Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

BACKGROUND: Potassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated. METHODS: We performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy. RESULTS: We identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition. CONCLUSIONS: SLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.

[Role of full-time nurses in reception of sperm donors in the sperm bank].

OBJECTIVE: To explore the necessity of reception of sperm donors by full-time nurses in the sperm bank by analyzing the efficiency of sperm donation before and after staffing Guangdong Sperm Bank (GSB) with full-time nurses. METHODS: We selected 9 712 qualified sperm donors in GSB from January 1, 2016 to December 31, 2021 and compared the efficiency of sperm donation before and after staffing GSB with full-time nurses. RESULTS: After staffing GSB with full-time nurses, the proportion of qualified sperm donors screened from the quasi-qualified ones was dramatically increased from 66.2% (1 230/1 858) to 77.1% (3 252/4 218) (P < 0.01), that of HIV re-examinees after 6-month suspension from sperm donation increased from 84.6% (137/162) to 93.4% (599/641) (P < 0.01), and that of the candidate donors lost to follow-up during the screening period decreased from 23.2% (831/3 583) to 21.3% (1 308/6 129) (P = 0.034). CONCLUSION: After Guangdong Sperm Bank was staffed with full-time nurses for reception of sperm donors, the proportion of the candidate donors lost to follow-up was reduced and the efficiency of sperm donation was significantly improved. Individualized psychological intervention for the sperm donors by the nurses could dispel the worries of the donors and improve the compliance and efficiency of sperm donation.

[Causes of failure in autologous sperm preservation in the human sperm bank and countermeasures].

OBJECTIVE: To analyze the causes of failure in autologous sperm cryopreservation (ASCP) in the human sperm bank and propose some countermeasures to improve the success rate of ASCP and promote it in human sperm banks. METHODS: We retrospectively analyzed the reasons for and causes of failure in ASCP 1 156 males undergoing ASCP in the Human Sperm Bank of Guangdong Province from January 1, 2016 to December 31, 2019. RESULTS: Of the 1 156 cases of ASCP, 857 (74.1%) succeeded and 299 (25.9%) failed, with a failure rate of 56.1% (96/171) in the microdissection testicular sperm extraction (micro-TESE) group, 29.9% (67/224) in the reproduction insurance group, 21.2% (27/100) in the non-tumor disease group and 17.2% (109/525) in the tumor group, with statistically significant difference between the four groups (χ2 = 109.926, P < 0.01). The causes of failed ASCP included failure to extract semen (14.0% ï¼»42/299ï¼½), failure to meet the standard of sperm cryopreservation (67.6% ï¼»202/299ï¼½), giving up ASCP for low semen quality (7.4% ï¼»22/299ï¼½), and giving up ASCP for some other reasons (11.0% ï¼»33/299ï¼½), including worry about the reliability of cryopreservation technology (6.0% ï¼»18/299ï¼½), suspicion about the complexity of the ASCP procedures (3.0% ï¼»9/299ï¼½) and expectation for fertility recovery after chemotherapy withdrawal (2.0% ï¼»6/299ï¼½). CONCLUSIONS: In view of different causes of failure in ASCP, human sperm banks can provide individualized cryopreservation schemes, including guidance with masturbation or the use of sperm extraction instrument in sperm extraction, strengthening sperm preservation-related education and the awareness of reproduction protection and earlier sperm preservation among cancer patients, promoting the cryopreservation of microsamples of motile sperm by microsample or single sperm freezing, and development of testis tissue cryopreservation to preserve the fertility of children with cancer.

High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris.

A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75°C, while recombinant protein encoded by the "wild type" xylanase gene xyn2, also expressed in Pichia, was 20°C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80°C for 10min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22°C, respectively. The maximum xylanase activity of Mxyn2p was 13,000nkat/mg (9.88g/l) in fed-batch cultivation after 168h induction with methanol in a 50l bioreactor.

Beneficial effect of residential greenness on sperm quality and the role of air pollution: A multicenter population-based study.

