Protective effect of butylated hydroxytoluene on sperm function in human spermatozoa cryopreserved by vitrification technique

Butylhydroxytoluene (BHT), a synthetic analogue of vitamin E, shows antioxidant and antiviral properties and has been successfully used for mammalian sperm cryopreservation. In this study, BHT was included in a vitrification solution to determine its cryoprotective effect on human spermatozoa. Spermatozoa were selected by swim‐up and vitrified in close sealed straw using either a combination of human tubal fluid (HTF), sucrose and BHT 1 mm (VMBHT), or only HTF and sucrose (VM). The optimal concentration of BHT was determined by the observation of preserved progressive sperm motility (PSM) after warming and detection of plasma membrane (PMI), membrane mitochondrial potential (ΔΨm) and DNA integrity. The presence of reactive oxygen species (ROS) was also detected. The PSM was significantly higher in the VMBHT group (80.86 ± 5.41%) compared with the VM group (68.9 ± 3.67%) (P < 0.05). Butylhydroxytoluene significantly preserved DNA integrity (4.0 ± 0.1% versus 6.1 ± 1.6%; P < 0.05) and reduced ROS production (5.5 ± 2.2 versus 8.6 ± 1.8%; P < 0.05). Plasma membrane and ΔΨm showed no statistical differences. One millimolar BHT effectively maintained cell function and due to its antioxidant and antiviral properties could be used in semen cryopreservation of patients with viral infections transmitted by seminal plasma.     

Protective effect of the superoxide dismutase mimetic MnTBAP during sperm vitrification process

Sperm cryopreservation is widely used in assisted reproduction and male infertility therapy; however, it induces oxidative stress affecting sperm quality. This work evaluated the effect of the antioxidant MnTBAP during vitrification steps in human spermatozoa. First, the effect of MnTBAP on viability and ROS production was evaluated. Then, the spermatozoa were vitrified in straws with the vitrification, warming and post-warming incubation media separately supplemented with MnTBAP. An untreated control was included. The sperm viability, ROS production, total and progressive motility were evaluated. The results showed that the direct exposure of spermatozoa to MnTBAP significantly decreases the ROS levels in comparison with the untreated control without affecting the viability. The supplementation of the vitrification medium with MnTBAP did not affect the parameters analysed. However, the supplementation of the warming and incubation post-warming media resulted in a decrease in ROS production and maintained viability and motility for 4 hr after warming with concentrations up to 100 μM of MnTBAP. Higher concentrations of MnTBAP caused a decrease in total motility. In conclusion, the use of MnTBAP during the warming or post-warming incubation media has beneficial effect decreasing ROS levels and maintaining the viability and motility during the vitrification procedure.     

不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏效果的比较

目的比较不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏的效果。方法将15份上游后的精液标本分别采用常规精子冷冻和不使用冷冻保护剂的冷冻环玻璃化法冷冻,比较精子复苏后的活力、形态及精子膜的完整性三项指标。结果冷冻前、前向活动精子百分率、正常精子形态百分率及精子膜完整率分别为(79±6.42)%、(34±9.36)%和(91±5.18)%;不采用冷冻保护剂的玻璃化法冷冻复苏后,三者分别为(42.20±8.35)%、(31.00±7.63)%和(50.00±9.34)%。常规冷冻法冷冻复苏后,三者分别为(38.00±15.80)%、(30.00±5.24)%和(47.00±13.67)%。冷冻前后前向活动精子百分率和精子膜完整率的差别有统计学意义,但两种冷冻方法相比差异无统计学意义。结论使用不加入冷冻保护剂的玻璃化方法冷冻人的精子是可行的,可取得与常规冷冻相同的效果。     

Canine sperm vitrification with sucrose: effect on sperm function

The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra‐rapid cryopreservation in canine sperm was investigated. Swim‐up selected spermatozoa of second‐fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m ) in proportion 1 : 1 v/v with HTF–BSA 1%. From each group, 30‐μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF–BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF–BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome‐reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra‐rapid cryopreservation.     

Vitrified sperm banks: the new aseptic technique for human spermatozoa allows cryopreservation at −86 °C

The vitrification technique is simple, quick, cost‐effective and has showed a significantly stronger cryoprotective effect in contrast to conventional freezing. The method is based on the rapid cooling of the cell by direct immersion in liquid nitrogen (LN ₂), thereby avoiding the formation of ice crystals, due to the lower risk of water thawing, which impairs cell function. The aim of this study was to evaluate the effect of storage at ?86 °C compared to the conventional ?196 °C (under LN ₂) on essential parameters of the functioning of aseptically vitrified human sperm. Sperm motility, integrity of mitochondrial membrane potential and the rate of DNA fragmentation were determined. The comparison of ?86 °C and ?196 °C demonstrated no statistical difference in sperm progressive motility (73% vs. 77%), integrity of mitochondrial membrane potential (71% vs. 74%) or DNA fragmentation (3.1% vs. 2.9%). In conclusion, aseptically vitrified sperm can be preserved at ?86 °C; eliminating the use of LN ₂ simplifies and significantly reduces the costs associated with storage in sperm banks by decreasing the time and space needed for storage, the effort in finding stored samples, and by improving safety for the operator. However, for prolonged storage further studies are needed.     

