Application of single-cell RNA sequencing on human testicular samples: a comprehensive review.

So far there has been no comprehensive review using systematic literature search strategies to show the application of single-cell RNA sequencing (scRNA-seq) in the human testis of the whole life cycle (from embryos to aging males). Here, we summarized the application of scRNA-seq analyses on various human testicular biological samples. A systematic search was conducted in PubMed and Gene Expression Omnibus (GEO), focusing on English researches published after 2009. Articles related to GEO data-series were also retrieved in PubMed or BioRxiv. 81 full-length studies were finally included in the review. ScRNA-seq has been widely used on different human testicular samples with various library strategies, and new cell subtypes such as State 0 spermatogonial stem cells (SSC) and stage_a/b/c Sertoli cells (SC) were identified. For the development of normal testes, scRNA-seq-based evidence showed dynamic transcriptional changes of both germ cells and somatic cells from embryos to adults. And dysregulated metabolic signaling or hedgehog signaling were revealed by scRNA-seq in aged SC or Leydig cells (LC), respectively. For infertile males, scRNA-seq studies revealed profound changes of testes, such as the increased proportion of immature SC/LC of Klinefelter syndrome, the somatic immaturity and altered germline autophagy of patients with non-obstructive azoospermia, and the repressed differentiation of SSC in trans-females receiving testosterone inhibition therapy. Besides, the re-analyzing of public scRNA-seq data made further discoveries such as the potential vulnerability of testicular SARS-CoV-2 infection, and both evolutionary conservatism and divergence among species. ScRNA-seq analyses would unveil mechanisms of testes' development and changes so as to help developing novel treatments for male infertility.

Cholesterol-Amino-Phosphate (CAP) Derived Lipid Nanoparticles for Delivery of Self-Amplifying RNA and Restoration of Spermatogenesis in Infertile Mice.

Male infertility caused by genetic mutations is an important type of infertility. Currently, there is no reliable method in the clinic to address this medical need. The emergence of mRNA therapy provides a possible strategy for restoring mutant genes in the reproductive system. However, effective delivery of mRNA to spermatocytes remains a formidable challenge. Here a series of cholesterol-amino-phosphate (CAP) lipids are reported by integrating three bioactive moieties into a geometric structure, which is favorable for mRNA delivery. The results demonstrate that CAP-derived lipid nanoparticles (CAP LNPs) can deliver RNA including traditional mRNA and self-amplifying RNA (saRNA) encoding DNA Meiotic Recombinase 1 (Dmc1) protein in spermatocytes and treat male infertility caused by the Dmc1 gene mutation. Notably, the delivery efficiency of CAP LNPs is significantly higher than that of the MC3 and ALC-0315 LNPs, which is consistent with the design of CAP molecules. More importantly, a single injection of CAP LNPs-saRNA can produce Dmc1 protein for an extended period, which restores the spermatogenesis in the Dmc1 gene knockout mouse model. Overall, this study proves the concept of LNPs for the delivery of mRNA to spermatocytes, which provides a unique method to probe male infertility caused by the genetic mutation.

SARS-CoV-2 and male infertility: from short- to long-term impacts.

PURPOSE: The coronavirus 2019 (COVID-19) pandemic-caused by a new type of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-has posed severe impacts on public health worldwide and has resulted in a total of > 6 million deaths. Notably, male patients developed more complications and had mortality rates ~ 77% higher than those of female patients. The extensive expression of the SARS-CoV-2 receptor and related proteins in the male reproductive tract and the association of serum testosterone levels with viral entry and infection have brought attention to COVID-19's effects on male fertility. METHODS: The peer-reviewed articles and reviews were obtained by searching for the keywords SARS-CoV-2, COVID-19, endocrine, spermatogenesis, epididymis, prostate, and vaccine in the databases of PubMed, Web of Science and Google Scholar from 2020-2022. RESULTS: This review summarizes the effects of COVID-19 on the male reproductive system and investigates the impact of various types of SARS-CoV-2 vaccines on male reproductive health. We also present the underlying mechanisms by which SARS-CoV-2 affects male reproduction and discuss the potentially harmful effects of asymptomatic infections, as well as the long-term impact of COVID-19 on male reproductive health. CONCLUSION: COVID-19 disrupted the HPG axis, which had negative impacts on spermatogenesis and the epididymis, albeit further investigations need to be performed. The development of vaccines against various SARS-CoV-2 variations is important to lower infection rates and long-term COVID risks.

