GLOBAL ANDROLOGY FORUM
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The Impact of Single- and Double-Strand DNA Breaks in Human Spermatozoa on Assisted Reproduction
Article #46: “The Impact of Single- and Double-Strand DNA Breaks in Human Spermatozoa on Assisted Reproduction”
Authors: Ashok Agarwal, Catalina Barbarosie, Rafael Ambar, Renata Finelli, International Journal of Molecular Sciences, 2020, 21, 3882
CAPSULE
Contributors: Carlo Giulioni, MD (Italy), and
Nikolaos Sofikitis, MD, PhD (Greece)
Commentary:
The integrity of sperm DNA represents a prerequisite for successful fertilization, optimal embryonic quality, and the development of healthy offspring. Male and female pronuclei development and extrusion of the second polar body are normally followed by zygotic divisions and further early embryonic development. Abnormalities in sperm DNA, due to the "late paternal effect," disrupt zygotic transcription impeding embryonic implantation. Various sperm DNA defects, including sperm DNA fragmentation (SDF), mitochondrial damage, and Y-chromosome microdeletions, may result in single- or double-strand breaks.
Methods to diagnose sperm DNA strand breaks (SDF) are available. Compromised sperm DNA compaction during spermatogenesis, involving protamine assembly, increases susceptibility to DNA damage, negatively affecting the final sperm reproductive capacity. Abortive apoptosis may lead to double-strand DNA breaks (DSBs). Oxidative stress, an imbalance between reactive oxygen species (ROS) and antioxidants, directly damages DNA causing SDF.
Diagnostic tests, such as TUNEL, SCSA, SCD (Halo test), Comet assay, and γH2AX immunodetection, employ diverse methodologies and yield variable outcomes. While the Comet assay distinguishes between single- and double-strand DNA breaks, γH2AX specifically identifies DSBs. It is essential to note that the outcome of these tests may not directly correlate, and the results are not easily comparable.
The section further examines the association between sperm DNA single-strand breaks (SSBs) and DSBs with their consequences on various reproductive outcomes, especially in assisted reproductive technology (ART). Limited evidence exists regarding the specific impact of SSBs and DSBs on ART outcomes. Several studies suggest a negative correlation between SSBs and fertilization rates. In fact, higher SSB percentages are associated with lower fertilization rates in in vitro fertilization (IVF). Additionally, elevated SSB profiles correlate with reduced implantation rates.
Conversely, intracytoplasmic sperm injection (ICSI) procedures may provide an alternative method to overcome the impact of sperm DNA damage on male reproductive capacity. DSBs exhibit a more pronounced negative influence on reproductive outcomes than SSBs. Evidence suggests that ICSI, involving the selection of sperm based on optimal motility and morphology, may partially overcome the consequences of sperm DNA damage compared to IVF trials.
Overall, DSBs appear to have a more substantial negative impact on reproductive outcomes than SSBs. This may be attributable to the separation of paternal and maternal DNA in early embryo development, limiting the probability of DSBs repair. The influence of sperm DNA damage is less pronounced with ICSI, where embryologists selectively choose spermatozoa, inadvertently favoring those with lower sperm DNA fragmentation rates due to the sperm selection method. In male germ cell development, DNA repair mechanisms operate until the third week of spermatogenesis, after which sperm DNA undergoes compaction and repair processes are downregulated.
However, concerning sperm genomic integrity, the oocyte can effectively repair paternal SSBs and DSBs when sperm DNA damage is below 8%. Male germ cells lack molecular mechanisms for single-strand break repair but employ base excision repair (BER) mechanisms during spermatid stages. For DSB repair, mechanisms include homologous recombination (HR) and non-homologous end joining (NHEJ), with an alternative NHEJ pathway active in spermatids, particularly in the absence of classical NHEJ components.
In conclusion, sperm DNA damage, affecting one or both strands, leads to a lower ability of the sperm nucleus to fertilize the female gamete and to trigger zygotic cleavage and early embryonic development appropriate to complete the implantation process with a subsequent normal fetal development. Current tests vary, with only the Comet assay distinguishing SSBs and DSBs. Repair potential decreases after early spermatogenesis, impacting reproductive outcomes. The role of ICSI in overcoming male infertility when high percentages of spermatozoa, with abnormalities in DNA, are present, needs further studies to be supported unequivocally.
