Conventional semen parameters have long been considered fundamental in male fertility analyses. However, doubts have been raised regarding the clinical utility of the assessment of spermatozoa (sperm) DNA damage. In this retrospective study, we investigated the potential correlation between conventional semen parameters and semen DNA fragmentation (SDF) assessed as sperm DNA damage, in 11,339 semen samples collected between January 2019 and June 2022. We observed significant negative correlations between the DNA fragmentation index (DFI) and sperm viability (correlation coefficient [r] = −0.514) as well as progressive sperm motility (r = −0.512, p < 0.05). Samples were categorized into three groups according to DFI levels (Groups A, B, and C: ≤15%, 15 < DFI ≤30%, and >30%, respectively). Furthermore, the percentage of semen samples with normal sperm conventional parameters in Groups A, B, and C was 76.7% (4369/5697), 61.4% (2351/3827), and 39.7% (721/1815), respectively. Moreover, according to the reference values of conventional sperm parameters, the samples were divided into Groups F, G, and H with all normal, only one abnormal, and > two abnormal parameters, respectively. In addition, the proportions of samples with abnormal DFI values (>30) in Groups F, G, and H were 9.7% (721/7441), 23.1% (618/2676), and 39.0% (476/1222), respectively. Multivariate logistic regression models demonstrated that sperm vitality, progressive sperm motility, normal sperm form, total sperm count, semen volume, age, and some sperm kinematics collectively improved the area under the receiver operating characteristic curve (AUROC) to 0.861, surpassing the predictive value of a single predictor of pathologically damaged sperm DNA. Our study suggests that samples with abnormal sperm parameters may have a higher likelihood of high DNA fragmentation. Furthermore, certain semen parameters could be potential indicators of sperm DNA fragmentation, aiding sperm selection in assisted reproductive procedures.
Male infertility has become an important issue of global concern. Semen analysis is the cornerstone of male fertility assessment. External quality assessment (EQA) of sperm concentration, motility, and morphology is widely recognized in the world. However, over the past 34 years, the implementation of EQA for semen analysis has varied across different countries, and there is no global consensus. The goal of this paper is to first explore the overall development of EQA during this period. Secondly, it aims to discuss the extent of difference of participating laboratories in different countries. Finally, the paper examines the differences in EQA programs developed by various EQA providers in order to seek a global standard. In total, 29 papers met the inclusion criteria and were included in this review. There is inconsistent in the implementation of EQA across different countries, and there is no global consensus. Policies for EQA of semen analysis vary from country to country. Some countries mandate laboratory participation, while others permit voluntary involvement. Different EQA organizers choose different ways to calculate assigned value and acceptance limits. The coefficient of variation (CV) for each EQA item was large. The CVs of concentration, motility, morphology, and viability were 12.7-138.0 %, 17.0-127.0 %, 7-375 %, and 6-41.1 %, respectively. The results of the semen analysis varied considerably among the participating laboratories. The collaborative efforts of national policymakers, EQA organizers, and all participating laboratories are essential to improving the current situation.
Preservation of human spermatozoa in vitro at normothermia or hypothermia maintaining their functions and fertility for several days plays a significant role in reproductive biology and medicine. However, it is well known that human spermatozoa left in vitro deteriorate over time irreversibly as the consequence of various stresses such as the change of osmolarity, energy deficiency, and oxidative damage, leading to substantial limitations including the need for semen examinations, fertility preservation, and assisted reproductive technology. These problems may be addressed with the aid of non-freezing storage techniques. The main and most effective preservation strategies are the partial or total replacement of seminal plasma with culture medium, named as extenders, and temperature-induced metabolic restriction. Semen extenders consist of buffers, osmolytes, and antioxidants, etc. to protect spermatozoa against the above-mentioned adverse factors. Extended preservation of human spermatozoa in vitro has a negative effect on sperm parameters, whereas its effect on ART outcomes remains inconsistent. The storage duration, temperature, and pre-treatment of semen should be determined according to the aims of preservation. Advanced techniques such as nanotechnology and omics have been introduced and show great potential in the lifespan extension of human sperm. It is certain that more patients will benefit from it in the near future. This review provided an overview of the current knowledge and prospects of prolonged non-freezing storage of human sperm in vitro.
High temperature induces structural and physiological damage to plants.However, studies on the effects of constant high temperature on climbing plant species are limited.To estimate the response of photosynthetic capacity of two creeper species, Parthenocissus tricuspidata (Sieb.et Zucc.) and Parthenocissus quinquefolia (L.) Planch, to constant high-temperature treatment at noon, we measured photosynthetic pigments, gas exchange, and chlorophyll fluorescence parameters at 35, 40, and 45 °C (25 °C was the control treatment).High temperature significantly reduced photosynthetic pigment content, whereas carotenoid content showed the opposite trend.Net photosynthetic rate, stomatal conductance, transpiration rate, maximal quantum yield of PSII photochemistry, actual quantum yield of PSII photochemistry, and the coefficient of photochemical quenching all showed a decreasing trend, with increasing stress duration, whereas the non-regulated thermal energy loss and regulated thermal energy loss indexes increased.As temperature increased, intercellular CO 2 concentration initially decreased and then increased.Non-stomatal restriction factors were the main cause of the decrease in photosynthetic rate when temperature exceeded 40 °C.These parameters recovered to pre-stress levels only in plants grown at 35 °C upon stress relief.P. quinquefolia showed higher photosynthetic heat resistance and resilience than P. tricuspidata.Our results revealed photosynthetic adaptation and recovery mechanisms in two creepers grown under high-temperature stress.Molecular and genetic approaches should be considered to gain deeper insight into the mechanism underlying high temperature adaptation in these two creepers.