The effect of cysteine and glutamine on human sperm functional parameters during vitrification

Assuming the adverse effects of reactive oxygen species (ROS) on sperm function, this study was conducted to assess the effects of cysteine and glutamine as effective antioxidants on human sperm parameters under vitrification. Twenty normozoospermic samples were used. The samples were subjected to a vitrification process and cysteine (5 and 10 mM) and glutamine (10 and 15 mM). The sperm motility parameters, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), DNA damage and intracellular ROS damage were assessed for each sample. Statistical analyses showed that motility, mitochondrial membrane potential and DNA damage decreased in the vitrified groups with cysteine 5, 10 mM and glutamine 10, 15 mM separately. Also intracellular ROS increased significantly compared to the fresh group (p < .05). No significant differences were observed for PMI compared with the fresh group (p > .05). Supplementation of cysteine and glutamine in both concentrations separately decreased intracellular ROS and DNA damage of spermatozoa with significant increase in PMI, MMP and progressive motility compared to vitrified control group (p < .05). The results showed no significant effect of a specific concentration in cysteine and glutamine on sperm parameters compared to other concentrations. Both amino acids have the potential to improve the harmful effects of freezing on sperm parameters.     

Morphometric analysis of human sperm morphology

Fourteen morphological forms of human spermatozoa were analysed morphometrically using semi-automated image analysis techniques. Five basic (area, perimeter, length, width and mass) and five derived (ratio, length minus width, ellipticity, form and total mass) parameters were considered. Statistical analysis showed differences among all 14 types of human sperm heads. Basic parameters describing the size and shape were enough to distinguish most of the categories, whereas derived parameters as well as parameters dependent on stain intensity, were demonstrated to be useful for the discrimination of some morphological categories. The fact that statistical analysis showed differences among all 14 sperm types provides evidence for the reliability of our morphological classification. These results show that morphometry can be used for the fine study of sperm morphology and may serve as a database for future work dealing with sperm classification. As this is a pilot study to assess methodology, further studies will be required to validate the method in terms of its application and usefulness in assessing the fertilizing potential of human spermatozoa.     

The influence of sperm density on the motility characteristics of washed human spermatozoa

To study the effects of sperm density on the results of computer-assisted semen analysis (CASA), 10 washed semen samples were diluted and measured with the CellTrak/S CASA system in a concentration range of 10–180×10⁶ spermatozoa/ ml. All sperm motility parameters were influenced to some extent by sperm density. The motility percentage was influenced significantly in 5 samples ( P< 0.005), the straight line velocity in all samples (P<0.0005 in 7 samples), the curvilinear velocity in 3 samples ( P< 0.005), the linearity in 9 samples (P<0.0005 in 6 samples) and the lateral head displacement in 9 samples (P< 0.005 in 6 samples). In general, the CellTrak/S data are influenced significantly if sperm density exceeds 50 × 10⁶ spermatozoa/ml.
The influence of sperm density on the motility parameters can be explained both by the accuracy of the CASA system and by actual changes in the motility of the spermatozoa. In the light of other published studies, it is concluded that sperm motility measurements with CASA systems should be assessed using 25–50 × 10⁶ spermatozoa/ml, especially in studies concerning lateral head displacement and the linearity, as in sperm hyperactivation studies.     

Semen analysis and sperm function testing

Despite controversy regarding the clinical value of semen analysis, male fertility investigation still relies on a standardized analysis of the semen parameters. This is especially true for infertility clinics in both developing and developed countries. Other optional tests or sophisticated technologies have not been widely applied. The current review addresses important changes in the analysis of semen as described in the new World Health Organization (WHO) manual for semen analysis. The most important change in the manual is the use of evidence-based publications as references to determine cutoff values for normality. Apart from the above mentioned changes, the initial evaluation and handling methods remain, in most instances, the same as in previous editions. Furthermore, the review evaluates the importance of quality control in andrology with emphasis on the evaluation of sperm morphology. WHO sperm morphology training programmes for Sub-Saharan countries were initiated at Tygerberg Hospital in 1995. The external quality control programme has ensured that the majority of participants have maintained their morphological reading skills acquired during initial training. This review reports on current sperm functional tests, such as the induced acrosome reaction, and sperm-zona pellucida binding assays, as well as the impact of sperm quality in terms of DNA integrity, and the relationship of sperm function tests to sperm morphology.     

