Male factors have accounted for nearly 50% of the causes of infertility. A variety of factors affect male fertility, among which sperm DNA damage has been one of the hot spots of research in the field of reproductive medicine in recent years. Studies have shown that infertile men with abnormal semen parameters have higher sperm DNA breakage index (DFI) than fertile men, and idiopathic infertility patients with normal semen parameters also have increased DFI; spontaneous pregnancy is almost impossible when DFI ≥ 30%; DFI is stable and can be used as a baseline indicator to predict male fertility. In (assisted reproductive technology) ART clinics, sperm DNA damage is one of the most important factors affecting its outcome and is receiving increasing attention from reproductive medicine practitioners. Testing sperm DNA breakage indicators (DFI) before performing ART and taking measures to obtain sperm with little or no DNA damage are necessary to improve ART outcomes. The purpose of this article is to review the causes, mechanisms, and countermeasures of sperm DNA damage and to highlight its relevance to male infertility. Tan Guangxing, Department of Urology, Wuxi Hospital of Traditional Chinese Medicine I. Causes of sperm DNA damage A range of environmental factors, contaminants and medical measures can affect the integrity of sperm DNA and lead to sperm DNA damage. The main ones are as follows: 1. Drugs and radiotherapy Young men with cancer (how to gold’s lymphoma and testicular cancer) tend to have poorer quality sperm and severe sperm DNA damage. The use of chemotherapy drugs, and with the accumulation of drug doses, can lead to absolute sterility, mainly because of the toxic effects of chemotherapy drugs on testicular spermatogenic cells. Damage to testicular spermatogonia and sperm DNA damage due to radiotherapy is related to the duration of treatment the patient receives and the dose of the drug. The recovery of spermatogenic function of testicular spermatogenic cells often occurs 1 month to 1 year after treatment, and studies have found that sperm DNA damage occurs throughout this process.2. Inflammation of the reproductive tract Infection and inflammation of the reproductive tract behind the testes (e.g., testicular epididymitis, prostatitis) can lead to the appearance of leukocytic spermatozoa, an enhanced oxidative stress response, and consequently to sperm DNA damage.3. High temperature in the testes Studies have demonstrated that The study has proven that heat-related illnesses can lead to an increase in the histone to ichthyosperm ratio of sperm ejected from the body, aggravating sperm DNA damage. This is also true for localized testicular heat, as some specific behaviors can increase the temperature of the scrotum, such as hot baths, sitting in front of a computer, and driving for long periods of time, which can lead to sperm DNA damage. 4, varicocele Varicocele is closely related to sperm DNA damage, and the extent of the damage is related to the high levels of oxidative stress found in the semen of patients with varicocele. Recent studies have shown that large numbers of developmentally arrested sperm cells with high levels of reactive oxygen products were found in the semen of patients with varicocele infertility and that these abnormally developed sperm cells were associated with DNA damage. Moreover, the integrity of sperm DNA was significantly improved after varicocele was treated.5. Hormone levels Experimental studies have shown that hormone abnormalities can lead to chromatin defects, and compared with wild mice, mice with knocked-out hormone receptors have lower sperm ichthyosperm protein levels, lower testosterone levels, impaired fertility, and elevated levels of DNA damage. Second, the mechanism of sperm DNA damage (a), sperm mitochondrial DNA damage Sperm mitochondrial DNA is naked DNA, lacking the protection of histones and DNA binding proteins; at the same time, its own ability to repair damage is very low, and it is extremely sensitive to various damage factors, especially reactive oxygen species (ROS). Therefore, spermatozoa are particularly susceptible to different forms of mitochondrial DNA damage during maturation, including deletion, mutation and polymorphic metamorphosis. The presence of a high prevalence of mitochondrial DNA damage in abnormal semen samples has been shown to be indicative of its role in male infertility. (ii) Nucleosomal DNA damage: [1] Oxidative stress: In various organs and tissues of the reproductive system, the production of ROS is a physiological phenomenon, and small amounts of appropriate ROS play an important role in sperm physiology, contributing