro studies. Out of three human studies on RSV, only two studies administered RSV alone, one of them reported a positive impact and the other reported no significant impact on semen parameters. Overall, the in-vitro studies have reported the ability of RSV to protect spermatozoa against damage due to freeze-thaw cycles during cryopreservation. Other in-vitro studies have reported positive effects of RSV in fresh samples and protective effects in cell lines. Available in-vivo studies are controversial with regard to the effect of RSV in improving semen parameters. In-vitro studies support the use of RSV before sperm cryopreservation. Further well-designed prospective studies on large sample sizes are needed to fully understand the role of RSV in the treatment of male infertility in vivo.

Phytochemical Screening, Antioxidant Effect and Sperm Quality of the Bomba ceiba Stamen Extracts on Charolais Cattle Sperm Induced by Ferrous Sulfate.

Orange Bombax ceiba (B. ceiba) is an indigenous plant, and its stamen is an important ingredient in traditional Lanna food. There are limitations in scientific reports on the effects of the biological activities of B. ceiba stamens on the male reproductive system. This study aims to investigate the phytochemical compounds of the orange B. ceiba stamen and its potential effect on the antioxidant properties and quality of cattle sperm treated with Fe. The orange BUE had the highest total phenolics, total tannins, total monomeric anthocyanins, and maximal antioxidant potential. The orange BAE had the highest concentration of total flavonoids. LC-QTOF/MS showed that the orange BUE contained the highest number of phytochemical compounds related to male reproductive enhancement. The orange BUE enhanced sperm motility, and both the orange BUE and the BAE enhanced sperm viability and normal sperm morphology via free radical scavenging. It might be suggested that B. ceiba stamens have benefits for sperm preservation, sperm quality, and increasing the economic value of local plants, and that they may be developed and used to guard against oxidative stress from cryodamage induced by frozen semen technology.