DNA damage in testicular germ cells and spermatozoa. When and how is it induced? How should we measure it? What does it mean?

This review surveys the causes and consequences of DNA damage in the male germ line from spermatogonial stem cells to fully differentiated spermatozoa. Within the stem cell population, DNA integrity is well maintained as a result of excellent DNA surveillance and repair; however, a progressive increase in background mutation rates does occur with paternal age possibly as a result of aberrant DNA repair as well as replication error. Once a germ cell has committed to spermatogenesis, it responds to genetic damage via a range of DNA repair pathways or, if this process fails, by the induction of apoptosis. When fully-differentiated spermatozoa are stressed, they also activate a truncated intrinsic apoptotic pathway which results in the activation of nucleases in the mitochondria and cytoplasm; however, the physical architecture of these cells prevents these enzymes from translocating to the nucleus to induce DNA fragmentation. Conversely, hydrogen peroxide released from the sperm midpiece during apoptosis is able to penetrate the nucleus and induce DNA damage. The base excision repair pathway responds to such damage by cleaving oxidized bases from the DNA, leaving abasic sites that are alkali-labile and readily detected with the comet assay. As levels of oxidative stress increase and these cells enter the perimortem, topoisomerase integrated into the sperm chromatin becomes activated by SUMOylation. Such activation may initially facilitate DNA repair by reannealing double strand breaks but ultimately prepares the DNA for destruction by nucleases released from the male reproductive tract. The abasic sites and oxidized base lesions found in live spermatozoa are mutagenic and may increase the mutational load carried by the offspring, particularly in the context of assisted conception. A variety of strategies are described for managing patients expressing high levels of DNA damage in their spermatozoa, to reduce the risks such lesions might pose to offspring health.

Revisiting the impact of varicocele and its treatments on male fertility.

Varicocele is one of the most common, yet treatable, causes of male infertility. Varicoceles are present in more than 40% of infertile men with primary infertility, a figure that increases with age. Varicoceles impair semen parameters and sperm DNA and are linked with lower pregnancy and live birth rates. Until recently, men had seldom been examined in male fertility workups. This is changing as urologists have become recognized as team members in infertility. Hence identification and treatment are available as never before. Furthermore, as men become aware that they are as likely as their female partners to be infertile, they want equal 'couple care', requesting urological referrals as they realize that they can improve their semen quality and chances of fatherhood without or before fertility treatment. There is now a greater understanding of the mechanisms of varicocele-induced damage by oxidative stress, using sperm DNA as a sensitive biomarker of sperm quality. There is a current consensus that varicocele is linked to poor semen and repair improves semen and sperm DNA quality. Evidence is strengthening to indicate that varicocele repair increases pregnancy and live birth rates in natural conception and following fertility treatment.

A Comprehensive Guide to Sperm Recovery in Infertile Men with Retrograde Ejaculation.

Retrograde ejaculation (RE) is a condition defined as the backward flow of the semen during ejaculation, and when present can result in male infertility. RE may be partial or complete, resulting in either low seminal volume or complete absence of the ejaculate (dry ejaculate). RE can result from anatomic, neurological or pharmacological conditions. The treatment approaches outlined are determined by the cause. Alkalinizing urinary pH with oral medications or by adding sperm wash media into the bladder prior to ejaculation may preserve the viability of the sperm. This article provides a step-by-step guide to diagnose RE and the optimal techniques to retrieve sperm.

Relevance of Leukocytospermia and Semen Culture and Its True Place in Diagnosing and Treating Male Infertility.

The current WHO 2010 manual for human semen analysis defines leukocytospermia as the presence of peroxidase-positive leukocytes at a concentration >1×106/mL of semen. Granular leukocytes when activated are capable of generating high levels of reactive oxygen species in semen resulting in oxidative stress. Oxidative stress has been correlated with poor sperm quality, increased level of sperm DNA fragmentation and low fertility potential. The presence of leukocytes and pathogens in the semen may be a sign of infection and/or localized inflammatory response in the male genital tract and the accessory glands. Common uro-pathogens including Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, Mycoplasma hominis, and Escherichia coli can cause epididymitis, epididymo-orchitis, or prostatitis. The relationship between leukocytospermia and infection is unclear. Therefore, we describe the pathogens responsible for male genital tract infections and their association with leukocytospermia. The review also examines the diagnostic tests available to identify seminal leukocytes. The role of leukocytospermia in male infertility and its management is also discussed.

Sperm Morphology Assessment in the Era of Intracytoplasmic Sperm Injection: Reliable Results Require Focus on Standardization, Quality Control, and Training.

