Serum levels of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) in relation to markers of reproductive function in young males from the general Swedish population

A time-related deterioration in male reproductive function caused by exposure to endocrine disrupters, including persistent organochlorines (POCs), has been hypothesized. In animal studies, POCs were found to have adverse effects on male reproductive function. However, little is known about the impact of POC exposure on reproductive parameters in men. In a study of 305 young Swedish men 18-21 years old from the general population, we correlated lipid-adjusted serum levels of 2,2',4,4',5,5' -hexachlorobiphenyl (CB-153)--an index substance for POC exposure--to markers of male reproductive function: testis size assessed by ultrasound, sperm concentration, total sperm count, sperm motility assessed manually and with a computer-aided sperm analyzer (CASA), and serum levels of follicle-stimulating hormone, inhibin B, testosterone, sexual hormone-binding globulin (SHBG), luteinizing hormone, and estradiol. We found weak but statistically significant, negative correlations between CB-153 levels and both the testosterone:SHBG ratio (r = -0.25, p < 0.001)--a measure of the biologically active free testosterone fraction--and CASA sperm motility (r = -0.13, p = 0.02). No statistically significant association with other seminal, hormonal, or clinical markers of male reproductive function was found. In previous studies of more highly POC-exposed groups of adult men, the correlation between POC exposure, including CB-153, and free testosterone levels was not statistically significant. The present study gives some tentative support for weak negative effects of CB-153 exposure on sperm motility and free testosterone levels in young men, but further semen studies on more highly exposed groups may give more firm conclusions on the hazard for male reproductive function from dietary POC exposure.     

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?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.     

Human Follicular Fluid Stimulates the Motility of Washed Human Sperm

Human follicular fluid (hFF) was collected by laparoscopic oocyte pickup during IVF to evaluate the effect of hFF on human sperm motility with a transmembrane migration method. Freshly ejaculated human sperm were washed with phosphate buffered saline (PBS) and mixed with either PBS or hFF. Amplitude of motility increases were 38% and 72% in washed fertile sperm and washed asthenozoo-spermic sperm when individual control motility was considered to be 100%. The stimulatory effect of hFF was lost when preheated at 100°C for 30 minutes. hFF collected from mature follicles stimulated sperm motility better than that collected from intermediate or immature follicles. hFF did not stimulate the motility of unwashed sperm in freshly ejaculated human semen. A heat labile factor(s) in hFF may stimulate the motility of washed human sperm. Whether this factor could be used to improve the success rate of IVF and artificial insemination awaits further investigation.     

Oocyte quality determines bovine embryo development after fertilisation with hydrogen peroxide-stressed spermatozoa

Exposure of gametes to specific stressors at sublethal levels can enhance the gametes' subsequent performance in processes such as cryopreservation. In the present study, bull spermatozoa were subjected to H?O? for 4 h at 100-, 200- and 500-μM levels; computer-assisted sperm analysis (CASA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay were used for evaluation of subsequent sperm motility and DNA integrity, respectively. Exposure of spermatozoa to H?O? did not affect sperm motility but DNA integrity was negatively affected by 500 μM H?O? compared with mock-exposed spermatozoa, whereas both motility and DNA integrity were affected compared with untreated spermatozoa. Nevertheless, insemination of oocytes with spermatozoa exposed to 200 μM H?O? increased fertilisation, cleavage and blastocyst rates (P < 0.05). Furthermore, the higher blastocyst yield after fertilisation of oocytes with spermatozoa exposed to 200 μM H?O? was related to oocyte diameter, with large-medium oocytes yielding higher blastocyst rates, while small-diameter oocytes consistently failed to develop into blastocysts. In conclusion, the results indicate that exposure of spermatozoa to 200 μM H?O? before sperm-oocyte interaction may enhance in vitro embryo production in cattle. However, this increased embryo production is largely dependent on the intrinsic quality of the oocytes.     

