Xét nghiệm phân mảnh DNA tinh trùng và khuyến cáo trong thực hành lâm sàng

Tinh dịch đồ là xét nghiệm đầu tay đánh giá khả năng sinh sản nam giới, nhưng xét nghiệm này không thể phản ánh chính xác những biến đổi vật chất di truyền trong nhân tinh trùng, cũng như không thể tiên lượng được kết cục điều trị trong hỗ trợ sinh sản. Tính toàn vẹn DNA tinh trùng đóng vai trò quan trọng cho sự phát triển của phôi cũng như là dấu hiệu sinh học đại diện cho một tinh trùng khỏe mạnh. Do đó, các kỹ thuật xét nghiệm phân mảnh DNA tinh trùng ngày càng được thực hiện phổ biến. Hiện nay, một số kỹ thuật thường được sử dụng để đánh giá phân mảnh DNA tinh trùng bao gồm TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling), Comet (Single cell gel electrophore sis), SCD (Sperm chromatin dispersion) và SCSA (Sperm chromatin structure assay). Cho đến nay, vẫn chưa có khuyến cáo cụ thể cho chỉ định thực hiện xét nghiệm phân mảnh DNA tinh trùng. Bài tổng quan nhằm giới thiệu về các kỹ thuật xét nghiệm phân mảnh DNA tinh trùng cũng như tổng hợp khuyến cáo cho chỉ định thực hiện xét nghiệm này.

Morphological, Gene, and Hormonal Changes in Gonads and In-Creased Micrococcal Nuclease Accessibility of Sperm Chromatin Induced by Mercury

Mercury is one of the most dangerous environmental pollutants. In this work, we analysed the effects of exposure of Mytilus galloprovincialis to 1, 10 and 100 pM HgCl2 for 24 h on the gonadal morphology and on the expression level of three stress genes: mt10, hsp70 and πgst. In this tissue we also evaluated the level of steroidogenic enzymes 3β-HSD and 17β-HSD and the expression of PL protein genes. Finally, we determined difference in sperm chromatin accessibility to micrococcal nuclease. We found alterations in gonadal morphology especially after exposure to 10 and 100 pM HgCl2 and hypo-expression of the three stress genes, particularly for hsp70. Furthermore, decreased labelling with both 3β-HSD and 17β-HSD antibodies was observed following exposure to 1 and 10 pM HgCl2 and complete absence at 100 pM HgCl2 exposure. Gonads of mussels exposed to all HgCl2 doses showed decreased expression of PL protein genes especially for PLIII. Finally, micrococcal nuclease digestions showed that all doses of HgCl2 exposure resulted in increased sperm chromatin accessibility to this enzyme, indicative of improper sperm chromatin structure. All of these changes provide preliminary data of the potential toxicity of mercury on the reproductive health of this mussel.

Hmga2 deficiency is associated with allometric growth retardation, infertility, and behavioral abnormalities in mice

The high mobility group AT-hook 2 (HMGA2) protein works as an architectural regulator by binding AT-rich DNA sequences to induce conformational changes affecting transcription. Genomic deletions disrupting HMGA2 coding sequences and flanking non-coding sequences cause dwarfism in mice and rabbits. Here, CRISPR/Cas9 was used in mice to generate the Hmga2 null allele that specifically disrupts only the coding sequence. The loss of one or both alleles of Hmga2 resulted in reduced body size of 20% and 60%, respectively, compared to wild-type littermates as well as an allometric reduction in skull length in Hmga2-/- mice. Both male and female Hmga2-/- mice are infertile, whereas Hmga2+/- mice are fertile. Examination of reproductive tissues of Hmga2-/- males revealed a significantly reduced size of testis, epididymis, and seminal vesicle compared to controls, and 70% of knock-out males showed externalized penis, but no cryptorchidism was observed. Sperm analyses revealed severe oligospermia in mutant males and slightly decreased sperm viability, increased DNA damage but normal sperm chromatin compaction. Testis histology surprisingly revealed a normal seminiferous epithelium, despite the significant reduction in testis size. In addition, Hmga2-/- mice showed a significantly reduced exploratory behavior. In summary, the phenotypic effects in mouse using targeted mutagenesis confirmed that Hmga2 is affecting prenatal and postnatal growth regulation, male reproductive tissue development, and presents the first indication that Hmga2 function is required for normal mouse behavior. No specific effect, despite an allometric reduction, on craniofacial development was noted in contrast to previous reports of an altered craniofacial development in mice and rabbits carrying deletions of both coding and non-coding sequences at the 5’part of Hmga2.