Key Takeaways (Carlo and Nikolaos):
Over the past 20 years, there has been a stable trend of ART and male infertility publications. Most of the research focused on the conventional approach compared to advanced techniques. More research will be needed for advanced techniques, particularly as it can be a salvage procedure for a few severe male infertility cases that still want to have a biological child. Azoospermia has been the most reported clinical scenario, but up until now, many controversies still exist. With the golden era of Andrology, more research can be done to fill the gap in the literature and improve the care for infertile men.
My Personal Viewpoint on Diagnostic Value of Advanced Semen Analysis
Dr. Carlo Giulioni and Prof. Nikolaos Sofikitis respond to questions by Ashok
Q. 1. How do single-strand breaks (SSBs) and double-strand breaks (DSBs) in sperm DNA specifically impact fertilization rates and embryonic development in the context of assisted reproductive technology (ART)?
A 1: Single-strand breaks (SSBs) and double-strand breaks (DSBs) in sperm DNA have been recognized as significant factors affecting fertilization rates and embryonic development, particularly in the context of assisted reproductive technology (ART). SSBs and DSBs can compromise the integrity of paternal genetic material transmitted to the oocyte during fertilization, with a significant association with an increased risk of chromosomal abnormalities and genomic instability within the developing embryo. Furthermore, SSBs and DSBs in sperm DNA may activate cellular pathways involved in apoptosis or senescence, further compromising embryo viability. These cellular responses to DNA damage can disrupt normal embryonic development and contribute to reduced implantation rates and increased rates of pregnancy loss in ART cycles.
The exact mechanisms by which SSBs and DSBs influence fertilization rates and embryonic development in the context of ART are unclear. However, sperm DNA damage exerts detrimental effects on reproductive outcomes by compromising the integrity and functionality of paternal genetic material transmitted to the embryo.
Q 2. What are the limitations of current diagnostic methods for detecting sperm DNA fragmentation (SDF), and how do these limitations affect the interpretation of test results in clinical practice)?
A 2. The contemporary diagnostic approaches for discerning sperm DNA fragmentation (SDF) possess several inherent limitations, which impinge upon the accurate interpretation of test outcomes within clinical settings. Firstly, one notable constraint lies in the diversity of methodologies employed across diagnostic platforms. Discrepancies in sample preparation techniques, assay protocols, and result quantification methodologies among laboratories render comparisons between studies challenging, thereby compromising the establishment of standardized diagnostic thresholds and benchmarks for clinical interpretation. Secondly, the lack of consensus regarding the optimal threshold for categorizing SDF levels as pathological further complicates the interpretation of test results. Divergent cutoff values across studies contribute to ambiguity in clinical decision-making, as clinicians grapple with discerning between normal and abnormal sperm DNA integrity. Moreover, the absence of universally accepted guidelines exacerbates the challenge of implementing consistent diagnostic criteria. Furthermore, inherent biological variability in sperm DNA fragmentation poses a significant obstacle to accurate diagnosis. SDF levels exhibit natural fluctuations within individuals over time. Finally, current diagnostic methods often lack the capability to discern the underlying causes of elevated SDF levels, thereby limiting their clinical relevance. Consequently, the absence of mechanistic insights hampers targeted interventions and personalized treatment strategies, thereby constraining the clinical utility of diagnostic tests for SDF.
Q 3. Considering the repair mechanisms for sperm DNA damage, how does the oocyte's capacity to repair paternal SSBs and DSBs influence the selection of sperm for ART procedures, particularly ICSI?
A3. The capacity of the oocyte to repair paternal single-strand breaks (SSBs) and double-strand breaks (DSBs) in sperm DNA significantly influences the selection of sperm for Assisted Reproductive Technology (ART) procedures, notably Intracytoplasmic Sperm Injection (ICSI). ART techniques such as ICSI bypass natural selection processes that typically occur during natural conception.
Unlike somatic cells, sperm lacks the cytoplasmic machinery required for DNA repair. Consequently, the oocyte's ability to repair DNA damage in sperm becomes a critical determinant in ensuring the genomic integrity of the resulting embryo.
The differential capacity of the oocyte to repair paternal SSBs and DSBs influences sperm selection strategies during ART procedures, especially ICSI. While the oocyte can partially repair DNA lesions, excessive sperm DNA damage can overwhelm its repair machinery, leading to impaired embryonic development, increased miscarriage rates, and potential long-term health consequences for offspring. Consequently, various sperm selection techniques have been developed to identify and isolate sperm with intact DNA, such as sperm DNA fragmentation assays and advanced sperm selection methods based on DNA integrity. By selecting sperm with minimal DNA damage, ART clinics aim to optimize embryo quality and improve reproductive outcomes for couples undergoing fertility treatments.