BACKGROUND: Poor sperm quality is a major cause of male infertility. However, evidence remains scarce on how greenness affects male sperm quality. OBJECTIVES: To assess the associations of residential greenness with male sperm quality and the modification effect of air pollution exposure on the relationship. METHODS: A total of 78,742 samples from 33,184 sperm donors from 6 regions across China during 2014-2020 were included and analyzed. Individual residential greenness exposures of study subjects were estimated using the Normalized Difference Vegetation Index (NDVI) during the entire (0-90 lag days) and two key stages (0-37, and 34-77 lag days) of sperm development. Contemporaneous personal exposure levels to air pollutants were estimated using a spatio-temporal deep learning method. Linear mixed models were employed to assess the impact of greenspace in relation to sperm quality. The modification effect of air pollution on the greenspace-sperm quality relationship was also estimated. RESULTS: Per IQR increment in NDVI exposure throughout spermatogenesis were statistically associated with increasing sperm count by 0.0122 (95 % CI: 0.0007, 0.0237), progressive motility by 0.0162 (95 % CI: 0.0045, 0.0280), and total motility by 0.0147 (95 % CI: 0.0014, 0.0281), respectively. Similar results were observed when the model added air pollutants (PM1, PM2.5 or O3) for adjustment. Additionally, specific air pollutants, including PM1, PM2.5, and O3, were found to modify this association. Notably, the protective effects of greenness exposure were more pronounced at higher concentrations of PM1 and PM2.5 and lower concentrations of O3 (all Pinteraction < 0.05). Statistically significant positive effects of NDVI were observed on sperm motility in early spermatogenesis and sperm count in late spermatogenesis. CONCLUSIONS: Exposure to residential greenness may have beneficial effects on sperm quality and air pollution modifies their relationship. These findings highlight the importance of adopting adaptable urban greenspace planning and policies to safeguard male fertility against environmental factors.

A retrospective study on sperm cryopreservation in 1034 patients diagnosed with cancer in southern China.

Sperm cryopreservation is an effective fertility preservation method for cancer patients before anticancer treatments. However, there are little data on fertility preservation in large cohorts of patients with cancer in southern China. This retrospective cross-sectional study aimed to assess the fertility preservation status of 1034 newly diagnosed male patients with cancer in the Human Sperm Bank of Guangdong Province in southern China (Guangzhou, China). Of these, 302 patients had reproductive system tumors, mostly testicular cancers (99.0%), and 732 had other tumors, including lymphoma (33.1%), gastrointestinal cancer (16.3%), nasopharyngeal carcinoma (15.7%), leukemia (7.7%), sarcoma (3.6%), and others (23.6%). Patients with reproductive system tumors had lower sperm concentration and prefreezing and post-thawing progressive motility than those with non-reproductive system tumors (all P < 0.001). Differences in sperm concentration, progressive motility, and normal morphology rate were observed between patients with and without anticancer surgery before sperm cryopreservation (all P < 0.05). As of April 30, 2022, 63 patients used their cryopreserved sperm for assisted reproductive technology treatments and 39 pregnancies were achieved. This study provides valuable data on the fertility preservation status in newly diagnosed cancer patients in southern China, demonstrating that patients with reproductive system tumors had poor sperm quality for their pretreatment fertility preservation.

Evaluating an external quality assurance program for semen analysis in China during 2009-2020.

INTRODUCTION: Semen analysis (SA) plays a key role in guiding treatments of male reproductive diseases and infertility due to male factors; however, it remains challenging to conduct an accurate SA due to lack of standardization, highly subjective assessments, and problems with automated procedures. Therefore, quality assurance (QA) and teaching courses are essential for making the laboratory results more consistent. MATERIALS AND METHODS: The external quality assurance (EQA) scheme was organized by national human sperm bank technology training bases in Guangdong province in China between 2009 and 2020. Until 2020, 124 laboratories from China participated in the EQA program. The EQA scheme per year has been organized involving two semen aliquots for sperm concentration, two video recordings for motility, and two smears for sperm morphology. All samples used in the EQA scheme were obtained from different healthy donors or patients. RESULTS: We estimated that the median coefficient of variation (CV) of sperm concentration, ignoring the method used, was 26.6%. Using a 100 µm deep counting chamber led to a decreasing CV of 13.6%. For sperm motility, the median CV of nonprogressive motility was high (50.8%), but the CV of progressive motility (13.2%), immotile sperm (14.3%), and total motility (11.8%) were acceptable. The morphology assessment revealed large variability (44.4%) irrespective of the classification criteria. DISCUSSION: The reduction of interlaboratory variability is still a challenge during SA in China. Therefore, it is critical to increase awareness of joining EQA schemes and establish standardized training centers to follow WHO-recommended procedures toward Chinese standards.