Multiomics analysis of male infertility†.

Infertility affects 8-12% of couples globally, and the male factor is a primary cause in ~50% of couples. Male infertility is a multifactorial reproductive disorder, which can be caused by paracrine and autocrine factors, hormones, genes, and epigenetic changes. Recent studies in rodents and most notably in humans using multiomics approach have yielded important insights into understanding the biology of spermatogenesis. Nonetheless, the etiology and pathogenesis of male infertility are still largely unknown. In this review, we summarized and critically evaluated findings based on the use of advanced technologies to compare normal and obstructive azoospermic versus nonobstructive azoospermic men, including whole-genome bisulfite sequencing, single-cell RNA-seq, whole-exome sequencing, and transposase-accessible chromatin using sequencing. It is obvious that the multiomics approach is the method of choice for basic research and clinical studies including clinical diagnosis of male infertility.

Changes in Spermatogenesis, Lipoperoxidation Processes, and Antioxidant Protection in Men with Pathozoospermia after COVID-19. The Effectiveness of Correction with a Promising Antioxidant Complex.

The indicators of spermatogenesis and the state of LPO and antioxidant protection in men with pathozoospermia after COVID-19 were assessed before and after treatment an antioxidant complex. Blood plasma served as the material for biochemical studies. In the examined patients, the parameters of spermatogenesis, as well as blood concentration of LPO components (diene conjugates and TBA-reactive substances) were analyzed. The total antioxidant activity of the blood was determined as an indicator characterizing the total activity of LPO inhibitors and determining its buffer capacity. In patients recovered from COVID-19, an increase in spermatogenesis disorders and shifts towards the predominance of prooxidant factors were observed. After a course (1 month) of antioxidant complex, patients showed increased sperm motility, decreased leukocyte count in the ejaculate, and restored balance in the prooxidant-antioxidant system towards antioxidant components. The effectiveness of correction of post-COVID disorders largely depends on the degree of damage to the structure and function of cell membranes caused by oxidative stress. The use of the antioxidant complex is a promising option, because it reduces the level of LPO, enhances antioxidant protection of the body, and also normalizes some parameters of spermatogenesis.

COVID-19 and varicocele: The possible overlap factors and the common therapeutic approaches.

Varicocele is recognized as one of the main attributable causes of male infertility which can affect spermatogenesis by various pathophysiological mechanisms. Recent studies have identified oxidative stress and reduction in antioxidant, hyperthermia, hypoxia, hormonal dysfunction, and inflammatory conditions as major factors in the pathophysiology of varicocele, all of which have known direct associations with the coronavirus disease 2019 (COVID-19) and can significantly increase the risk of detrimental COVID-19-related outcomes. Emerging data have shown an association between COVID-19 and inflammation, overproduction of cytokine, and other pathophysiological processes. The present review, summarizes the current understanding of the pathophysiology of varicocele and investigates the potential correlation between the severity of COVID-19 and the varicocele disease. In addition, various possible treatments which can be effective in both diseases were examined. Despite numerous challenges associated with the prevalence of COVID-19 in healthcare systems in infected countries, special attention should be given to maintaining a high level of care for complex patients with a pre-existing disease such as varicocele and providing appropriate practical advice for optimal control of the COVID-19 disease.

Perspectives into the possible effects of the B.1.1.7 variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on spermatogenesis.

