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Case - Sperm DNA fragmentation associated with COVID-19 infection

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Multiorgan dysfunction is the main characteristic of severe COVID-19 patients and the involvement of male reproductive system may occur among these patients. Although there is a limited evidence to confirm the orchitis and virus presence in the semen of patients, there are concerns about the transmission of virus through the semen. In addition, reduced fertility or infertility can be seen as consequences of severe COVID-19 in recovered subjects. In this study, we aimed to review articles related to COVID-19 and male reproductive system to find the possible underlying mechanisms of SARS-CoV-2 in affecting male fertility. The following keywords of SARS-CoV-2, COVID-19, testis, orchitis, semen, angiotensin-converting enzyme 2 (ACE2), hypothalamic–pituitary–testicular (HPT) axis, Hypothalamus, etc., were defined to find the related publications from standard search engines, e.g., PUBMED, SCOPUS, Google Scholar. According to studies, COVID-19 occurs in severe patients as respiratory disease, along with multi-organ failure. The most important mechanisms are classified as direct and indirect pathogenesis of SARS-CoV-2. The presence of ACE2 on the cell surface of various cells in testis increases the risk of direct infection by this virus. SARS-CoV-2 also affects the testis through the cytokine storm. In addition, the important role of HPT axis dysregulation through impaired Leydig cells and hypothalamus should be considered. Using antiviral and immunomodulatory therapy can be harmful for testis function. Further investigations are required to investigate potential mechanisms of male infertility in survivals of COVID-19. Since involvement of testis is essential for fertility, increasing the knowledge of health system may improve the outcomes.
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The novel coronavirus was recognised in December 2019 and caught humanity off guard. The virus employs the angiotensin-converting enzyme 2 (ACE2) receptor for entry into human cells. ACE2 is expressed on different organs, which is raising concern as to whether these organs can be infected by the virus or not. The testis appears to be an organ enriched with levels of ACE2, while the possible mechanisms of involvement of the male reproductive system by SARS-CoV-2 are not fully elucidated. The major focus of the present studies is on the short-term complications of the coronavirus and gains importance on studying the long-term effects, including the possible effects of the virus on the male reproductive system. The aim of this review was to provide new insights into different possible mechanisms of involvement of male gonads with SARS-CoV-2 including investigating the ACE2 axis in testis, hormonal alterations in patients with COVID-19, possible formation of anti-sperm antibodies (ASA) and subsequently immunological infertility as a complication of SARS-CoV-2 infection. Finally, we suggest measuring the sperm DNA fragmentation index (DFI) as a determiner of male fertility impairment in patients with COVID-19 along with other options such as sex-related hormones and semen analysis. Invasion of SARS-CoV-2 to the spermatogonia, Leydig cells and Sertoli cells can lead to sex hormonal alteration and impaired gonadal function. Once infected, changes in ACE2 signalling pathways followed by oxidative stress and inflammation could cause spermatogenesis failure, abnormal sperm motility, DNA fragmentation and male infertility.
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We herein summarise the evidence concerning the impact of sperm DNA fragmentation in various clinical infertility scenarios and the advances on sperm DNA fragmentation tests. The collected evidence was used to formulate 41 recommendations. Of these, 13 recommendations concern technical aspects of sperm DNA fragmentation testing, including pre-analytical information, clinical thresholds and interpretation of results. The remaining 28 recommendations relate to indications for sperm DNA fragmentation testing and clinical management. Clinical scenarios like varicocele, unexplained infertility, idiopathic infertility, recurrent pregnancy loss, intrauterine insemination, in vitro fertilisation/intracytoplasmic sperm injection, fertility counselling for men with infertility risk factors and sperm cryopreservation have been contemplated. The bulk evidence supporting the recommendations has increased in recent years, but it is still of moderate to low quality. This practice guideline provides clinicians with advice on best practices in sperm DNA fragmentation testing. Also, recommendations are provided on possible management strategies to overcome infertility related to sperm DNA fragmentation, based on the best available evidence. Lastly, we identified gaps in knowledge and opportunities for research and elaborated a list of recommendations to stimulate further investigation concerning sperm DNA fragmentation testing and management of sperm DNA fragmentation-related infertility conditions.