Relationship of acrosin activity to sperm function tests

Summary.  Acrosin activity of spermatozoa from infertile patients and fertile volunteers was measured. Acrosin activity of spermatozoa from asthenozoospermic patients and patients with unexplained infertility was lower than that from fertile volunteers. We utilize the zona-free hamster egg sperm penetration test to select candidates for conventional in vitro fertilization among unexplained infertile patients. The zona-free hamster egg sperm penetration test, however, requires several hours and special equipment which are not used in the clinical setting. It is preferable that other sperm function tests or a combination of them can replace this test. Thus three distinct tests of sperm function, namely, acrosin activity, Penetrak® test, hypo-osmotic swelling test, were compared with the hamster test, using spermatozoa from patients with unexplained infertility.
A combination of the Penetrak® test and measurement of acrosin activity could predict the results of the zona-free hamster egg sperm penetration test with 88.2% accuracy. Thus, the hamster test should be done when either Penetrak® test or measurement of acrosin activity showed abnormal values.     

伊红Y水试验法应用研究进展

基于体外活体染色技术、低渗肿胀试验和水试验原理建立的伊红Y水试验方法可同步检测精子头部和尾部、精子膜结构和功能的完整性,在中国已被临床广泛应用。伊红Y水试验方法学上的3个特点包括:1同时对精子头部和尾部进行检测,全面评估精子膜各部位损伤的内在联系,便于观察;2用蒸馏水代替常用的配方溶液,避免不同渗透压或不同比例糖和电解质溶液对精子膜有不同作用影响水分子通过精子膜,既简化了方法,又使反应标准化;3检测时间短,可以重复检测,适合治疗前后反复检测。本文综述伊红Y水试验法的基本方法及其改进,在精子功能检查、男性不育常规精液分析、睾丸穿刺精子质量评估、精液冷冻保存程序研究、受微波辐射男性精子膜完整性等相关研究方面的应用。     

Ultrastructural categorization of human sperm cryopreserved in glycerol and in TESTCY

The motility of human sperm after thawing and the ultrastructural integrity of their acrosomal region was evaluated in 5 semen donors after utilizing 2 cyproprotective extenders--glycerol and TESTCY. Sperm motility in the fresh control semen fraction (65.5% +/- 6.1) was significantly (P less than 0.05) better than the immediate post-thaw motility in both cyprotected fractions (38.8% +/- 2.0 for glycerol and 31.0% +/- 2.9 for TESTCY), and there was no significant difference between the two cyprotective treatments. The overall % of normal intact sperm heads was significantly less than in controls (P less than 0.05) following cypropreservation in glycerol, but values for sperm stored in TESTCY were not significantly different from controls or glycerol. However, when expressed as % change from controls values, sperm preservation was significantly better (P less than 0.05) in TESTCY than in glycerol. TESTCY is considered a better cryoprotective agent for human sperm.     

Factors affecting sperm motility VIII. Velocity and survival of human spermatozoa as related to temperatures above zero

The effect of temperature on motility and survival of ejaculated human spermatozoa was investigated with the aid of the multiple exposure photography (MEP) method for objective sperm motility determination. Fresh specimens from healthy donors were analyzed while being heated or cooled gradually, or during their storage at various temperatures from 0 to 48°C. Sperm velocity increased steadily from zero to 50.4 nm/sec between freezing point and body temperature. Thereafter, their activity dropped dramatically and total immobilization occurred at 45°C. The induced immobilization was reversible providing exposure to those extreme temperatures was short enough to prevent permanent damage. Sperm survived up to 24–48 h when stored at 23°C, while at body temperature, their survival in vitro was much shorter and rarely extended beyond 12 h. Their longevity was still shorter at higher or lower temperatures, especially when approaching 48°C. With the aid of the combined supravital staining and MEP methods it was found that temperatures of extreme levels induced mainly immobilization rather than a spermicidic effect. The possible mechanism of thermal effect on sperm motility and some of its practical implications are discussed.     