to sperm capacitation and acrosome response. However, when the presence of large amounts of ROS exceeds the scavenging capacity of the antioxidant system and the defense capacity of the unique compact structure of the sperm nucleus, it leads to single- or double-strand breaks in sperm DNA, resulting in sperm DNA damage. Moreover, because sperm membranes contain high concentrations of unsaturated fatty acids and low levels of purifying enzymes in the plasma membrane, they are prone to lipid peroxidation under the attack of excess reactive oxygen species, which causes fatty acids to lose their double bonds, and then the sperm membrane loses its fluidity and changes the internal environment, finally leading to sperm nucleus DNA mutation or breakage. In addition, the sperm DNA matrix is susceptible to oxidative damage. It has been reported that DNA breakage increases significantly when spermatozoa are exposed to exogenous (artificial) ROS in the form of all base pattern, generation of base-free sites, deletion, DNA cross-linking and chromosomal reorganization. [2] Sperm chromatin assembly: abnormal sperm chromatin assembly leads to sperm single- and double-stranded DNA breaks and DNA damage in the sperm nucleus, the main link of which is abnormal sperm protein substitution for histones. During spermatogenesis, chromatin assembly requires the involvement of endogenous nucleases (topoisomerase II) to establish and connect DNA gaps, which helps to release torsionalstress and chromatin reorganization during the replacement of histones by fish sperm proteins, but may also cause abnormal spermatogenesis or DNA damage. Abnormalities in chromatin assembly leading to DNA damage in the sperm nucleus may also be due to abnormal DNA double-strand breaks, as DNA double-strand breaks can occur naturally during spermatogenesis in the preparation for chromatin recombination and during chromatin assembly. [3] Apoptosis: During spermatogenesis, apoptosis controls the level of spermatogenesis and proliferation to match the supporting capacity of supporting cells and maintain the balance of spermatozoa in number, morphology and function. The spermatogenic cell surface protein Fas initiates sperm apoptosis, and in combination with Fas ligand (FasL) or excitatory anti-Fas antibody agonists can initiate the apoptotic program to kill spermatogenic cells. Support cells express FasL, thereby limiting excessive proliferation and clearing Fas-positive sperm. The percentage of Fas-positive sperm in fertile men is small, while the percentage of Fas-positive sperm in those with abnormal semen parameters is as high as 50%, suggesting that their apoptosis may be abnormal and their ability to clear DNA-damaged sperm is insufficient [1]. Another mechanism of abnormal apoptosis leading to sperm DNA damage is related to photogate proteases. Blockage of FasL/Fas in endogenous mitochondrial membranes leads to activation of photogate proteases 8 and 9, which conduct photogate protease receptor signals, which in turn lead to activation of deoxyribonuclease (DNA breakage factor 40) and ultimately to sperm DNA breakage. The rate of Fas positivity was higher in spermatozoa of fertile men. Numerous studies have confirmed that weak and malformed spermatozoa have higher levels of DNA damage, again demonstrating that apoptosis is closely related to sperm DNA damage. [4] Fisetin defects: When fisetin is defective, the disulfide bonds are abnormal, making it unable to bind tightly to DNA, which leads to a loose chromatin structure, unstable double-stranded structure, breakage or denaturation to single-stranded in an acidic environment, eventually leading to sperm DNA damage. aoki et al. confirmed that fisetin-1 (P1) or fisetin -2 (P2) concentrations were significantly higher in infertile patients, and that sperm DNA damage was significantly higher at reduced P1/P2 values than at normal and increased P1/P2 values [. Therefore, fisetin defects are also a cause of sperm DNA damage. Third, sperm DNA damage detection methods Currently, the main methods for detecting sperm DNA damage are: Comet technique (Comet), end-transferase-mediated deoxyuridine triphosphate (dUTP) end-labeling (TUNEL), sperm chromatin structure analysis (SCSA), maiden orange test (AOT) and in situ incision translation (NT). They can be used to detect defects in chromatin structure and DNA integrity in spermatozoa, with TUNEL and Comet detecting single and double strand breaks, SCSA detecting chromatin structure abnormalities, and NT detecting DNA single strand breaks. These are not yet used as routine methods in clinical practice, and studies with a sufficient number of samples are needed to