B.1.1.7 is a recently discovered variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) associated with increased transmissibility. Recent findings indicate that this variant has a propensity to infect adolescents and children at higher rates than adults. The virus gains entry into various body cells utilizing angiotensin-converting enzyme 2 (ACE-2) and basigin (CD147) as receptors. The virus mainly affects type II pneumocytes of lungs, endothelial cells, enterocytes, and renal tubular cells. It is reported to affect testes, causing testicular pain, and producing histopathological changes, as observed in some autopsies. The B.1.1.7 variant can also affect various cells in the testes. This raises a major concern regarding the long-term effects of the viral infection on spermatogenesis and highlights the pressing need for a robust database of serum samples from infected male children.

[Impact of SARS-CoV-2 on fertility, gametes' quality and Assisted Reproduction Technology]. / Impact du SARS-CoV-2 sur la fertilité, les gamètes et l'Assistance médicale à la procréation.

The current pandemic context raises questions about COVID-19 consequences on Assisted Reproduction Technology (ART). Indeed, according to the first Biomedicine Agency recommendations, ART centers suspended their activities in March 2020 during the first wave of Covid-19. However, SARS-CoV-2 direct and indirect effects on gametes, fertility, pregnancy and neonatal health are still debated. The aim of this review is to assess the available data on this subject, to inform patients in care and adapt daily practice. Most recent studies are based on the effects of the infectious syndrome, on hormonal factors as well as on the expression of viral entry proteins (ACE2 and TMPRSS2) in cells involved in gametogenesis, to assess the impact of COVID-19. So far, no effect on female gametes was highlighted. More studies are needed to confirm this hypothesis. Mother to children transmission couldn't be proven, yet neonatal infection remains possible. However, men are more susceptible to be infected by SARS-CoV-2, to be symptomatic, and spermatogenesis is likely to be affected. Presence of the virus in semen is infrequently reported, but all of these parameters should be taken into account in ART.

Impact of the SARS-CoV-2 virus on male reproductive health.

The coronavirus 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 160 million infections and 3.5 million deaths globally. Men are disproportionately affected by COVID-19, having more severe disease with higher mortality rates than women. Androgens have been implicated as the underlying cause for more severe disease, as the androgen receptor has been noted to upregulate the cell surface receptors that mediate viral cell entry and infection. Unfortunately, despite testosterone's potential role in COVID-19 prognosis, androgen deprivation therapy is neither protective nor a treatment for COVID-19. Interestingly, the male reproductive organs have been found to be vulnerable in moderate to severe illness, leading to reports of erectile dysfunction and orchitis. COVID-19 viral particles have been identified in penile and testis tissue, both in live patients who recovered from COVID-19 and post mortem in men who succumbed to the disease. Although sexual transmission remains unlikely in recovered men, moderate to severe COVID-19 infection can lead to germ cell and Leydig cell depletion, leading to decreased spermatogenesis and male hypogonadism. The objective of this review is to describe the impact of SARS-CoV-2 on male reproductive health. There are still many unanswered questions as to the specific underlying mechanisms by which COVID-19 impacts male reproductive organs and the long-term sequelae of SARS-CoV-2 on male reproductive health.

COVID-19 disrupts spermatogenesis through the oxidative stress pathway following induction of apoptosis.