Plant growth and microbial change in iron mine tailings were detected under controlled conditions in laboratory. Three crops, soybean, corn and grain sorghum, were used in the experiment for testing the adaptation of three crops on iron mine tailings. Cow manure vermicompost (25%, V:V) was added into iron mine tailings for improve the poor fertility of mine tailings. The results showed that corn and grain sorghum germinated faster than soybean in iron mine tailings. It took 3 days for corn and grain sorghum germination and 7 days for soybean to 80% germination. There were different in shoot weight of seedlings of three crops. For the CK(iron mine tailings without vermicompost) and V(iron mine tailing with vermicompost), the shoot weight of soybean seedling, both fresh weight and dry weight, was the most one, and that of grain sorghum was the least one. So did the root weight. The microbial populations of three cultural microbes, bacteria, fungi and actinomyces, in rhizosphere of three crops were significant difference. The population of bacteria and actinomyces in corn rhizosphere were the most in three crops and those of soybean were the least one. The fungi population in soybean rhizosphere was the least one which was benefit for plant resistance to pathogen. It was conclude that soybean and corn were the better plants for the revegetation and agricultural utilization of iron mine tailing. The plant growth improved microbial environment in iron mine tailings.
The expression of exogenous DNA in Sertoli cells is essential for studying its functional genomics, pathway analysis, and medical applications. Electroporation is a valuable tool for nucleic acid delivery, even in primarily cultured cells, which are considered difficult to transfect. In this study, we developed an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation under different conditions. After transfection of plasmid into Sertoli cells, enhanced green fluorescent protein (EGFP) expression could be easily detected by fluorescent microscopy, and cell survival was evaluated by dye exclusion assay using Trypan blue. In terms of both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping the voltage constant (250 V), relatively high cell survival (76.5% ± 3.4%) and transfection efficiency (30.6% ± 5.6%) were observed with a pulse length of 20 μm. The number of pulses significantly affected cell survival and EGFP expression (P < 0.001). Cell survival clearly decreased following one to three pulses, from 83.9% ± 6.1% to 3.2% ± 1.1%, with EGFP expression increasing from 41.8% ± 9.4% to 66.7% ± 5.2%. The yield of positive cells increased with increasing concentration of plasmid DNA (range, 10–50 μg/ml), from 14.0% ± 2.8% to 35.0% ± 6.3%, but cell viability steadily decreased following 20 μg/ml plasmid DNA, from 73.1% ± 4.9% to 57.0% ± 6.6%. Compared with two popular cationic lipid transfection methods, the transfection efficiency of electroporation (21.5% ± 5.7%) was significantly higher than those of Lipofectamine 2000 (2.9% ± 1.0%) and Effectene (1.9% ± 0.8%) in this experiment (P < 0.001). We describe the process of optimizing electroporation conditions, and the successful electroporation of plasmid DNA into primarily cultured Sertoli cells. Our results indicate that the method of electroporation is more suitable than other approaches for the transfection of Sertoli cells.
Abstract Steroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ 4 structure, such as testosterone, androstenedione and progesterone, could be catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. Abnormal activities of SRD5As will lead to benign prostatic hyperplasia, alopecia, prostatic cancer or infertility due to the poor quality of sperms. However, the detailed reduction mechanisms of SRD5As remain elusive. Here we report the crystal structure of PbSRD5A, which shares 60.6% and 51.5% sequence similarities with human SRD5A1 and −2 respectively, from Proteobacteria bacterium in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic cavity for steroids substrates binding, whereas TM5-7 coordinate with cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and −2, together with extensive biochemical characterizations, for the first time unveiled the substrate recognition of SRD5As and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ 4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible. One Sentence Summary Structural and biochemical characterizations decipher the evolutionarily conserved mechanism in steroid 5α-reductases catalyzing NADPH mediated steroids reduction.
Abstract Background As one of the most common congenital abnormalities in male births, cryptorchidism has been found to have a polygenic etiology according to previous studies of common variants. However, little is known about genetic predisposition of rare variants for cryptorchidism, since rare variants have larger effective size on diseases than common variants. Methods In this study, a cohort of 115 Chinese probands with cryptorchidism was analyzed using whole-genome sequencing (WGS), alongside 19 parental controls and 2136 unaffected men. Additionally, CRISPR-Cas9 editing of a conserved variant was performed in a mouse model, with MRI screening utilized to observe the phenotype. Results In 30 of 115 patients (26.1%), we identified four novel genes ( ARSH , DMD , MAGEA4 , and SHROOM2 ) affecting at least five unrelated patients and four known genes ( USP9Y , UBA1 , BCORL1 , and KDM6A ) with the candidate rare pathogenic variants affecting at least two cases. Burden tests of rare variants revealed the genome-wide significances for newly identified genes ( p < 2.5×10 -6 ) under the Bonferroni correction. Surprisingly, novel and known genes were mainly from X chromosome (seven on X and one on Y) and all rare X-chromosomal segregating variants exhibited a maternal inheritance rather than de novo origin. CRISPR-Cas9 mouse modeling of a splice donor loss variant in DMD (NC_000023.11:g.32454661C>G), which resides in a conserved site across vertebrates, replicated bilateral cryptorchidism phenotypes, confirmed by Magnetic resonance imaging (MRI) at 4 and 10 weeks. Conclusion Our results revealed the role of the DMD gene mutation in causing cryptorchidism. The results also suggest that maternal-X inheritance of pathogenic defects could have a predominant role in the development of cryptorchidism.
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