Risks associated with cryopreserved semen in a human sperm bank during and after the COVID-19 pandemic.

RESEARCH QUESTION: What are the risks associated with cryopreserved semen collected during and after the coronavirus disease 2019 (COVID-19) pandemic wave in Wuhan, China? DESIGN: Retrospective cohort study involving young adult men who were qualified sperm donors at the Hunan Province Human Sperm Bank (China) during the pandemic wave (1 January 2020 to 30 January 2020) and after the wave and return to work (7 April 2020 to 30 May 30 2020). One hundred paired semen and blood specimens from 100 donors were included. One-step single-tube nested quantitative real-time polymerase chain reaction (OSN-qRT-PCR) was used to detect SARS-CoV-2. Moreover, to control the unacceptable risk of false-negative results, a second round of screening was performed with pooled RNA from negative semen samples using crystal digital PCR (cd-PCR). RESULTS: For individual blood and semen samples, the target genes, namely the nucleocapsid protein (N) and open reading frame (ORF-1ab) genes, tested negative in all of the 100 paired samples. Further, as per cd-PCR results, there were >20,000 droplets per well in the RNA for each combined sample and no positive droplets were present for either of the aforementioned target genes. A total of 100 paired semen and blood samples from these two groups tested negative for SARS-CoV-2. CONCLUSIONS: Cryopreserved semen at the Hunan Province Human Sperm Bank during and after the COVID-19 pandemic wave was free of SARS-CoV-2 and was judged safe for external use in the future.

Intra-Cytosplamic sperm injection outcomes with fresh and cryopreserved human epidydimal sperm from patients with obstructive azoospermia.

Techniques for the cryopreservation of epididymal sperm was are widely used in clinical practice. However, given the unique characteristics of sperm from patients with obstructive azoospermia, epididymal sperm cryopreservation is more difficult because of low count and weak motility; therefore, conventional methods of sperm cryopreservation may not result in the best outcomes. We used the micro-straw method to store small quantities of sperm obtained from patients with severe oligozoospermia or azoospermia and achieved successful deliveries in the previous study. This retrospective study of ICSI cycles included the first ICSI cycles of fresh or frozen/thawed epididymal sperm that were performed in patients suffering from obstructive azoospermia who were admitted to the CITIC-Xiangya Hospital of Reproduction and Genetics of China from June 1, 2015 to June 31, 2019. A total of 2441 patients with obstructive azoospermia were divided according to the use of fresh (n = 2342) or frozen/thawed (n = 99) epididymal sperm. The results showed that the fertilisation rate was higher with fresh epididymal sperm than that with frozen/thawed epididymal sperm (85.14% vs. 79.26%, respectively; p = 0.000). However, the rates of embryo cleavage, high-quality embryos, clinical pregnancy, miscarriage, singletons and birth defect were similar between fresh and frozen/thawed epididymal sperm (98.28% vs. 99.13%, 60.34% vs. 57.29%, 67.90% vs. 70.51%, 8.12% vs. 10.91%, 57.76% vs. 49.09%, 1.59% vs. 1.45%respectively; p = 0.088, 0.109, 0.628, 0.462,0.203 and 0.686). In addition, the short-term cryostorage of small quantities of epididymal sperm did not affect clinical outcomes. The results indicated that in cases of obstructive azoospermia, cryostorage of small quantities epididymal sperm is a reliable option.

Sperm donor lifestyle survey: modifiable risk factors for potential sperm donors.

OBJECTIVES: To examine the association between modifiable lifestyle factors and the main semen parameter values, the number of qualified sperm donors, and to provide some sensible guidance for sperm donors. METHODS: Healthy men screened as potential sperm donors were recruited in the Hunan Province Human Sperm Bank of China from March 2019 to December 2019. Participants were invited to complete interviewer-assisted questionnaires on eleven items of information. Univariate and multivariate analyses were conducted to analyze which lifestyle factors collected by the questionnaire had an impact on the eligibility and main semen parameters of sperm donors. RESULTS: The eligibility of men as sperm donors was strongly influenced by the duration of abstinence (P = 0.002). The rate of eligibility sperm donors increased significantly with the number of days of abstinence. In addition, semen volume increased with abstinence time (P = 0.000). Exercise frequency (P = 0.025) and abstinence time (P = 0.000) were positively correlated with sperm concentration, and masturbation frequency was negatively correlated with sperm concentration (P = 0.013). Progressive sperm motility was significantly affected by abstinence time (P = 0.000) and bedtime (P = 0.047). CONCLUSIONS: Abstinence time was highly associated with semen parameters and donor qualification. Increase the abstinence time before donation may be meaningful in improving the proportion of eligible sperm donors.