In Vitro Studying the Effect of Adding Autologous Platelet Rich Plasma (PRP) to the Human Semen on the Sperm DNA Integrity

For conception and the development of healthy embryos, sperm DNA integrity is crucial. According to a growing body of studies, there is a strong correlation between sperm DNA damage and male infertility. Among the new medicines being developed in the medical field, the application of Platelet Rich Plasma (PRP) in human reproduction has yet to be examined. A total of 100 semen samples were used in the current experimental investigation. From November 2020 to June 2021, the research was conducted at the High Institute for Infertility Diagnosis and Assisted Reproductive Technologies. Masturbation was used to get an ejaculated semen sample. After semen analysis, the samples were separated into two equal parts, one without autologous PRP and the other with 2% autologous PRP, with the DNA fragmentation assessed using the Sperm Chromatin Dispersion Test. There was highly significant reduction in DNA fragmentation index (p < 0.001). The mean sperm DNA integrity was reduced after adding PRP (33.85±16.73 vs 38.55±16.64), Mean (± SE). PRP has been shown to improve human sperm DNA integrity.

Sperm chromatin condensation as an in vivo fertility biomarker in bulls: a flow cytometry approach

Background Genetic selection in cattle has been directed to increase milk production. This, coupled to the fact that the vast majority of bovine artificial inseminations (AI) are performed using cryopreserved sperm, have led to a reduction of fertility rates over the years. Thus, seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency. In humans, sperm chromatin condensation evaluated through chromomycin A3 (CMA3) has recently been purported to be a powerful biomarker for sperm functional status and male infertility. The objectives of the present study were: a) to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability, and b) to test whether this parameter could be used as a predictor of in vivo fertility in bulls. The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males. Reproductive outcomes of each sire were determined by non-return rates, which were used to classify bulls into two groups (highly fertile and subfertile). Results Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry. Sperm quality parameters (morphology, viability, total and progressive motility) were also assessed. Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility. Sperm morphology, viability and total motility presented an area under the ROC curve (AUC) of 0.54, 0.64 and 0.68, respectively (P > 0.05), and thus were not able to discriminate between fertile and subfertile individuals. Alternatively, while the percentage of progressively motile sperm showed a significant predictive value, with an AUC of 0.73 (P = 0.05), CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls. Specifically, the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility, with an AUC of 0.78 (P = 0.02). Conclusions Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility, with a potential ability to maximising the efficiency of dairy breeding industry.

Beneficial effects of hypotaurine supplementation in preparation and freezing media on human sperm cryo-capacitation and DNA quality

Background Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal (n = 19) or abnormal (n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with (“H+” arm) or without (“H-” arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus. Results Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p < 0.001), progressive and total motility (+ 39.9% and + 21.6% respectively, p < 0.005) than spermatozoa from the control “H-” arm. Hypotaurine also reduced the non-specific phosphorylation of the capacitation protein markers P110 and P80 (p < 0.01), indicating a decrease in cryo-capacitation. Hypotaurine supplementation reduced chromatin decondensation, measured by chromomycin A3 (− 16.1%, p < 0.05), DNA fragmentation (− 18.7%, p < 0.05) and nuclear vacuolization (− 20.8%, p < 0.05). Conclusion Our study is the first to demonstrate beneficial effects of hypotaurine supplementation in preparation and freezing procedures on human spermatozoa sperm fertilization capacity and nucleus quality. Hypotaurine supplementation limited cryo-capacitation, increased the proportion of live and progressively motile spermatozoa and reduces the percentage of spermatozoa showing chromatin decondensation, DNA fragmentation and nuclear vacuolation. Trial registration Clinical Trial, NCT04011813. Registered 19 May 2019 - Retrospectively registered.