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Purpose: Since its discovery in December 2019, the novel coronavirus SARS-CoV-2 has spread globally, causing the current COVID-19 (coronavirus disease-19) pandemic. As there is an increase of infections in the male population, concerns have emerged about the potential impact of COVID-19 on male reproductive organs and male fertility. Therefore, this study systematically investigates the current evidence of SARS-CoV-2 impact on male reproduction and pregnancy outcomes, discussing them in light of the evidence published on other coronaviruses. Materials and methods: Literature search was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A total of 24 original articles were included for the analysis, investigating the effects of the infection on semen parameters, male reproductive hormones, and pregnancy outcomes. Further, a Strengths-Weaknesses-Opportunities-Threats (SWOT) analysis was conducted based on the available evidence linking the virus with male reproduction and conception. Results: Although there is limited data, viral mRNA has been identified in semen of infected men, with some evidence of altered seminal parameters. Low testosterone and dihydrotestosterone with raised luteinizing hormone has been reported as well as preterm delivery in pregnant women; however, data regarding vertical transmission remains contradictory and inconclusive. Conclusions: The recent literature provides evidence that male gonads may be potentially vulnerable to SARS-CoV-2 infection, recommending caution to pregnant women and couples planning natural pregnancy or assisted reproduction.
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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.
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Studies have explored the influence of DNA damage in assisted reproductive technology (ART), but the outcome remains controversial. To determine whether sperm DNA fragmentation index (DFI) has any effect on ART outcomes, we collected detailed data regarding 1,333 IVF cycles performed at our centre, and the data of our retrospective cohort study were extracted for this meta‐analysis. We searched PubMed, Web of Science, EMBASE and Google Scholar and performed a systemic review and meta‐analysis. Primary meta‐analysis of 10 studies comprising 1,785 couples showed that live birth rate was no significantly different between low‐DFI group and high‐DFI group (p > 0.05). Secondary meta‐analysis of 25 studies comprising 3,992 couples showed a higher miscarriage rate in high‐DFI group than in low‐DFI group (RR=1.57 [1.18, 2.09], p < 0.01). Meta‐analysis of eight studies comprising 17,879 embryos revealed a lower good‐quality embryo rate (RR=0.65 [0.62, 0.68], p < 0.01). Meta‐analysis of 23 studies comprising 6,771 cycles showed that the high‐DFI group had a lower clinical pregnancy rate than low‐DFI group (RR=0.85 [0.75, 0.96], p < 0.01). Heterogeneity of included studies weakened our conclusions. Our study showed that DFI has adverse effects on ART outcome. More well‐designed studies exploring the association between DFI and ART outcome are desired.
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Study question: Is there an association between high levels of sperm DNA damage and miscarriage? Summary answer: Miscarriage rates are positively correlated with sperm DNA damage levels. What is known already: Most ejaculates contain a subpopulation of sperm with DNA damage, also referred to as DNA fragmentation, in the form of double or single-strand breaks which have been induced in the DNA prior to or following ejaculation. This DNA damage may be particularly elevated in some subfertile men, hence several studies have examined the link between sperm DNA damage levels and conception and miscarriage rates. Study design, size, duration: A systematic review and meta-analysis of studies which examined the effect of sperm DNA damage on miscarriage rates was performed. Searches were conducted on MEDLINE, EMBASE and the Cochrane Library without any language restrictions from database inception to January 2012. Participants/materials, setting, methods: We used the terms 'DNA damage' or 'DNA fragmentation' combined with 'miscarriage', 'abortion' or 'pregnancy' to generate a set of relevant citations. Data extraction was performed by two reviewers. Study quality was assessed using the Newcastle-Ottawa Scale. Meta-analysis of relative risks of miscarriage was performed with a random effects model. Subgroup analyses were performed by the type of DNA damage test, whether the sperm examined were prepared or from raw semen and for pregnancies resulting from IVF or ICSI treatment. Main results and the role of chance: We identified 16 cohort studies (2969 couples), 14 of which were prospective. Eight studies used acridine orange-based assays, six the TUNEL assay and two the COMET assay. Meta-analysis showed a significant increase in miscarriage in patients with high DNA damage compared with those with low DNA damage [risk ratio (RR) = 2.16 (1.54, 3.03), P < 0.00001)]. A subgroup analysis showed that the miscarriage association is strongest for the TUNEL assay (RR = 3.94 (2.45, 6.32), P < 0.00001). Limitations, reasons for caution: There is some variation in study characteristics, including the use of different assays and different thresholds for DNA damage and the definition of pregnancy loss. Wider implications of the findings: The use of methods which select sperm without DNA damage for use in assisted conception treatment may reduce the risk of miscarriage. This finding indicates that assays detecting DNA damage could be considered in those suffering from recurrent pregnancy loss. Further research is necessary to study the mechanisms of DNA damage and the potential therapeutic effects of antioxidant therapy. Study funding/competing interest(s): None.
