Sperm DNA damage and its clinical relevance in assessing reproductive outcome

The routine examination of semen, which assesses sperm concentration, percentage motility and morphology,does not identify subtle defects in sperm chromatin architecture. The focus on the genomic integrity of the male gamete has intensified recently due to the growing concern that genetic diseases may be transmitted via assisted reproductive techniques (ART). Accordingly, the intent of this review is to describe the details of the informationpertaining to mitochondfial/nuclear sperm DNA damage with an emphasis on its clinical significance and its relationship with male infertility. Assessment of sperm DNA damage appears to be a potential tool for evaluating semen samples prior to their use in ART. Testing DNA integrity may help select spermatozoa with intact DNA or with the least amount of DNA damage for use in assisted conception. In turn, this may alleviate the financial, social and emotional problems associated with failed ART attempts.     

Sperm preparation: state-of-the-art--physiological aspects and application of advanced sperm preparation methods

For assisted reproduction technologies (ART), numerous techniques were developed to isolate spermatozoa capable of fertilizing oocytes. While early methodologies only focused on isolating viable, motile spermatozoa, with progress of ART, particularly intracytoplasmic sperm injection (ICSI), it became clear that these parameters are insufficient for the identification of the most suitable spermatozoon for fertilization. Conventional sperm preparation techniques, namely, swim-up, density gradient centrifugation and glass wool filtration, are not efficient enough to produce sperm populations free of DNA damage, because these techniques are not physiological and not modeled on the stringent sperm selection processes taking place in the female genital tract. These processes only allow one male germ cell out of tens of millions to fuse with the oocyte. Sites of sperm selection in the female genital tract are the cervix, uterus, uterotubal junction, oviduct, cumulus oophorus and the zona pellucida. Newer strategies of sperm preparation are founded on: (i) morphological assessment by means of 'motile sperm organelle morphological examination (MSOME)'; (ii) electrical charge; and (iii) molecular binding characteristics of the sperm cell. Whereas separation methods based on electrical charge take advantage of the sperm's adherence to a test tube surface or separate in an electrophoresis, molecular binding techniques use Annexin V or hyaluronic acid (HA) as substrates. Techniques in this category are magnet-activated cell sorting, Annexin V-activated glass wool filtration, flow cytometry and picked spermatozoa for ICSI (PICSI) from HA-coated dishes and HA-containing media. Future developments may include Raman microspectrometry, confocal light absorption and scattering spectroscopic microscopy and polarization microscopy.     

精子DNA损伤与辅助生殖技术

随着辅助生殖技术的广泛开展,精子评估已由传统的精液常规分析向细胞、分子水平深入发展。精子DNA损伤是反映男性生育力的一个新指标。精子DNA损伤的发生机制包括精子染色质包装与分离异常、氧化应激、细胞凋亡异常等。精子染色质结构分析是目前检测精子DNA损伤最常用的方法之一。精子DNA损伤可能与辅助生殖技术治疗结局、复发性自然流产、增加ICSI后代遗传风险相关。采取口服抗氧化药物、取睾丸精子行ICSI、预冻存精子、去除病因以及中医中药等治疗对策可能会降低精子DNA损伤程度,进而提高辅助生殖技术成功率。本文主要就精子DNA损伤的机制与检测方法、DNA损伤与生殖结局以及辅助生殖技术中与DNA损伤相关的治疗对策作一综述。     

Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation

Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze‐thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m  ml^?1. The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short‐term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART.     