Q4. Given the evidence suggesting a more pronounced negative impact of DSBs over SSBs on reproductive outcomes, what future research directions are critical for improving ART success rates in cases of significant sperm DNA damage?
A4. The discernible evidence indicating a heightened detrimental influence of Double Strand Breaks (DSBs) compared to Single Strand Breaks (SSBs) on reproductive outcomes underscores the imperative for delineating pivotal future research trajectories aimed at enhancing the success rates of Assisted Reproductive technology (ART) in instances marked by substantial sperm DNA impairment. Several critical avenues for future investigation emerge in this context.
Primarily, comprehensive elucidation of the mechanistic underpinnings governing the divergent impact of DSBs vis-à-vis SSBs on reproductive outcomes constitutes a fundamental research imperative. This necessitates in-depth exploration into the distinct molecular pathways and cellular processes implicated in the repair mechanisms of DSBs and SSBs within the context of sperm DNA damage. Moreover, prospective research endeavors should prioritize the development and validation of refined diagnostic modalities capable of accurately discerning the extent and nature of sperm DNA damage, with a particular emphasis on distinguishing between DSBs and SSBs.
Furthermore, there exists a compelling imperative to explore innovative therapeutic strategies aimed at mitigating the adverse effects of DSBs on sperm function and fertility outcomes. In this regard, the exploration of pharmacological agents targeting key molecular effectors involved in the repair and mitigation of DSBs holds significant therapeutic potential. Concurrently, the integration of emerging biotechnological approaches such as genome editing technologies may offer novel avenues for the precise manipulation and correction of DNA lesions, thereby ameliorating sperm DNA integrity and enhancing ART success rates.
Carlo Giulioni, MD: Short Biography
Carlo Giulioni, MD
Urology consultant
Urology Unit
Casa di Cura Villa Igea
Ancona, Italy
E-mail: carlo.giulioni9@gmail.com
ORCID ID: 0000-0001-9934-4011
Dr. Carlo Giulioni is a distinguished urologist and andrologist, specializing in male fertility and minimally invasive surgical techniques. After graduating with distinction in Medicine and Surgery in 2017, he pursued a specialization in Urology and Andrology. In 2022, he undertook a Fellowship program focusing on robotic and laparoscopic procedures at Clinique Saint Augustin, Bordeaux (France). His residency program was completed with honors in 2023. Throughout his academic journey, Carlo Giulioni has dedicated his research efforts to areas such as male infertility, uro-oncology, and endourology. Carlo has been a member of the Global Andrology Forum and serves as the co-leader of its Research Team 3 under the leadership of Dr. Rossella Cannarella. Currently, he works as a consultant at Casa di Cura Villa Igea, located in Ancona, Italy.
Nikolaos Sofikitis, MD, Ph.D., D.M.Sci: Short Biography
Nikolaos Sofikitis, MD, PhD Professor and Chair of Urology
Department of Urology
Ioannina University School of Medicine,
Ioannina, Greece
Email: v.sofikitis@hotmail.com
ORCID id: 0000-0003-1528-4029
Professor Nikolaos Sofikitis
graduated from the Athens University School of Medicine in 1986, he then embarked on an illustrious career that spans decades and continents. He earned his first Ph.D. diploma in 1993 from the Graduate School of Athens University School of Medicine and secured a second Ph.D. from the Graduate School of Tottori University School of Medicine, Yonago, Japan, the same year. Between 1989 and 1993, he contributed to the field of urology as a teacher at Tottori University School of Medicine, before transitioning into roles such as Assistant Lecturer and Director of the Reproductive Physiology Unit within the same department, positions he held until 2000. His academic journey also took them to the United States, where he served as a Research Instructor at Tulane University School of Medicine and as an Assistant Professor at Cornell University Medical Center. In 2001, he was board-certified as a urologist in Greece and since then, has been the Professor and Chair of the Department of Urology at Ioannina University School of Medicine in Greece. His leadership and expertise were further recognized through his role as the Chair of the European Section of Andrological Urology (ESAU) of the European Association of Urology from 2016 to 2024 and as a board member for various prestigious European urology programs. He has published extensively and has 262 research articles in PubMed-indexed journals; a citation count of 561 and an h-index of 39 (source: Scopus). Niko is a member of the Global Andrology Forum.