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The novel coronavirus disease (COVID-19) pandemic is emerging as a global health threat and shows a higher risk for men than women. Thus far, the studies on andrological consequences of COVID-19 are limited. To ascertain the consequences of COVID-19 on sperm parameters after recovery, we recruited 41 reproductive-aged male patients who had recovered from COVID-19, and analyzed their semen parameters and serum sex hormones at a median time of 56 days after hospital discharge. For longitudinal analysis, a second sampling was obtained from 22 of the 41 patients after a median time interval of 29 days from first sampling. Compared with controls who had not suffered from COVID-19, the total sperm count, sperm concentration, and percentages of motile and progressively motile spermatozoa in the patients were significantly lower at first sampling, while sperm vitality and morphology were not affected. The total sperm count, sperm concentration, and number of motile spermatozoa per ejaculate were significantly increased and the percentage of morphologically abnormal sperm was reduced at the second sampling compared with those at first in the 22 patients examined. Though there were higher prolactin and lower progesterone levels in patients at first sampling than those in controls, no significant alterations were detected for any sex hormones examined over time following COVID-19 recovery in the 22 patients. Although it should be interpreted carefully, these findings indicate an adverse but potentially reversible consequence of COVID-19 on sperm quality.
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Purpose: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a surge of research to help better understand the breadth of possible sequelae. However, little is known regarding the impact on semen parameters and fertility potential. We sought to investigate for presence of viral RNA in semen of men with SARS-CoV-2 infection and to evaluate its effect on semen parameters in ejaculate. Materials and methods: We prospectively recruited thirty men diagnosed with acute SARS-CoV-2 infection using real-time reverse transcriptase polymerase chain reaction (RT-PCR) of pharyngeal swab specimens. Semen samples were collected from each individual using mailed kits. Follow-up semen samples were done with mailed kits or in-person in office setting. Semen analysis and PCR was performed after samples were received. Results: Thirty semen samples from recovered men were obtained 11-64 days after testing positive for SAR-CoV-2 infection. The median duration between positive SAR-CoV-2 test and semen collection was 37 days (interquartile range [IQR]=23). The median total sperm number (TSN) in ejaculate was 12.5 million (IQR=52.1). When compared with age-matched SARS-CoV-2(-) men, TSN was lower among SARS-CoV-2(+) men (p=0.0024). Five men completed a follow-up sperm analysis (median 3 months) and had a median TSN of 18 million (IQR=21.6). No RNA was detected by means of RT-PCR in the semen in 16 samples tested. Conclusions: SARS-CoV-2 infection, though not detected in semen of recovered men, can affect TSN in ejaculate in the acute setting. Whether SARS-CoV-2 can affect spermatogenic function long-term remains to be evaluated.
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In the past several months, the outbreak of SARS‐CoV‐2‐associated infection (coronavirus disease 2019, COVID‐19) developed rapidly and has turned into a global pandemic. Although SARS‐CoV‐2 mainly attacks respiratory systems, manifestations of multiple organs have been observed. Great concern was raised about whether COVID‐19 may affect male reproductive functions. In this study, we collected semen specimens from twelve male COVID‐19 patients for virus detection and semen characteristics analysis. No SARS‐CoV‐2 was found in semen specimens. 8 out of 12 patients had normal semen quality. We also compared the sex‐related hormone levels between 119 reproductive‐aged men with SARS‐CoV‐2 infection and 273 age‐matched control men. A higher serum luteinizing hormone (LH) and a lower ratio of testosterone (T) to LH were observed in the COVID‐19 group. Multiple regression analysis indicated that serum T:LH ratio was negatively associated with white blood cell counts (WBC) and c‐reactive protein (CRP) level in COVID‐19 patients. It's the first report about semen assessment and sex‐hormone evaluation in reproductive‐aged male COVID‐19 patients. Although further study is needed to clarify the reasons and underlying mechanisms, our study presents an abnormal sex hormone secretion among COVID‐19 patients, suggesting that attention should be paid to reproductive function evaluation in the follow‐up. This article is protected by copyright. All rights reserved.
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This article provides an overview of infectious and inflammatory conditions associated with male infertility. These conditions may affect several components of the male reproductive tract and therefore have the ability to potentially alter sperm function. The effect of these conditions on male fertility is poorly understood and often underestimated.
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To standardize the TUNEL assay by establishing inter- and intraobserver variability, interassay variability, cutoff values, sensitivity and specificity of the assay, and studying the distribution of the DNA damage in a population of infertile men referred to a clinical andrology laboratory. Seminal ejaculates from 25 healthy male volunteers (controls) and 194 infertile men (with male factor infertility) referred to an andrology laboratory were examined for DNA damage by TUNEL assay using flow cytometric analysis. Both the inter- and intraobserver variability and interassay variability was small (<10%). DNA damage in the controls was 11.9 ± 6.8% vs. 29.5 ± 18.7% in patients (P <.001). The cut-off value of 19.25% maximized the observed sensitivity (64.9%) and specificity (100%) of the assay. The distribution of DNA damage in the patients was as follows: 14.9% (29 of 194) with DNA damage between 0% and 10%; 22.7% (44 of 194) between 10% and 20%; 8.8% (17 of 194) between 20% and 30%; and 17.5% (34 of 194) between 30% and 40%. Finally, 27.3% (53 of 194) had TUNEL values >40%. We report a detailed standardization of the TUNEL assay for clinical use, as well as reference ranges for DNA damage in normal healthy donors and infertile men. A cut-off of 19.25% with observed 100% specificity established in our program can differentiate infertile men with DNA damage from healthy men. This test can be offered to infertile patients who are idiopathic, have severe oxidative stress-related abnormal semen quality, and contribute to the infertility problem of the couple who are considering assisted reproductive techniques.