EFFECT OF SEMINAL PLASMA CALCITONIN LEVELS ON SPERM MOTILITY

This study investigated the effect of the seminal and blood plasma calcitonin levels on the sperm motility in idiopathic infertile patients. The number of sperm cells and their motility were evaluated in the spermiograms of 52 idiopathic infertile patients. The levels of seminal plasma calcitonin were studied with double antibody technique using a DPC kit. Fifty-two patients were divided into 2 groups according to the motility rates of sperm and 20 healthy volunteers were assigned to a control group. The difference between the groups was evaluated by using Kruskall-Wallis and Mann-Whitney U tests, and the correlation of seminal and blood calcitonin levels with sperm motility were determined. The difference in motility rates between the 3 groups was statistically significant ( p =. 000, p <. 05). Blood plasma calcitonin levels were in normal ranges in all cases and no significant difference was found among the 3 groups ( &#104 2 = 2.7219, p =. 2589, p >. 05). While sperm motility was correlated with seminal calcitonin levels ( r =. 8581), blood calcitonin levels did not show a correlation with sperm motility rate ( r = -.0265). Moreover, there was no correlation between seminal and blood plasma levels of calcitonin ( r = -.0010). Motility rates decreased in the patients with low seminal calcitonin levels and seminal calcitonin levels had a significant effect on sperm motility.     

丙烯腈对小鼠精子生成的影响

目的　研究丙烯腈对小鼠精子生成的影响。方法　经皮下注射丙烯腈３５ 天, 检测雄性小鼠的精子数量、活动度、精子畸形率。结果　剂量达６ｍｇ·ｋｇ －１ｂｗ 时, 小鼠精子活动度即受影响, 且随染毒剂量增加直线运动精子数明显减少（ｒ ＝ － ０－５７ , Ｐ＜ ０－０１） , 静止精子数增加（ ｒ＝ ０－３０ , Ｐ＜ ０－０５） ; 精子畸形率亦增高, 且存在剂量效应关系（ｒ ＝ ０－５２ ; Ｐ＜ ０－０１） 。结论　丙烯腈对小鼠的精子生成和发育具有一定的损害作用。     

Treated Wastewater Effluent Reduces Sperm Motility Along an Osmolality Gradient

Many toxic effects of treated wastewater effluent on organismal and reproductive health have been documented. However, the physicochemical environment of treated wastewater effluent frequently differs considerably from that of its receiving waters and may affect organismal function independently of toxic effects. Teleost sperm, for example, may be affected by the higher osmolality of treated wastewater, as this sperm is activated for a brief period of time following ejaculation due to the sudden decrease in osmolality of its surrounding environment. In this study, we examined the effects of treated wastewater effluent on sperm motility to test the hypothesis that the higher osmolality of effluent compared to river water will adversely affect sperm activation in a concentration-dependent relationship. Treated wastewater effluent was collected on 5 days from the outflow of the Metropolitan Wastewater Treatment Plant, St. Paul, Minnesota, and from an upstream site on the Mississippi River. Milt aliquots collected from goldfish were diluted in an isotonic extender solution and subsequently activated in either deionized water, 100%, 50%, or 10% effluent, a synthetic ion mixture, or river water. Sperm motility and velocity were assessed at 15-s intervals for 1 min using a computer assisted sperm analyzer. Significant differences in performance parameters were found only at 15 s, with sperm motility and velocity declining rapidly at later sampling times. Predictably, deionized water resulted in the greatest activation of sperm motility, while motility exhibited a concentration-dependent decline in 10%, 50%, and 100% treated wastewater effluent. Interestingly, Mississippi River water and a synthetic ion mixture with an osmolality comparable to 50% effluent both resulted in the least amount of sperm activation. However, sperm activation in river water varied between collection days during the study. River water and 100% effluent both had low sperm activation characteristics despite a 10-fold difference in osmolality between these two treatments (1 and 10 mOsmol kg⁻¹, respectively). Results of this study indicate a concentration-dependent decrease in sperm motility in treated wastewater effluent as well as significant fluctuations of sperm activation in Mississippi River water. This study illustrates the complexity of assessing the effects of treated wastewater effluents and the difficulty of determining appropriate reference sites for such studies.     

Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa

In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved samples staining positive with propidium iodide, Hoechst H33258 and Trypan blue; these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1 : 6 or 1 : 20) with or without 0.1-5.0 mM theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800 g were associated with a significant reduction in motility (mean 56 +/- 3% decreasing to 27 +/- 3%), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures; osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates; and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mM theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.     