To evaluate the incidence of apoptosis within the testes of patients who died from severe acute respiratory syndrome coronavirus 2 (COVID-19) complications, testis tissue was collected from autopsies of COVID-19 positive (n = 6) and negative men (n = 6). They were then taken for histopathological experiments, and RNA extraction, to examine the expression of angiotensin-converting enzyme 2 (ACE2), transmembrane protease, serine 2 (TMPRSS2), BAX, BCL2 and Caspase3 genes. Reactive oxygen species (ROS) production and glutathione disulfide (GSH) activity were also thoroughly examined. Autopsied testicular specimens of COVID-19 showed that COVID-19 infection significantly decreased the seminiferous tubule length, interstitial tissue and seminiferous tubule volume, as well as the number of testicular cells. An analysis of the results showed that the Johnsen expressed a reduction in the COVID-19 group when compared to the control group. Our data showed that the expression of ACE2, BAX and Caspase3 were remarkably increased as well as a decrease in the expression of BCL2 in COVID-19 cases. Although, no significant difference was found for TMPRSS2. Furthermore, the results signified an increase in the formation of ROS and suppression of the GSH activity as oxidative stress biomarkers. The results of immunohistochemistry and TUNEL assay showed that the expression of ACE2 and the number of apoptotic cells significantly increased in the COVID-19 group. Overall, this study suggests that COVID-19 infection causes spermatogenesis disruption, probably through the oxidative stress pathway and subsequently induces apoptosis.

Short-term effects of COVID-19 on semen parameters: A multicenter study of 69 cases.

OBJECTIVE: COVID-19, which is known to be caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global health problem that can cause multiorgan damage because of its use of the angiotensin-converting enzyme 2 (ACE2) receptor in its pathophysiology. We aimed to investigate whether SARS-CoV-2 had a short-term effect on spermatogenesis, which plays an important role in male reproductive health as it has abundant ACE2 expression in testicular tissue. MATERIAL AND METHODS: This multicenter study included 69 patients aged 20-45 years, who admitted to our hospitals between April 2020 and October 2020 with a history of a positive test result for SARS-CoV-2 based on the nasopharyngeal or oropharyngeal swab samples and had recovered from the disease at least three months earlier and who had undergone a spermiogram test in the hospital database within the last year before the onset of disease. The patients were divided into two groups according to their COVID-19 symptoms being mild or moderate, depending on whether they had received home treatment or required hospitalization for oxygen therapy. Semen samples taken before and after COVID-19 were compared within and between the groups in terms of sperm parameters. RESULTS: The mean age of the patients included in the study was 30.4±4.8 years in the mild symptomatic COVID-19 group and 31.06±4.2 years in the moderate symptomatic group. When the spermiogram samples of the patients before and after COVID-19 were evaluated, it was found that motility and vitality significantly decreased in the mild symptomatic group, while the decrease in all semen parameters was statistically significant in the moderate symptomatic group. CONCLUSION: Although the mechanism by which COVID-19 causes testicular involvement remains uncertain, its short-term results on spermatogenesis reveals that COVID-19 negatively affects sperm parameters.

The COVID-19 pandemic and reproductive health.

This introduction tees off an outstanding collection of Views and Reviews articles on the effects of the SARS-CoV-2 and COVID-19 on human reproductive health. These articles written by the experts in the field review the current literature on COVID-19 and male reproductive health, female reproductive health, and the assisted reproductive technology laboratory. Despite the prolonged nature of the pandemic and the number of people infected worldwide, there still are limited data on the effects of the virus and infection on human reproductive health and human fertility. The investigators distill a vast and often conflicting series of reports into a digestible summary to guide patient counseling and institute the safest practices into the assisted reproductive technology laboratory.

COVID-19 and human reproduction: A pandemic that packs a serious punch.