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Severe acute respiratory syndrome (SARS) coronavirus has been known to damage multiple organs; however, little is known about its impact on the reproductive system. In the present study, we analyzed the pathological changes of testes from six patients who died of SARS. Results suggested that SARS caused orchitis. All SARS testes displayed widespread germ cell destruction, few or no spermatozoon in the seminiferous tubule, thickened basement membrane, and leukocyte infiltration. The numbers of CD3+ T lymphocytes and CD68+ macrophages increased significantly in the interstitial tissue compared with the control group (P < 0.05). SARS viral genomic sequences were not detected in the testes by in situ hybridization. Immunohistochemistry demonstrated abundant IgG precipitation in the seminiferous epithelium of SARS testes, indicating possible immune response as the cause for the damage. Our findings indicated that orchitis is a complication of SARS. It further suggests that the reproductive functions should be followed and evaluated in recovered male SARS patients.
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Determination of sperm DNA fragmentation, as assessed by the sperm chromatin structure assay (SCSA), has become an important tool for the evaluation of semen quality. The aim of the present study was to describe the biological variation of sperm DNA fragmentation in men attending an andrology clinic and to identify clinical correlates of the biological variation of sperm DNA fragmentation. For this study, two consecutive semen samples from 100 patients attending our andrology outpatient clinic were subjected to semen analysis, performed in parallel according to WHO guidelines and by SCSA. A good agreement between pairs of samples was found for SCSA-derived variables, as indicated by a significantly lower median coefficient of variation (CV) of the DNA Fragmentation Index (DFI) and the high DNA stainability (HDS) compared with WHO semen parameters. In half of the men attending our andrology clinic, however, the individual biological variation of DFI and HDS, expressed as CV of two samples, exceeded 10%. Dysregulation of spermatogenesis, as seen as testicular insufficiency or varicocele, was not associated with increased variability of DFI or HDS. A backward multiple linear regression analysis, however, indicated that the biological variation of DFI may be more profound in men with characteristics of normal spermatogenesis. In conclusion, we confirm previous reports that sperm DNA fragmentation has a lower biological variability than classical semen parameters. We hypothesize that the sperm chromatin structure may be more influenced in patients with normal spermatogenesis, whereas in men with disturbed spermatogenesis, the chromatin structure may be already so impaired that the effect of unidentified factors leading to variability of sperm DNA fragmentation in time may not be as profound.
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To report parameters in semen samples and sperm deoxyribonucleic acid integrity in a fertile volunteer presenting a 2-day fever of 39 degrees -40 degrees C. Case report. University-affiliated teaching hospital. None. Semen samples from a fertile volunteer of proven fertility were obtained and analyzed before the febrile illness episode and at days 15, 37, 58, 79, and >180 after the fever. Semen parameters (total sperm count, motility a+b, and vitality), sperm protamination state, measured by sperm chromatin structure assay (SCSA) and apoptotic activities, measured by terminal uridine nick-end labeling (TUNEL) assay. Total sperm count significantly decreased at days 15, 37, and 58 after the fever and returned to normal by day 79 after the fever. The percentage of motility significantly decreased at days 15 and 37 after the fever and returned to normal by day 58. Vitality score also showed a slight, although not statistically significant, decrease after the fever. The DNA fragmentation index (DFI, a SCSA parameter), which defines abnormal chromatin structure, significantly increased by 24% and 36% at days 15 and 37 after the fever, respectively, and decreased to 15% and 8% when reaching days 58 and 79 after the fever. High DNA stainability (HDS, a SCSA parameter) also significantly increased at day 37 after the fever. On the other hand, sperm DNA fragmentation, as measured by TUNEL assay, increased up to 23% by day 15 after the fever but this was not statistically significant. This report demonstrates that a febrile episode can have marked effects on semen parameters and sperm DNA integrity. These results are particularly important for the counseling of infertile couples and in relation to assisted reproductive techniques (ART).
Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations
  • S C Esteves
  • A Zini
  • R M Coward
Esteves SC, Zini A, Coward RM, et al. Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations. Andrologia 2021;53:e13874-e. https://doi.org/10.1111/and.13874