Intracytoplasmic sperm injection in the bovine induces abnormal [Ca2+]i responses and oocyte activation

Fertilisation by intracytoplasmic sperm injection (ICSI), a technique that bypasses the membrane fusion of the gametes, has been widely used to produce offspring in humans and mice. Success with this technique has lent support to the hypothesis that in mammalian fertilisation, a factor from the sperm, the so-called sperm factor, is responsible for oocyte activation and that the fusion process is not involved in the generation of the hallmark [Ca2+]i signalling seen following fertilisation. However, the success of ICSI has largely eluded large domestic species, such as the bovine, porcine and equine, casting doubt on the current model of oocyte activation at fertilisation in these species. Using Ca2+ imagery and a series of treatments to manipulate the chemical structure of the sperm, we have investigated the early events of oocyte activation in response to ICSI in the bovine. Our results demonstrate, for the first time, that following ICSI, the majority of bovine oocytes are unable to mount [Ca2+]i oscillations, although, in few cases, the initiation of [Ca2+]i oscillations can occur in a manner indistinguishable from in vitro fertilisation. We also show that bull sperm possess a full complement of sperm factor. However, either the release and/or activation of the sperm factor are compromised after ICSI, leading to the delivery of a defective Ca2+ stimulus, which results in premature termination of embryo development.     

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte(-1) h(-1)), in vitro- matured (0.93 pmol oocyte(-1) h(-1)) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte(-1) h(-1)) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17-19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.     

十一酸睾酮对人精浆蛋白的影响

本文采用SDS—PAGE法,测定7例健康育龄男子在肌肉注射十一酸睾酮(TU)前,用药后l、2、3个月及停药后精子计数恢复正常时的精浆蛋白浓度和组份变化,并结合血清测定激素水平及常规精液分析,以探讨TU对上述指标的影响及其相互关系。结果显示:用药早期精浆总蛋白浓度开始逐步降低,在停药后精子计数恢复正常时仍未达到正常水平。精浆总蛋白浓度的变化与精子活动率、前向运动率及LH呈正相关(r值分别为0.8975、0.7776、0.6406,P<0.01或0.05)。精浆蛋白组份也发生变化,其中51KD、37KD的蛋白条带变化最为明显,其变化除与精子活动率相关外(r=0.9094,P<0.01),其中51KD蛋白条带的变化还与精子前向运动率相关(r=0.7660,P<0.05)。上述结果提示TU可能在抑制生精之前即通过改变精浆蛋白而影响精子的功能,说明单用睾酮(T)的避孕机理是多因素的。     

Osmolality of preterm formulas supplemented with nonprotein energy supplements

BACKGROUND: Addition of energy supplements to preterm formulas is an optional strategy to increase the energy intake in infants requiring fluid restriction, in conditions like bronchopulmonary dysplasia. This strategy may lead to an undesirable increase in osmolality of feeds, the maximum recommended safe limit being 400 mOsm/kg. The aim of the study was to measure the changes in osmolality of several commercialized preterm formulas after addition of glucose polymers and medium-chain triglycerides. METHODS: Osmolality was measured by the freezing point depression method. Six powdered formulas with concentrations of 14 g/100 ml and 16 g/100 ml, and five ready-to-feed liquid formulas were analyzed. All formulas, were supplemented with 10% (low supplementation) or 20% (high supplementation) of additional calories, respectively, in the form of glucose polymers and medium chain triglycerides, maintaining a 1:1 glucose:lipid calorie ratio. Inter-analysis and intra-analysis coefficients of variation of the measurements were always < 3.9%. RESULTS: The mean osmolality (mOsm/kg) of the non-supplemented formulas varied between 268.5 and 315.3 mOsm/kg, increasing by 3-5% in low supplemented formulas, and by 6-10% in high supplemented formulas. None of the formulas analyzed exceeded 352.8 mOsm/kg. CONCLUSION: The supplementation of preterm formulas with nonprotein energy supplements with up to 20% additional calories did not exceed the maximum recommended osmolality for neonatal feedings.     