Sperm evaluation in cryopreserved bovine semen recovered by two selection methods

Previous experiments have established that various semen manipulation techniques are able to increase the qualitative features of the spermatozoa used in different techniques of assisted reproduction, but practically no comparative data on frozen-thawed bovine semen have been found. The aim of this study was to compare the efficacy of two sperm selection methods: centrifugation on Percoll gradient and filtration through a Sephadex ion-exchange column, to improve the recovery of motile and morphologically normal spermatozoa, without inducing sperm damage, from cryopreserved bovine semen samples. Semen samples were thawed and centrifuged on a discontinuous Percoll gradient, or were filtered through a Sephadex G-15-120 column with the addition of ion exchangers. Sperm concentration, percentages of motile spermatozoa, acrosome integrity, superoxide dismutase activity and lipid peroxidation were evaluated in recovered samples and controls. The motility of spermatozoa obtained by Sephadex ion-exchange filtration (88.87 +/- 6.37%) and by Percoll gradient centrifugation (83.00 +/- 6.21%) were significantly greater than that of control samples (60.14 +/- 8.44%). Other results disclosed that both sperm selection methods significantly increased the percentage of intact acrosome and superoxide dismutase activity. In both cases, the number of recovered spermatozoa diminished significantly versus untreated samples. Although the number of recovered spermatozoa was low, these methods were effective to select viable sperm from cryopreserved bovine semen.     

Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay （SCSA） parameters, that is, DNA fragmentation index （%DFI） and high DNA stainability （%HDS）, were assessed on the flesh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation （DGC） was followed by swimup treatment in comparison with DGC alone （P 〈 0.01）. Although the %DFI increased in a time-dependent manner with incubation both at room temperature （RT） and at 37℃ in air, the %DFI after 24 h at RT was significantly lower than that at 37℃ （P 〈 0.05）. Incubation with 5% CO2 was effective in maintaining sperm motility （P 〈 0.01）; however, it induced further elevation of %DFI （P 〈 0.001）. Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity.     

Association of Zinc deficiency,oxidative stress and increased double-stranded DNA breaks in globozoospermic infertile patients and its implication for the assisted reproductive technique

BackgroundSperm DNA fragmentation and its adverse impact on outcomes of assisted reproductive techniques (ART) in globozoospermic infertile patients has been previously reported. However, the association of Zinc element with DNA damage and intracytoplasmic sperm injection (ICSI) outcome in globozoospermic infertile patients remains unclear.MethodsUsing flame atomic absorption spectrophotometer and superoxide dismutase (SOD) assay, the levels of Cu, Fe, Mn, Zn and SOD activities in seminal plasma from both globozoospermic infertile patients and fertile volunteers were tested respectively. Using sperm chromatin dispersion (SCD) test and Comet assay, the DNA damages in their semen samples from the two groups was detected. In addition, using Aniline Blue staining, their sperm nucleus maturations were also examined.ResultsThe levels of seminal Zinc and SOD activities were lower in the globozoospermic infertile patients and the double-stranded break DFI (DSB-DFI) were significantly higher than that in the fertile controls. Antioxidative insufficiency of SOD with a low Zn level might be responsible for oxidative stress, which may lead to DNA damage in globozoospermic spermatozoa. Zn deficiency might also have influence on the chromatin stabilization of globozoospermic spermatozoa during spermiogenesis, causing its more vulnerable to oxidative attack.ConclusionsSerious DSBs in globozoospermia and antioxidative insufficiency due to Zinc element deficiency in spermatozoa might be responsible for the failure of ICSI in globozoospermia.     

Standardisation of a novel sperm banking kit – NextGen® – to preserve sperm parameters during shipment

Many male patients diagnosed with cancer are within their reproductive years. These men are advised to freeze their spermatozoa prior to the start of cancer treatment. Very often, sperm banking facilities may not be readily available and patients may be required to travel to distant sperm bank centres. Our objective was to design and standardise a remote home shipping sperm kit that allows patients to collect a semen sample at home and ship it overnight to a sperm bank. A total of 21 semen samples and two transport media (refrigeration media and human tubal fluid) and five different combinations of ice packs were tested for maintaining desired shipping temperature. Ten semen samples were assessed for pre‐ and post‐shipment changes in sperm motility, membrane integrity, total motile spermatozoa and recovery of motile spermatozoa. Even though motility, membrane integrity and total motile spermatozoa declined both in samples examined under simulated shipped conditions and in overnight‐shipped samples, the observed motility and total motile spermatozoa were adequate for use with assisted reproductive techniques. Using refrigeration media, cooling sleeve and ice packs, adequate sperm motility can be maintained utilising NextGen^® kit and these spermatozoa can be used for procreation utilising ART techniques such as intracytoplasmic sperm injection.     