Semen analysis is the first, and frequently, the only step in the evaluation of male fertility. Although the laboratory procedures are conducted according to the World Health Organization (WHO) guidelines, semen analysis and especially sperm morphology assessment is very difficult to standardize and obtain reproducible results. This is mainly due to the highly subjective nature of their evaluation. ICSI is the choice of treatment when sperm morphology is severely abnormal (teratozoospermic). Hence, the standardization of laboratory protocols for sperm morphology evaluation represents a fundamental step to ensure reliable, accurate and consistent laboratory results that avoid misdiagnoses and inadequate treatment of the infertile patient. This article aims to promote standardized laboratory procedures for an accurate evaluation of sperm morphology, including the establishment of quality control and quality assurance policies. Additionally, the clinical importance of sperm morphology results in assisted reproductive outcomes is discussed, along with the clinical management of teratozoospermic patients.

Sperm DNA fragmentation is a novel biomarker for early pregnancy loss.

RESEARCH QUESTION: Spontaneous pregnancy loss affects 10-15% of couples, with 1-2% suffering recurrent pregnancy loss and 50% of miscarriages remaining unexplained. Male genomic integrity is essential for healthy offspring, meaning sperm DNA quality may be important in maintaining a pregnancy. Does sperm DNA fragmentation measured by alkaline Comet assay act as a biomarker for early pregnancy loss? DESIGN: Sperm DNA fragmentation was measured by alkaline Comet test in 76 fertile donors and 217 men whose partners had recently experienced miscarriage. Couples were divided into five groups for analysis: one miscarriage after spontaneous conception; two or more miscarriages after spontaneous conception; one miscarriage after fertility treatment; two or more miscarriages after fertility treatment and biochemical pregnancy. RESULTS: Receiver operator characteristic curve analysis was used to determine ability of the average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) to diagnose miscarriage and develop clinical thresholds comparing men whose partners have miscarried with men with recently proven fertility. Male partners of women who had miscarried had higher sperm DNA damage (ACS 33.32 ± 0.57%) than fertile men (ACS 14.87 ± 0.66%; P < 0.001). Average Comet score, HCS and LCS all have promise as being highly predictive of sporadic and recurrent miscarriage using clinical thresholds from comparisons with fertile men's spermatozoa: receiver operating characteristic curve AUC for ACS ≥26%, 0.965; LCS ≤70%, 0.969; HCS ≥2%, 0.883; P <0.0001. CONCLUSIONS: Sperm DNA damage measured by the alkaline Comet has promise as a robust biomarker for sporadic and recurrent miscarriage after spontaneous or assisted conception, and may provide novel diagnoses and guidance for future fertility pathways.

SARS-CoV-2 pandemic and repercussions for male infertility patients: A proposal for the individualized provision of andrological services.

The prolonged lockdown of health facilities providing non-urgent gamete cryopreservation-as currently recommended by many reproductive medicine entities and regulatory authorities due to the SARS-CoV-2 pandemic will be detrimental for subgroups of male infertility patients. We believe the existing recommendations should be promptly modified and propose that the same permissive approach for sperm banking granted for men with cancer is expanded to other groups of vulnerable patients. These groups include infertility patients (eg, azoospermic and cryptozoospermic) undergoing medical or surgical treatment to improve sperm quantity and quality, as well as males of reproductive age affected by inflammatory and systemic auto-immune diseases who are about to start treatment with gonadotoxic drugs or who are under remission. In both scenarios, the "fertility window" may be transitory; postponing diagnostic semen analysis and sperm banking in these men could compromise the prospects of biological parenthood. Moreover, we provide recommendations on how to continue the provision of andrological services in a considered manner and a safe environment. Our opinion is timely and relevant given the fact that fertility services are currently rated as of low priority in most countries.

Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations.

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 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.

Novel use of COMET parameters of sperm DNA damage may increase its utility to diagnose male infertility and predict live births following both IVF and ICSI.

STUDY QUESTION: Do the Comet parameters of the proportions of sperm with low or high DNA damage improve the power of the test in the diagnosis of male infertility and/or prediction of IVF and ICSI live birth rates? SUMMARY ANSWER: The mean Comet score and the scores for proportions of sperm with high or low DNA damage were useful in diagnosing male infertility and provided additional discriminatory information for the prediction of both IVF and ICSI live births. WHAT IS KNOWN ALREADY: Sperm DNA damage impacts adversely on male fertility and IVF outcomes. STUDY DESIGN, SIZE, DURATION: A retrospective study was performed involving a total of 457 participants (381 patients and 76 fertile donors). Data was collected from a fertility clinic between 2015 and 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 381 consecutive male partners of couples attending for ART and 76 fertile donors were included in the study. DNA fragmentation was measured by the alkaline Comet assay. Receiver operator characteristic curve analysis (area under the ROC curve (AUC)) was used to determine the value of average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) to diagnose male factor infertility. In total, 77 IVF and 226 ICSI cycles were included to determine thresholds for each parameter (AUC analysis) and to compare live birth rates (LBRs) following each ART. MAIN RESULTS AND THE ROLE OF CHANCE: ACS, HCS and LCS were predictive of male infertility (AUC > 0.9, P < 0.0001). IVF LBRs declined once DNA damage exceeded the threshold levels. HCS showed the sharpest decline. Following ICSI, the highest LBRs were in men whose DNA damage levels approached the fertile range. Trends differed in IVF. LBRs decreased as damage increased whereas in ICSI the LBRs decreased but then remained stable. LIMITATIONS, REASONS FOR CAUTION: Since this is the first study to show the impact of sperm DNA damage on ICSI live births, a prospective study should be performed (stratifying patients to IVF or ICSI based on these thresholds) to validate this study. WIDER IMPLICATIONS OF THE FINDINGS: Our study presents novel information towards elucidating the genetic basis of male infertility and secondly on relevance of the extent of DNA damage as an impending factor in both IVF and ICSI success. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Examenlab Ltd, The Lister Clinic, Cryos International and Imperial College London NHS Trust. No external funding was obtained for this study. SL and KL are employees of Examenlab Ltd, a university spin-out company with a commercial interest in sperm DNA damage. No other author has a conflict of interest to declare. TRIAL REGISTRATION NUMBER: Non-applicable.