A perspective on the role of emerging technologies for the propagation of companion animals, non-domestic and endangered species

Assisted reproductive technologies (ART) have been used successfully in humans, domestic and laboratory species for many years. In contrast, our limited knowledge of basic reproductive physiology has restricted the application of ART in companion animal, non-domestic and endangered species (CANDES). Although there are numerous benefits, and in some cases a necessity, for applying ART for the reproductive and genetic management of CANDES, the challenges encountered with even the most basic procedures have limited the rate of progress. In this foreword we discuss the status of conventional ART, such as artificial insemination and in vitro fertilisation, as well as their benefits and inherent difficulties when applied to CANDES. It is upon these techniques, and ultimately our knowledge of basic reproductive physiology, that the success of emerging technologies, such as those described in this special issue, are dependent for success.     

职业接触拟除虫菊酯工人精子浓度的调查

目的 调查拟除虫菊酯暴露与工人体内氧化应激状态和精子浓度的关联性,为制定拟除虫菊酯职业暴露引起男性职业性生殖毒性损害的诊断标准提供依据。方法 2020年9—10月,选取武汉市某拟除虫菊酯类农药工厂一线岗位234名成年男性工人为暴露组,117名成年男性行政后勤人员为对照组,收集调查对象的一般人口学信息并采集尿液5 mL,检测尿中3-苯氧基苯甲酸（3-PBA）、8-羟氧基苯甲酸（8-OHdG）浓度;采集调查对象禁欲3 d后的精液,测量精子浓度。同时检测该工厂生产工人主要接触的化学有害因素浓度。结果 生产工人主要接触的化学有害因素为：溴氰菊酯、氯氰菊酯、高效氯氰菊酯和顺式氯氰菊酯。暴露组人员的3-PBA浓度、8-OHdG浓度均高于对照组,精子浓度低于对照组,差异均有统计学意义（P <0.01）。经Person相关性检验,351名研究对象3-PBA浓度与8-OHdG浓度呈正相关（r=0.511,P <0.01）,与精子浓度呈负相关（r=-0.546,P <0.01）;8-OHdG浓度（经自然对数转换）与精子浓度呈负相关（r=-0.113,P <0.05）。线性回归模型分...     

醋酸铅对小鼠生精功能的影响

小鼠灌胃染毒醋酸铅 10mg/kg、2 0mg/kg和 40mg/kg ,1次 /d ,连续染毒 5d ,测定精子数、精子活动度、精子畸形率和初级精母细胞染色体畸变率。结果显示染毒 2 0mg/kg以上时 ,精子计数、直线运动精子数、活精子率随染毒剂量增加而下降 ,而静止不动精子数、精子畸形率和初级精母细胞染色体畸变率随剂量增加而升高 ,各指标与阴性对照组比较差异均有显著性或高度显著性 (P <0 0 5 ,P <0 0 1) ,并呈明确的剂量 反应关系 ,r值依次为 -0 7866,-0 8949,-0 780 9,0 780 9,0 5 5 81,0 493 2 (P <0 0 1,P <0 0 5 )。说明染毒醋酸铅≥ 2 0mg/kg可损害小鼠的生精功能。     

Sublethal effects of phenol on spermatogenesis in sea urchins (Anthocidaris crassispina)

Adult sea urchins, Anthocidaris crassispina, were exposed to 0.1 and 10 mgL(-1) phenol for 4 weeks. Abnormal sperm development was clearly evident in phenol-treated sea urchins, although no mortality was found throughout the exposure period. Occurrences of multinucleate sperm cells (including spermatocytes to spermatozoa) showed a significant increase from 0.07% in the control to 10.7% and 43.3% in the 0.1- and 10-mgL(-1) treatments, respectively (P<0.01). Likewise, sperm with electron dense, dark tails increased significantly from 8% in the control to 36.6% and 43.4% in the 0.1- and 10-mgL(-1) phenol-treated sea urchins, respectively (P<0.01). In addition, disruption of cytoplasmic membranous structures such as loss of mitochondrial cristae and distortion of mitochondrial membranes and nucleus envelope were commonly found in phenol-treated spermatogonia and spermatocytes. Previous studies have clearly demonstrated motility impairment and a concomitant reduction of fertilization capability in sea urchin sperm with dark tails and/or distorted mitochondria. Our current findings therefore suggest that chronic exposure to phenol at 0.1 mgL(-1) could lower the quality of sperm and reproductive success in sea urchins, which may threaten the survival of these ecologically important species. The observed impairment of spermatogenesis by phenol might also occur in other invertebrate species.     

Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus)

Research was conducted to develop sperm sorting and novel sperm preservation methodologies for sex predetermination in the bottlenose dolphin (Tursiops truncatus) using artificial insemination. In Study 1, the effect of seminal plasma (SP), sperm concentration and freezing rate (FR) on in vitro sperm quality of liquid-stored, non-sorted spermatozoa was examined. There was no effect (P > 0.05) of prefreeze SP addition on post-thaw quality (progressive motility, kinetic rating, sperm motility index (SMI), viability and acrosome integrity). Post-thaw motility parameters and viability were higher (P < 0.05) for slow FR than fast FR samples. In Study 2 investigating the effects of liquid storage and sorting on sperm quality, motility and SMI after sorting and centrifugation were lower (P < 0.05) than those of the initial ejaculate. The sort rate for enrichment (91 +/- 4% purity) of X- and Y-bearing spermatozoa was 3400 +/- 850 spermatozoa sex(-1) s(-1). In Study 3, compared with a modified straw method, directional freezing resulted in enhanced in vitro quality of sorted and non-sorted spermatozoa derived from liquid-stored semen (P < 0.05). In Study 4, endoscopic insemination of three dolphins with sorted, frozen-thawed X-bearing spermatozoa resulted in one conception and the birth of a female calf. High-purity sorting of dolphin spermatozoa, derived from liquid-stored semen, can be achieved with minimal loss of in vitro sperm quality and samples are functional in vivo.     

Interactions of spermatozoa with the female reproductive tract: inspiration for assisted reproduction

Artificial insemination with sexed semen, in vitro fertilisation and intracytoplasmic sperm injection have been used to reproduce animals, but often not as successfully as natural mating. Learning more about how spermatozoa normally interact with the female tract can provide inspiration for developing improvements in assisted reproduction. The present review focuses on Bos taurus, because more is known about this species than others. At coitus, bull spermatozoa are deposited into the anterior vagina, where they rapidly enter the cervix. Cervical mucus quickly filters out seminal plasma from spermatozoa, unlike most assisted reproduction protocols. Spermatozoa that reach the uterus may require certain cell surface proteins to swim through the uterotubal junction. Shortly after passing through the junction, most spermatozoa are trapped in a storage reservoir by binding to oviducal epithelium, in the case of cattle via bovine seminal plasma (BSP) proteins coating the sperm head. As ovulation approaches, spermatozoa capacitate and shed BSP proteins. This reduces sperm binding to the epithelium and releases them from storage. Motility hyperactivation assists spermatozoa in leaving the storage reservoir, swimming through oviducal mucus and the cumulus oophorus, and penetrating the oocyte zona pellucida. Chemotactically regulated switching between asymmetrical (i.e. hyperactivated) and symmetrical flagellar beating may also guide spermatozoa to the oocyte.     

CORRELATION OF SPERM PARAMETERS WITH APOPTOSIS ASSESSED BY DUAL FLUORESCENCE DNA INTEGRITY ASSAY

Failed fertilization after intracytoplasmic sperm injection or miscarriages occurs in cases involving apoptotic and necrotic sperm. Identifying normal sperm is important for successful assisted reproductive technologies (ART) procedures. The study was conducted to correlate sperm parameters with intact sperm with normal DNA assessed by the dual stain assay in 118 separate individuals. The results showed differences in percent DNA intact sperm in individuals with normal W.H.O. sperm features (62 ± 1.1; mean ± S.E.M.) compared with oligoasthenoteratozoospermia patients (38 ± 5.3). Individuals whose sperm had fertilizing capacity had higher percentages of intact DNA (60 ± 1.3 versus 47 ± 2.4). The percentages of intact DNA sperm were significantly correlated to total motility in semen (R = 0.7), post-wash motility (R = 0.6), rapid progression (R = 0.6), intact acrosome (R = 0.5), and strict morphology (R = 0.5). There were no correlations with the remaining parameters. The dual stain assay identified sperm with normal physiology and fertilizing capacity. The dual stain assay measures DNA integrity and is a promising method to select normal sperm for ART.