Detrimental effects of cerium oxide nanoparticles on testis, sperm parameters quality, and in vitro fertilization in mice: An experimental study

Background: Cerium oxide nanoparticles (CeO2 NPs) as an important nanomaterial have a wide range of applications in many fields and human beings’ exposure to this nanomaterial is unavoidable. The effects of CeO2 NPs on the male reproductive system are controversial. Objective: To determine the effects of the administration of CeO2 NPs on the testis tissue, sperm parameters, and in vitro fertilization (IVF) in mice. Materials and Methods: Twenty-four male mice were divided into three groups (n = 8/each): one control and two experimental groups receiving CeO2 NPs at doses of 50 and 100 mg/kg body weight, respectively, for 35 days. At the end of the experiment, the diameter of seminiferous tubules (SNTs), epithelial height of SNTs, spermiogenesis index in testes, sperm parameters (count, motility, viability, and morphology), sperm chromatin condensation, DNA integrity, and IVF assays were analyzed. Results: Histological results showed that the tubular diameter, the epithelial height of the SNTs, and the spermiogenesis index were significantly decreased in the experimental groups receiving CeO2 NPs. All sperm parameters in the experimental groups were significantly reduced and, additionally, the percentages of immature sperms and sperms with DNA damage were significantly increased in groups treated with CeO2 NPs compared to the control. Furthermore, the rates of IVF and in vitro embryo development were decreased. Conclusion: Collectively, the current study showed that oral administration of CeO2 NPs in mice had detrimental effects on the male reproductive system through inducing testicular tissue alterations, decreasing sperm parameters quality, and also diminishing the IVF rate and in vitro embryonic development. Key words: Cerium oxide, Testis, Sperm, Fertilization, Mice.

Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and mediates chromatin hyper-condensation, together with PRM1. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation and the prevention of DNA damage. Yet, the role of cP2 is not yet understood. Using CRISPR-Cas9 mediated gene editing, we generated mice lacking the cP2 domain while the mP2 is still expressed. We show that deletion of one allele of the cP2 domain is sufficient to render male mice infertile. cP2 deficient sperm show incomplete PRM2 incorporation, retention of transition proteins and a severely altered protamine ratio. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation, inviability and immotility of mature sperm. The cP2 domain therefore seems to be necessary for the complex crosstalk leading to the successive and complete removal of transition proteins and complete protamination of sperm chromatin. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.

Seminal Plasma Protein N-Glycan Peaks Are Potential Predictors of Semen Pathology and Sperm Chromatin Maturity in Men

Background: Male infertility is increasingly becoming a health and demographic problem. While it may originate from congenital or acquired diseases, it can also result from environmental exposure. Hence, the complexity of involved molecular mechanisms often requires a multiparametric approach. This study aimed to associate semen parameters with sperm DNA fragmentation, chromatin maturity and seminal plasma protein N-glycosylation. Methods: The study was conducted with 166 participants, 20–55 y old, 82 normozoospermic and 84 with pathological diagnosis. Sperm was analyzed by Halosperm assay and aniline blue staining, while seminal plasma total protein N-glycans were analyzed by ultra-high-performance liquid chromatography. Results: Sperm DNA fragmentation was significantly increased in the pathological group and was inversely correlated with sperm motility and viability. Seminal plasma total protein N-glycans were chromatographically separated in 37 individual peaks. The pattern of seminal plasma N-glycan peaks (SPGP) showed that SPGP14 significantly differs between men with normal and pathological semen parameters (p < 0.001). The multivariate analysis showed that when sperm chromatin maturity increases by 10%, SPGP17 decreases by 14% while SPGP25 increases by 25%. Conclusion: DNA integrity and seminal plasma N-glycans are associated with pathological sperm parameters. Specific N-glycans are also associated with sperm chromatin maturity and have a potential in future fertility research and clinical diagnostics.