Semen processing by density gradient centrifugation is useful in selecting sperm with higher double-strand DNA integrity

The objective of this study was to determine the effects of density gradient centrifugation on sperm cell DNA integrity and to correlate any detected DNA damage with semen analysis parameter. A total of 40 semen samples were collected from nonazoospermic men presenting for infertility evaluation at our department. Individual samples were divided into two parts: one part of the semen was washed and the remainder was prepared using the PureSperm density gradient centrifugation. Sperm DNA fragmentation as evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay, was monitored in the initially washed sample and in the different layers of the density gradient centrifugation. No significant correlations were observed between sperm DNA fragmentation, age of patient, concentration and motility. However, a significant correlation existed with strict spermatic morphology. Following density gradient centrifugation, the proportion of spermatozoa with DNA fragmentation decreased significantly when compared with whole semen. In addition, we found that spermatozoa isolated in the 90% layer possessed a significantly lower percentage of DNA damage when compared with those remaining in the 70% and 50% layers. These results demonstrate that semen processing by the PureSperm gradient is useful in selecting sperm with higher double-strand DNA integrity.     

Implications of cryopreservation on structural and functional attributes of bovine spermatozoa: An overview

Sperm cryopreservation is an important adjunct to assisted reproduction techniques (ART) for improving the reproductive efficiency of dairy cattle and buffaloes. Improved understanding of mechanisms and challenges of bovine semen cryopreservation is vital for artificial insemination on a commercial basis. Although cryopreservation of bovine spermatozoa is widely practiced and advanced beyond that of other species, there are still major gaps in the knowledge and technology. Upon cryopreservation, disruption of spermatozoal plasma membrane configuration due to alterations in metabolic pathways, enzymes and antioxidants activity add to lower efficiency with loss of sperm longevity and fertilising ability. Therefore, the effective amalgamation of cryo-variables like ambient temperature, cooling and thawing rates, nucleation temperature, type and concentration of the cryoprotectant, seminal plasma composition, free radicals and antioxidant status are required to optimise cryopreservation. Novel strategies like supplementation of cholesterol-loaded cyclodextrins (CLC), nanovesicles, osteopontin, antioxidants, etc., in an extender and recent techniques like nano-purification and modified packaging have to be optimised to ameliorate the cryodamage. This article is intended to describe the basic facts about the sperm cryopreservation process in bovine and the associated biochemical, biophysical, ultra-structural, molecular and functional alterations.     

异黄酮辅剂对解冻精子特征的影响

本研究评价了使用异黄酮作为辅剂的解冻剂对解冻精子精液参数的影响。我们分析了辅剂在解冻过程中对精子活力、精子获能能力（膜脂功能紊乱）、活性氧生成、精子核浓缩及DNA损害的影响。根据初步的数据,辅剂可能会改善解冻过程,减少细胞损伤。我们已经证实异黄酮在精子解冻过程中有抗氧化作用、能够减少活性氧的生成,有利于精子活力轻微提高,降低膜脂功能紊乱和由冷冻而引起的DNA破坏。结果显示异黄酮作为辅剂有助于提高精子功能,这对于辅助生育中使用解冻精子很有意义。我们的发现有必要进一步研究来确证并评价临床应用的可能性。     

精子DNA损伤与辅助生殖技术

精子DNA损伤与辅助生殖技术(ART)结局密切相关,精子DNA损伤影响到ART中的受精比率、胚细胞发育、妊娠率和子代的健康。同时,精子低温冷冻保存和诸多体外处理技术均能影响精子DNA的完整性。检测精子DNA断裂指标(DFI),采取适当措施获得DNA无损伤或损伤较小的精子,对改善ART结局十分必要。现就精子DNA损伤与ART相关性的研究做一综述。     