The COVID-19 pandemic has led to a worldwide health emergency that has impacted 188 countries at last count. The rapid community transmission and relatively high mortality rates with COVID-19 in modern times are relatively unique features of this flu pandemic and have resulted in an unparalleled global health crisis. SARS-CoV-2, being a respiratory virus, mainly affects the lungs, but is capable of infecting other vital organs, such as brain, heart and kidney. Emerging evidence suggests that the virus also targets male and female reproductive organs that express its main receptor ACE2, although it is as yet unclear if this has any implications for human fertility. Furthermore, professional bodies have recommended discontinuing fertility services during the pandemic such that reproductive services have also been affected. Although increased safety measures have helped to mitigate the propagation of COVID-19 in a number of countries, it seems that there is no predictable timeline to containment of the virus, a goal likely to remain elusive until an effective vaccine becomes available  and widely distributed across the globe. In parallel, research on reproduction has been postponed for obvious reasons, while diagnostic tests that detect the virus or antibodies against it are of vital importance to support public health policies, such as social distancing and our obligation to wear masks in public spaces. This review aims to provide an overview of critical research and ethics issues that have been continuously emerging in the field of reproductive medicine as the COVID-19 pandemic tragically unfolds.Abbreviations: ACE2: angiotensin- converting enzyme 2; ART: Assisted reproductive technology; ASRM: American Society for Reproductive Medicine; CCR9: C-C Motif Chemokine Receptor 9; CDC: Centers for Disease Control and Prevention; COVID-19: Coronavirus disease 2019; Ct: Cycle threshold; CXCR6: C-X-C Motif Chemokine Receptor 6; ELISA: enzyme-linked immunosorbent assay; ESHRE: European Society of Human Reproduction and Embryology; ET: Embryo transfer; FSH: Follicle Stimulating Hormone; FFPE: formalin fixed paraffin embedded; FYCO1: FYVE And Coiled-Coil Domain Autophagy Adaptor 1; IFFS: International Federation of Fertility Societies; IUI: Intrauterine insemination; IVF: In vitro fertilization; LH: Luteinizing Hormone; LZTFL1: Leucine Zipper Transcription Factor Like 1; MAR: medically assisted reproduction services; MERS: Middle East Respiratory syndrome; NGS: Next Generation Sequencing; ORF: Open Reading Frame; PPE: personal protective equipment; RE: RNA Element; REDa: RNA Element Discovery algorithm; RT-PCR: Reverse=trascriptase transcriptase-polymerase chain reaction; SARS: Severe acute respiratory syndrome; SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2; SLC6A20: Solute Carrier Family 6 Member 20; SMS: Single Molecule Sequencing; T: Testosterone; TMPRSS2: transmembrane serine protease 2; WHO: World Health Organization; XCR1: X-C Motif Chemokine Receptor.

Evaluating the impact of COVID-19 on male reproduction.

Invasion or damage of the male reproductive system is one of the reported outcomes of viral infection. Current studies have documented that SARS-CoV-2, which causes COVID-19, can damage the male reproductive system in large part by inflammatory damage caused by a cytokine storm. However, whether SARS-CoV-2 can infect the human testis directly and enter semen is controversial. Other adverse effects of SARS-CoV-2 on male reproduction are also of concern and require comprehensive evaluation. Here, we analyze the invasiveness of SARS-CoV-2 in the testis and examine reported mechanisms by which SARS-CoV-2 interferes with male reproduction. Long-term implications of SARS-CoV-2 infection on male reproduction are also discussed. It should be emphasized that although COVID-19 may induce testicular damage, a substantial decrease in male reproductive capacity awaits clinical evidence. We propose that there is an urgent need to track male COVID-19 patients during their recovery. The development of suitable experimental models, including human reproductive organoids, will be valuable to further investigate the viral impact on reproduction for current and future pandemics.

SARS-COV-2 (Covid-19) and male fertility: Where are we?

Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), a single-stranded RNA virus, was found to be the causal agent of the disease called coronavirus disease. During December 2019, China informed the World Health Organization (WHO) of an outbreak of cases of pneumonia of unknown etiology, which caused severe-acute respiratory distress. The disease was termed coronavirus disease 2019 (Covid-19). Due to alarming levels of spread and severity, on the 11th of March 2020, the WHO declared the outbreak as a global pandemic. As of September 14, 2020, more than 29 million cases have been reported, with over 900,000 deaths globally. Since the outbreak, although not conclusive, discoveries have been made regarding the understanding of the epidemiology, etiology, clinical features, clinical treatment, and prevention of the disease. SARS-COV-2 has been detected in saliva, respiratory fluids, blood, urine, and faeces. Findings are however controversial regarding its presence in the semen or the testis. Hence, this review aimed to further analyse the literature concerning (i) the effects of previously identified human coronaviruses on male fertility (ii) the impact of Covid-19 on male fertility and (iii) the implication for general health in terms of infection and transmission.