Long-term storage modifies the microRNA expression profile of cryopreserved human semen.

The global practice of cryopreservation of human semen is commonplace in Assisted Reproductive Technology (ART) labs and sperm banks. However, information on the effects of long-term cryopreservation on semen is limited to clinical data summaries and descriptions. For this study, we prepared 4 semen specimens of fresh semen, 4 specimens cryostored for at least 1 year, 3 specimens cryostored for at least 5 years, 4 specimens cryostored for at least 10 years, and 3 specimens cryostored for at least 15 years. Total RNA was extracted from each sample, amplified, labeled, and mapped to the known primary microRNA (miRNA) in the miRBase database, enabling the prediction of novel miRNAs. We found that cryopreservation can lead to changes in miRNA expression, and with the increase in storage time, these changes became more pronounced. Meanwhile, the expression of let-7d-3p, let-7c-5p and let-7i-3p miRNAs changed dynamically over cryostorage time in frozen-thawed human sperm. Furthermore, we analyzed the time-dependent dynamics of cryostorage-expressed miRNAs and their target mRNAs and found that half of the target genes were expressed in oocytes. These intersection genes were mainly enriched in cancer and cytoskeletal signaling pathways. Our findings showed that the miRNA expression profile of cryopreserved human semen is modified by long-term storage. Furthermore, as the storage time increases, the impact on human sperm becomes more pronounced in terms of miRNAs, which may have an effect on subsequent fertilization and embryonic development.

Global status of research on fertility preservation in male patients with cancer: A bibliometric and visual analysis.

Background: Recently, male fertility preservation before cancer treatment has become more prevalent. The research in this field has progressed over time, with some studies having a major impact and providing guidance for further research. However, the trends and hotspots of research on fertility preservation in male cancer patients may have changed; exploring them is essential for relevant research progress. Design: We extracted relevant studies from the Web of Science Core Collection database, capturing information on the countries of study, affiliations, authors, keywords, as well as co-citations of references and journals. To identify publication trends, research strengths, key subjects, prominent topics, and emerging areas, we conducted a bibliometric analysis using CiteSpace. Results: We included 3201 articles on fertility preservation in male cancer patients published over January 1999 to December 2023 were included. Although the relevant research growth rate was slow initially, the number of publications increased annually. Of all study countries, the United States, Germany, and Japan reported the earliest studies; the United States published the highest number of relevant studies. The US institutions remained at the forefront for all 25 years, and the US researcher Ashok Agarwal published the most articles. Literature co-citation analyses indicated a transformation in the study participants; they comprised a younger demographic (i.e., a large number of adolescent male patients underwent fertility preservation); moreover, fertility preservation techniques evolved from sperm cryopreservation to testicular tissue cryopreservation. Research on reproductive outcomes of sperm cryopreservation was the recent hotspot in male fertility preservation research, and the impact of immunotherapy and checkpoint inhibitors on male fertility requires further research. Conclusions: Male fertility preservation will be a major future research focus, with closer connections and collaborations between countries and organizations. Our results present the historical data on the development of research on male fertility preservation in cancer patients, providing relevant insights for future research and development in this study area.

Exploring the impact of pericentric inversion of chromosome 9 on fertility in sperm donors.

Pericentric inversion of chromosome 9 (inv[9]) is a common chromosomal structural variant, but its impact on clinical outcomes remains debated. The screening criteria of sperm banks are rarely mentioned to individuals with inv(9). In this study, we evaluated the fertility of sperm donors with inv(9) who met eligibility criteria for sperm banks (inv[9]-eligible donors). From March 2004 to May 2022, chromosomal analysis of 16 124 sperm donors at CITIC-Xiangya Human Sperm Bank in Hunan Province (Changsha, China) found that 251 (1.6%) had chromosome variations, with inv(9) being the most prevalent at 1.1%. All 169 inv(9)-eligible donors were contacted to collect fertility outcome data, along with 206 eligible donors without inv(9) as controls. In addition, semen samples from inv(9)-eligible donors and eligible donors underwent assessments of sperm fluorescence in situ hybridization (FISH), mitochondrial membrane potential, DNA fragmentation index, acrosome integrity, reactive oxygen species (ROS), and sperm morphology. Results showed that inv(9) did not significantly increase reproductive risks overall. Despite detecting ROS level differences, the clinical impact may be insignificant. This study provides new data on the inv(9) population that can serve as a valuable reference for decision-making by sperm banks as well as for genetic counseling and clinical guidance for individuals carrying inv(9) variant.