精子DNA完整性在男性不育中的作用

精子DNA完整性对自然受精或人工授精 ,胚胎、胎儿和婴儿的发育至关重要。研究表明 ,夫妇中丈夫精子DNA受损率超过 30 %时 ,自然受精率很低。精子DNA完整性被认为是优于传统精液参数的男性不育指标 ,已经有多种方法可用于检测 ,而且监测精子DNA完整性可用于评估欲行辅助生殖技术 ,如卵细胞胞质内单精子注射 (ICSI)治疗的夫妇。最后对精液冷冻和制备技术对精子DNA完整性的影响进行了总结。     

精子冻融技术及其研究进展

精子冻融是辅助生殖技术的基础,冻融可能造成精子结构损伤和活力功能丧失,因此优化冻融方法十分必要。提高冷冻前精液质量、进行精子优选、添加合适的冷冻保护剂、选择适宜的冷冻复苏方法等均可有效地减少冻融损伤,有利于保存男性生育力。本文对精子冻融的基本原理、方法改良、针对特殊质量精液的冷冻方法及冷冻后精子质量评估进行了综述。     

Effects of post‐density gradient swim‐up on apoptosis signalling in human spermatozoa

The inclusion of apoptotic spermatozoa during assisted reproductive techniques (ART) may be one reason for suboptimal success rates. The aim of our study was to evaluate the potential of routine semen preparation to eliminate spermatozoa with activated apoptosis signalling. Semen samples from 20 infertility patients scheduled for ART procedures were investigated. Following density gradient centrifugation (DGC) and swim‐up, aliquots were taken from each sample to analyse motility, Caspase‐3 activation (CP3) and integrity of the mitochondrial membrane potential (MMP) using flow cytometry. Aliquots from the neat semen served as controls. Semen samples of patients contained 53.8 ± 17.7% spermatozoa with disrupted MMP and 51.8 ± 14.9% with active CP3. Preparation by DGC and swim‐up resulted in improvement of progressive motility (+43.5%) and reduction of spermatozoa with disrupted MMP (?34.3%) and activated CP3 (?25.7%, P < 0.01). Minimal reduction of spermatozoa with disrupted MMP and active CP3 was 6.0% and 0.7%, maximum reduction was 65.5% (disrupted MMP) and 49.3% (CP3). Semen samples of subfertile patients contain high levels of spermatozoa with activated apoptosis signalling. Although there was a reduction in the majority of the samples, profound interindividual differences in the separation effect demand further development of innovative molecular‐based separation methods to deplete apoptotic spermatozoa.     

ART success and in vivo sperm cell selection depend on the ultramorphological status of spermatozoa

Management of male infertility has recently shifted from treatment of the subfertile man towards techniques of assisted reproduction (ART). This study aimed to evaluate the possible role of the ultramorphological status of the spermatozoon with respect to sperm selection in vivo and prediction of ART success. Ultramorphological sperm parameters were assessed retrospectively for 92 males with sufficient sperm density (10⁷ spermatozoa ejaculate⁻¹) whose wives conceived following a stepwise discarding of the female genital tract barriers, using intra-uterine insemination (IUI) ( n =26), in vitro fertilization (IVF) ( n =45) or intracytoplasmic sperm injection (ICSI) ( n =21). In parallel, sperm samples of 71 fertile males were examined. Normal ultramorphology of all head and tail subcellular organelles was found to be essential for the ability of spermatozoa to pass the lower female genital tract. The ultramorphological migration threshold for this barrier is apparently higher than that essential for oocyte fertilization. No specific indication associated with passage through the upper genital tract was found. A high prevalence of axonema defects was found to impair the ability of sperm cells to penetrate the oocyte investment. The natural fertility index, based on routine sperm parameters and the ultrastructural status of the spermatozoon's subcellular organelles was confirmed to be beneficial for directing patients to ART. A discriminative score based on axonema integrity was found to contribute additional information for the first choice decision between conventional ART and ICSI (75% prediction ability). Thus it may be helpful in finding the simplest and least expensive procedure with the greatest long-term chance for pregnancy.     