Pathological Findings in the Testes of COVID-19 Patients: Clinical Implications.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), involves multiple organs. Testicular involvement is largely unknown. OBJECTIVE: To determine the pathological changes and whether SARS-CoV-2 can be detected in the testes of deceased COVID-19 patients. DESIGN, SETTING, AND PARTICIPANTS: Postmortem examination of the testes from 12 COVID-19 patients was performed using light and electron microscopy, and immunohistochemistry for lymphocytic and histiocytic markers. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the virus in testicular tissue. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Seminiferous tubular injury was assessed as none, mild, moderate, or severe according to the extent of tubular damage. Leydig cells in the interstitium were counted in ten 400× microscopy fields. RESULTS AND LIMITATIONS: Microscopically, Sertoli cells showed swelling, vacuolation and cytoplasmic rarefaction, detachment from tubular basement membranes, and loss and sloughing into lumens of the intratubular cell mass. Two, five, and four of 11 cases showed mild, moderate, and severe injury, respectively. The mean number of Leydig cells in COVID-19 testes was significantly lower than in the control group (2.2 vs 7.8, p < 0.001). In the interstitium there was edema and mild inflammatory infiltrates composed of T lymphocytes and histiocytes. Transmission EM did not identify viral particles in three cases. RT-PCR detected the virus in one of 12 cases. CONCLUSIONS: Testes from COVID-19 patients exhibited significant seminiferous tubular injury, reduced Leydig cells, and mild lymphocytic inflammation. We found no evidence of SARS-CoV-2 virus in the testes in the majority (90%) of the cases by RT-PCR, and in none by electron microscopy. These findings can provide evidence-based guidance for sperm donation and inform management strategies to mitigate the risk of testicular injury during the COVID-19 disease course. PATIENT SUMMARY: We examined the testes of deceased COVID-19 patients. We found significant damage to the testicular parenchyma. However, virus was not detected in testes in the majority of cases.

Single-cell transcriptome analysis of the novel coronavirus (SARS-CoV-2) associated gene ACE2 expression in normal and non-obstructive azoospermia (NOA) human male testes.

Being infected by SARS-CoV-2 may cause damage to multiple organs in patients, such as the lung, liver and heart. Angiotensin-converting enzyme 2 (ACE2), reported as a SARS-CoV-2 receptor, is also expressed in human male testes. This suggests a potential risk in human male reproductive system. However, the characteristics of ACE2-positive cells and the expression of other SARS-CoV-2 process-related genes are still worthy of further investigation. Here, we performed singlecell RNA seq (scRNA-seq) analysis on 853 male embryo primordial germ cells (PGCs) and 2,854 normal testis cells to assess the effects of the SARS-CoV-2 virus on the male reproductive system from embryonic stage to adulthood. We also collected and constructed the scRNA-seq library on 228 Sertoli cells from three non-obstructive azoospermia (NOA) patients to assess the effects at disease state. We found that ACE2 expressing cells existed in almost all testis cell types and Sertoli cells had highest expression level and positive cells ratio. Moreover, ACE2 was also expressed in human male PGCs. In adulthood, the level of ACE2 expression decreased with the increase of age. We also found that ACE2 positive cells had high expressions of stress response and immune activation-related genes. Interestingly, some potential SARS-CoV-2 process-related genes such as TMPRSS2, BSG, CTSL and CTSB had different expression patterns in the same cell type. Furthermore, ACE2 expression level in NOA donors' Sertoli cells was significantly decreased. Our work would help to assess the risk of SARS-CoV-2 infection in the male reproductive system.