Comparison of Clinical Outcomes, Risks, and Costs for 20,910 Donor In Vitro Fertilization and 16,850 Donor Artificial Insemination Treatment Cycles: A Retrospective Analysis in China.

PURPOSE: To evaluate the effectiveness of donor in vitro fertilization (IVF-D) and donor artificial insemination (AI-D) in clinical outcomes, risks, and costs. METHODS: This study analyzed the cycle changes and clinical outcomes in 20,910 IVF-D and 16,850 AI-D cycles between 2013 and 2021 in the Reproductive and Genetic Hospital of CITIC-Xiangya. A cost-effectiveness analysis was performed to evaluate the costs per couple and per live birth cycle in the two treatment groups. RESULTS: IVF-D had higher pregnancy and live birth rates than AI-D (p < 0.001). The cumulative pregnancy and live birth rates for three AI-D cycles were 41.01% and 32.42%, respectively, higher than the rates for one or two AI-D cycles. The multiple birth and birth defect rate of AI-D was lower than that of IVF-D significantly. IVF-D mean cost per couple was higher than that of AI-D (CNY32,575 vs. CNY11,062, p < 0.001), with a mean cost difference of CNY21,513 (95% confidence interval, CNY20,517-22,508). The mean costs per live birth cycle for IVF-D and AI-D were CNY49,411 and CNY31,246, respectively. CONCLUSION: AI-D is more cost-effective and poses a lower risk for infertility couples than IVF-D, and patients should undergo three AI-D cycles to obtain the highest success rate.

Micro-straw: An efficient cryopreservation carrier for rare human spermatozoa.

BACKGROUND: Many cryopreservation carriers have been introduced to freeze rare human spermatozoa, however, these carriers relative attributes and comparative effectivenesses have not yet been systematically studied. OBJECTIVES: Is the micro-straw cryopreservation carrier more effective for cryopreserving rare human spermatozoa compared with the Cryoplus and a new micro-straw (LSL straw) carriers? MATERIALS AND METHODS: This study involves 93 samples from healthy sperm donors and 40 samples from patients diagnosed with oligospermia, asthenospermia, oligoasthenospermia, or obstructive azoospermia. We determined the optimal freeze-thaw protocol for the Micro-straw carrier. The post-thaw survival rate, normal sperm morphology, acrosome integrity, and DNA fragmentation for Micro-straw, Cryoplus, and LSL carriers were then determined. Finally, we verified the effects of freezing using these carriers by comparing the qualities of post-thaw spermatozoa from patients. RESULTS: The highest total motility (TM) and progressive motility (PR) survival rates were obtained by placing the Micro-straw at 1 cm above the LN2 surface for 70 s during freezing and in a 42°C water bath for 40 s during thawing. No differences were observed in the PR survival rate, acrosome integrity, and DNA fragmentation of the post-thaw spermatozoa from the three carriers. However, the normal morphology rate of spermatozoa frozen using the Micro-straw carrier was higher than for the Cryoplus carrier (p < 0.05), and the TM survival rate of spermatozoa frozen with the Micro-straw was higher than that for the LSL carrier (p < 0.01). In verification tests, there were no significant differences in the quality of post-thaw spermatozoa cryopreserved using these carriers for both rare spermatozoa or epididymal sperm. DISCUSSION AND CONCLUSION: Micro-straw, Cryoplus, and LSL carriers are all efficient means of freezing rare human spermatozoa. However, the Micro-straw carrier is more economical, safe, and user-friendly.

Influence of sperm morphology on pregnancy outcome and offspring in in vitro fertilization and intracytoplasmic sperm injection: a matched case-control study.