Freezing-free preservation of human spermatozoa--a pilot study

This study tested a method for maintaining human spermatozoa without freezing for subsequent use in intracytoplasmic sperm injection (ICSI). We demonstrated that human sperm stored in electrolyte-free solution maintain their motility and viability for at least 4 and 6 weeks, respectively. We also have shown that preserved spermatozoa are fully functional in ICSI. Sperm chromosome analysis after injection of human sperm into mouse oocytes revealed that two weeks of storage does not negatively affect sperm DNA integrity. A mouse model was used to analyze the ability of preserved sperm to participate in normal embryogenesis. Mouse sperm preserved in electrolyte-free solution in a similar manner as human sperm maintained motility for up to 3 weeks. When mouse spermatozoa stored for 1 week were injected into the oocytes by ICSI, they yielded normal blastoctysts and normal viable fetuses. The results of the study bear significance for human assisted reproduction technologies (ART) and provide clinicians and infertile patients with a new method that can simplify sperm preparation for ICSI, assisting men who are unable to provide semen on the day of assisted fertilization.     

Negative biomarker-based male fertility evaluation: sperm phenotypes associated with molecular-level anomalies

Biomarker-based sperm analysis elevates the treatment of human infertility and ameliorates reproductive performance in livestock. The negative biomarker-based approach focuses on proteins and ligands unique to defective spermatozoa, regardless of their morphological phenotype, lending itself to analysis by flow cytometry (FC). A prime example is the spermatid specific thioredoxin SPTRX3/TXNDC8, retained in the nuclear vacuoles and superfluous cytoplasm of defective human spermatozoa. Infertile couples with high semen SPTRX3 are less likely to conceive by assisted reproductive therapies (ART) and more prone to recurrent miscarriage while low SPTRX3 has been associated with multiple ART births. Ubiquitin, a small, proteolysis-promoting covalent posttranslational protein modifier is found on the surface of defective posttesticular spermatozoa and in the damaged protein aggregates, the aggresomes of spermiogenic origin. Semen ubiquitin content correlates negatively with fertility and conventional semen parameters, and with sperm binding of lectins LCA (Lens culinaris agglutinin; reveals altered sperm surface) and PNA (Arachis hypogaea/peanut agglutinin; reveals acrosomal malformation or damage). The Postacrosomal Sheath WWI Domain Binding Protein (PAWP), implicated in oocyte activation during fertilization, is ectopic or absent from defective human and animal spermatozoa. Consequently, FC-parameters of PAWP correlate with ART outcomes in infertile couples and with fertility in bulls. Assays based on the above biomarkers have been combined into multiplex FC semen screening protocols, and the surface expression of lectins and ubiquitin has been utilized to develop nanoparticle-based bull semen purification method validated by field artificial insemination trials. These advances go hand-in-hand with the innovation of FC-technology and genomics/proteomics-based biomarker discovery.     

Testicular versus ejaculated spermatozoa in ICSI cycles of normozoospermic men with high sperm DNA fragmentation and previous ART failures

As a part of male assessment, conventional sperm parameters including morphologic features have been dedicated as major factors influencing fertilisation and pregnancy rates in assisted reproductive technology (ART). Genomic integrity of spermatozoa has also been found to influence fertility prognosis, and hence, sperm DNA fragmentation index (DFI) has been adopted by many centres to document this entity. Despite several suggested approaches, there is lack of universal consensus on optimising fertility outcomes in males with high sperm DFI. In this context, the results from cycles using testicular spermatozoa (TESA) obtained by aspiration were compared with those of ejaculated spermatozoa (EJ) in normozoospermic subjects with high sperm DFI and previous ART failures. Clinical (41.9% versus 20%) and ongoing pregnancy rates (38.7% versus 15%) were significantly better and miscarriages were lower in TESA group when compared to EJ group. Sperm DFI should be a part of male partner's evaluation following unsuccessful ART attempts. When high DFI is detected (>30%), ICSI using testicular spermatozoa obtained by TESA seems an effective option particularly for those with repeated ART failures in terms of clinical, ongoing pregnancies and miscarriages even though conventional sperm parameters are within normal range.