confirm their usefulness and significance. IV. Sperm DNA damage and assisted reproductive technology In the past 10 years, assisted reproductive technology (ART) has made breakthroughs, especially the application of single sperm intracytoplasmic injection (ICSI) technology, which has led to a major breakthrough in the treatment of severe oligospermia, weakness, and malformations. However, numerous studies have shown that in ART, sperm DNA damage affects fertilization rates, embryonic cell development, and pregnancy probability, although the mechanisms are not fully understood, which has led to increasing attention to the relationship between sperm DNA damage and ART.1. Sperm source In patients with obstructive azoospermia undergoing ICSI, a significantly higher DFI was found when sperm were extracted from the epididymis and testis. This may be due to the prolonged retention of sperm in the obstructed reproductive ducts or to incomplete depolymerization of sperm chromatin DNA, which is susceptible to damage and toxic substances. At the same time, it has been found that the level of sperm DNA damage in the testis of patients with obstructive azoospermia is significantly lower than that of sperm near the epididymis. Ermanno et al. compared the DNA damage level of testicular sperm with that of ejaculated sperm as samples and performed ICSI with them separately. The pregnancy rate after ICSI was also significantly higher in the former than in the latter, although the difference in the chance of fertilization was not significant. It was concluded that ICSI using testicular sperm is the most effective means of ART in patients with high levels of sperm DNA damage. However, Bukulmez and Zhao et al. compared the outcomes of ICSI with ejaculated sperm, epididymal sperm and testicular sperm without assessing sperm DNA integrity and found no statistical differences in fertilization rate, oogenesis rate, quality embryo rate, clinical pregnancy rate and early miscarriage rate. Therefore, further studies are needed to investigate the effects of different sources of sperm and sperm DNA damage on the outcome of ICSI. 2. Intrauterine injection Numerous studies have confirmed that sperm DNA damage affects the outcome of intrauterine injection (IUI). Among these studies, DFI was assessed using sperm chromatin structure analysis (SCSA), and the chance of fertilization in IUI was close to zero when it was greater than 30%. In addition, pregnancy cannot occur when the DFI is > 12%, as assessed by the terminal transferase-mediated dUTP end-labeling (TUNEL) method, and results in miscarriage when it reaches between 10% and 12%. Therefore, in IUI, assessment of DFI has a better prognostic role.3. In vitro fertilization In in vitro fertilization (IVF), sperm DNA damage affects the chance of fertilization and blastocyst development. Also, embryo quality is a better indicator of pregnancy in IVF compared to conventional sperm parameters, and there is a significant negative correlation between sperm DNA damage and embryo quality. henkel et al. reported a significant decrease in pregnancy rate in IVF when DFI >36.5%, and tomlinson et al. showed that a DFI threshold of 27% in IVF is necessary for a successful pregnancy. Therefore, DFI may be a better prognostic assessment in IVF.4. Single sperm intracytoplasmic injection In ICSI, although sperm DNA damage does not prevent fertilization and postnuclear somatic forms from occurring, and pregnancy can even be achieved using sperm with higher DNA damage, there is still a significant negative correlation between DNA damage and fertilization and pregnancy rates.Virro et al. found in performing ICSI that when DFI Virro et al. found that when ICSI was performed at > 30%, low blastocyst rates were likely to occur and pregnancy did not occur. Clinically, the successful fertilization rate of ICSI is usually not higher than 65%-80%, probably due to DNA defects in sperm. When ICSI is performed with DNA-deficient sperm, which are injected directly into the egg, it is important that the genetic material of the sperm is intact, as it bypasses natural selection and can affect embryonic cell and embryo development as well as the health of the offspring. Therefore, it is essential that sperm DNA be evaluated before ICSI is performed.5. Cryopreservation techniques Numerous reports have shown that freezing and thawing of sperm can compromise sperm DNA integrity in many sperm cryopreservation techniques. Moreover, the results of sperm cryoprotection tests in the absence of sperm cryoprotection have shown that freezing and thawing of spermatozoa can impair the integrity of sperm DNA compared to fresh sperm (19±16%, p