Sperm morphology was once believed as one of the most predictive indicators of pregnancy outcome in assisted reproductive technology (ART). However, the impact of teratozoospermia on in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcomes and its offspring remains inconclusive. In order to evaluate the influence of teratozoospermia on pregnancy outcome and newborn status after IVF and ICSI, a retrospective study was conducted. This was a matched case-control study that included 2202 IVF cycles and 2574 ICSI cycles and was conducted at the Reproductive and Genetic Hospital of CITIC-Xiangya in Changsha, China, from June 2013 to June 2018. Patients were divided into two groups based on sperm morphology: teratozoospermia and normal sperm group. The pregnancy outcome and newborn outcome were analyzed. The results indicated that couples with teratozoospermia had a significantly lower optimal embryo rate compared to those with normal sperm morphology in IVF (P = 0.007), while there were no statistically significant differences between the two groups in terms of the fertilization rate, cleavage rate, implantation rate, and pregnancy rate (all P > 0.05). Additionally, teratozoospermia was associated with lower infant birth weight in multiple births after IVF. With regard to ICSI, there was no significant difference in both pregnancy outcome and newborn outcome between the teratozoospermia and normal groups (both P > 0.05). Furthermore, no increase in the risk of birth defects occurred in the teratozoospermia group after IVF/ICSI. Consequently, we believe that teratozoospermia has limited predictive value for pregnancy outcomes in IVF/ICSI, and has little impact on the resulting offspring if multiple pregnancy is avoided.

MKK7-mediated phosphorylation of JNKs regulates the proliferation and apoptosis of human spermatogonial stem cells.

BACKGROUND: Human spermatogonial stem cells (SSCs) are the basis of spermatogenesis. However, little is known about the developmental regulatory mechanisms of SSC due to sample origin and species differences. AIM: To investigates the mechanisms involved in the proliferation of human SSC. METHODS: The expression of mitogen-activated protein kinase kinase 7 (MKK7) in human testis was identified using immunohistochemistry and western blotting (WB). MKK7 was knocked down using small interfering RNA, and cell proliferation and apoptosis were detected by WB, EdU, cell counting kit-8 and fluorescence-activated cell sorting. After bioinformatic analysis, the interaction of MKK7 with c-Jun N-terminal kinases ( JNKs ) was verified by protein co-immunoprecipitation and WB. The phosphorylation of JNKs was inhibited by SP600125, and the phenotypic changes were detected by WB, cell counting kit-8 and fluorescence-activated cell sorting. RESULTS: MKK7 is mainly expressed in human SSCs, and MKK7 knockdown inhibits SSC proliferation and promotes their apoptosis. MKK7 mediated the phosphorylation of JNKs, and after inhibiting the phosphorylation of JNKs, the phenotypic changes of the cells were similar to those after MKK7 downregulation. The expression of MKK7 was significantly downregulated in patients with abnormal spermatogenesis, suggesting that abnormal MKK7 may be associated with spermatogenesis impairment. CONCLUSION: MKK7 regulates the proliferation and apoptosis of human SSC by mediating the phosphorylation of JNKs.

Novel micro-straw for freezing small quantities of human spermatozoa.

OBJECTIVE: To evaluate a novel micro-straw as an efficient, simple method for freezing a small number of human spermatozoa for intracytoplasmic sperm injection (ICSI). DESIGN: Prospective cohort study. SETTING: Sperm bank. PATIENT(S): Men with severe oligozoospermia or azoospermia undergoing a total of 143 ICSI cycles at the CITIC-Xiangya Hospital of Reproduction and Genetics from June 1, 2015, to June 31, 2019, and 20 donors at the Hunan Province Human Sperm Bank from 2001 to 2016. INTERVENTION(S): Analysis of sperm samples and clinical outcomes after sperm use. MAIN OUTCOME MEASURE(S): Clinical information, including number of motile sperm before and after freezing, freeze-thaw survival rates, two-pronuclear fertilization rates, clinical pregnancy, and early pregnancy loss rates after sperm use. RESULT(S): In the feasibility experiment using the micro-straw, we found a freeze-thaw survival rate of 73% ± 8.3% and no difference in normal sperm morphology, normal acrosome integrity, or DNA fragmentation index between the micro-straw and 1.8-mL cryotubes. The prospective cohort included 1,325 cases, and we collected sperm from testicular, epididymis, and ejaculation sources. We observed motile sperm in 1,294 (97.6%) of 1,325 frozen-thawed samples. Postthaw sperm were available for ICSI in 140 (97.9%) of 143 of cycles. The fertilization, cleavage, and high-quality embryo rates were 1,007 (81.7%) of 1,233; 995 (98.8%) of 1,007; and 537 (53.9%) of 995, respectively. Sixty-nine (49%) clinical pregnancies were achieved, and the miscarriage rate was 6 (8.6%) of 69. CONCLUSION(S): The micro-straw is suitable and clinically useful for the cryopreservation of small numbers of spermatozoa.