Should sperm DNA fragmentation testing be included in the male infertility work-up?

A response to the editorial 'Are we ready to incorporate sperm DNA fragmentation testing into our male infertility work-up? A plea for more robust studies' by Erma Drobnis and Martin Johnson.

The paternal genome and the health of the assisted reproductive technology child.

As a number of children born by assisted reproductive technology (ART) are increasing each year across the developed world, the health of such offspring is a matter of public concern. Does the integrity of the paternal genome impact on offspring health? In societal terms, as birth rates fall, and the Western population become unsustainable, do the benefits outweigh the costs of creating and providing for this ART conceived subpopulation? There are little data to date to answer these questions. The long-term health of such children has largely been ignored, and success measured only by early (prebirth) outcomes such as embryo quality or pregnancy. However, there are powerful paradigms such as ageing and smoking that give vital clues as to the potential impact of unhealthy spermatozoa on disease risk, mental and physical health, fertility and mortality of these offspring.

Sperm DNA fragmentation and base oxidation.

Sperm DNA damage has been shown to be a valuable diagnostic and prognostic biomarker for male infertility and assisted reproductive treatment (ART) outcome. It is linked to every fertility checkpoint from reduced fertilization rates, lower embryo quality and pregnancy rates to higher rates of spontaneous miscarriage and childhood diseases. It is more robust than conventional semen parameters.The aim of this chapter is to provide an overview of current laboratory tests and relationships between sperm DNA damage and clinical outcomes. The conclusion is that sperm DNA damage is an important indicator of semen quality, and its routine use in the fertility clinic would improve ART success rates.

The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment.

Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths, weaknesses and clinical applicability of current sperm DNA tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. DNA damage in human spermatozoa is an important attribute of semen quality. It should be part of the clinical work up and properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency. Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. With all of these fertility check points, it shows more promise than conventional semen parameters from a diagnostic perspective. Despite this, few infertility clinics use it routinely. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths and weaknesses and clinical applicability of current sperm DNA fragmentation tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of increased oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. As those working in this field of clinical research, we conclude that DNA damage in human spermatozoa is an important attribute of semen quality which should be carefully assessed in the clinical work up of infertile couples and that properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency.

Differences in the endocannabinoid system of sperm from fertile and infertile men.

Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) at cannabinoid (CB(1), CB(2)) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and 2-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for 2-AG), as well as of their binding receptors CB(1), CB(2) and TRPV1. Our findings show a marked reduction of AEA and 2-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB(1) and CB(2) receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems.

Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes.

The exchange of histones with protamines in sperm DNA results in sperm chromatin compaction and protection. Variations in sperm protamine expression are associated with male infertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content (P1-DNA, P2-DNA, P1+P2-DNA) decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content (P1-DNA, P2-DNA, P1+P2-DNA) or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates.

Sperm DNA damage or progressive motility: which one is the better predictor of fertilization in vitro?

Sperm progressive motility has been reported to be one of the key factors influencing in vitro fertilization rates. However, recent studies have shown that sperm DNA fragmentation is a more robust predictor of assisted reproductive outcomes including reduced fertilization rates, embryo quality, and pregnancy rates. This study aimed to compare the usefulness of sperm progressive motility and DNA damage as predictive tools of in vitro fertilization rates. Here, 136 couples provided 1,767 eggs with an overall fertilization rate of 64.2%. The fertilization rate in vitro correlated with both sperm progressive motility (r² = 0.236; P = 0.002) and DNA fragmentation (r² = -0.318; P < 0.001). The relative risk of a poor fertilization rate was 9.5 times higher in sperm of men with high DNA fragmentation (>40%) compared with 2.6 times in sperm with poor motility (<40%). Further, sperm DNA fragmentation gave a higher specificity (93.3%) in predicting the fertilization rate than progressive motility (77.8%). Finally, the odds ratio to determine fertilization rate (>70%) was 4.81 (1.89-12.65) using progressive motility compared with 24.18 (5.21-154.51) using DNA fragmentation. This study shows that fertilization rates are directly dependent upon both sperm progressive motility and DNA fragmentation, but sperm DNA fragmentation is a much stronger test.