Detection of progesterone receptors in human spermatozoa and their correlation with morphological and functional properties

In previous reports, it has been demonstrated that progesterone (P) stimulates capacitation, hyperactivation of human sperm motility and initiates the acrosome reaction (AR). This last effect has been related to the presence of non-genomic receptors for the steroid, localized on the sperm head plasma membrane. These receptors can be detected after treating spermatozoa with the non-permeable conjugate Progesterone - 3-(O-carboxymethyl) oxime: bovine serum albumin-fluorescein isothiocyanate (P-BSA-FITC). In the present study, the presence of progesterone receptors was determined in a selected sperm population with normal morphology and high progressive motility. In addition, other parameters such as the AR, hypo-osmotic swelling test, stability of chromatin and capacitating effect of P were evaluated. The percentage of P-BSA-FITC positive-spermatozoa present in the selected sperm population was higher than in total seminal spermatozoa. Furthermore, spermatozoa incubated with P showed a higher percentage motility and AR than did control spermatozoa. The HOS test indicated that membrane integrity of P-treated spermatozoa was not different to that found in the control sperm suspensions. Unexpectedly, the total sperm population treated with P showed a marked susceptibility to nuclear decondensation with reducing agents. According to these results, the selected sperm population of this study, able to respond to P, may be similar to that with good motility and normal morphology selected in the female reproductive tract, before fertilization.     

去透明带地鼠卵穿透试验与精子DNA荧光染色、精子尾部低渗肿胀试验相关性分析

本研究对６６例不明原因的原发不育及３４例继发不育男性病人的精液标本同时进行去透明带地鼠卵穿透试验（ＨＯＰ）、精子尾部低渗肿胀试验（ＨＯＳ）与ＤＮＡ荧光染色的有效精子计数（ＥＳＣ）的检测和统计分析。结果在１００例中ＨＯＳ和ＨＯＰ以及ＥＳＣ与ＨＯＰ的符合率分别达到７８％与７４％；ＨＯＳ与ＥＳＣ与ＨＯＰ三者的符合率亦达到６４％，表明ＨＯＳ、ＥＳＣ与ＨＯＰ一样也有反映精子受精能力的作用，并认为ＨＯＳ与ＥＳＣ的标准分别采用≥６０％与≥２０×１０￣６较为合适。此外，继发不育组的ＨＯＳ与ＨＯＰ符合率、ＥＳＣ与ＨＯＰ符合率以及ＨＯＳ与ＥＳＣ与ＨＯＰ三者符合率还明显高于原发不育组。     

The human sperm nucleus takes up zinc at ejaculation

Ejaculated and vasal sperm were obtained from men referred for vasectomy, and sperm nuclear elements were determined by X-ray microanalysis. Sperm head zinc concentrations, expressed as the ratio Zinc to Sulphur, were significantly higher in ejaculated than in vasal sperm. A physiological sperm nuclear zinc uptake is discussed in relation to sperm chromatin decondensation.     

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.     

Relationship between human sperm lipid peroxidation, comprehensive quality parameters and IVF outcome

The membranes of human spermatozoa contain an extremely high concentration of polyunsaturated fatty acids and are therefore susceptible to lipid peroxidation damage. The aim of this study was to retrospectively determine the association between the lipid peroxidation levels of washed spermatozoa, as indicated by thiobarbituric-acid-reactive substance concentration, and: (a) semen quality evaluated by basic routine, biochemical, cytological and quantitative ultramorphological analyses; (b) IVF fertilization rate. Semen samples from 45 male partners of couples who had been referred for IVF treatment due to a female infertility factor were evaluated for quality as well as for thiobarbituric-acid-reactive substance concentrations. The latter were found to have a negative correlation with total sperm count, semen volume, zinc/fructose ratio, and the integrity of sperm acrosome and axonema. It was suggested that lipid peroxidation has a deleterious effect on the ultramorphological status of the sperm cells and, thereby, on the male fertilization potential. The content of the seminal fluid, about 30% of which is produced by the prostate, may protect spermatozoa from this destructive process. A negative correlation was also found between thiobarbituric-acid-reactive substance concentrations and IVF fertilization rate. When the patients were subdivided into fertilizing (fertilization rate > 0%) and nonfertilizing (fertilization rate = 0%) subgroups (n = 33 and n = 12, respectively), the former exhibited significantly lower thiobarbituric-acid-reactive substance concentrations than the latter. A new IVF fertilization index based on the lipid peroxidation level was established. This index had a predictive power of 93% (94% sensitivity and 92% specificity). The clinical value of this index should be further verified.     

The presence of human papillomavirus in semen does not affect the integrity of sperm DNA

It remains unknown whether human papillomaviruses (HPVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen samples of 22 normozoospermic patients undergoing infertility treatment, nine fertile donors and seven fertile men with a risk of HPV infection (genital warts or condylomas) were included in the study. The samples were examined by an INNO‐LiPA test PCR‐based reverse hybridisation array that identifies 28 types of HPVs as simple or multiple infections. Sperm DNA integrity was determined by sperm chromatin dispersion assay (SCD). Our preliminary findings demonstrate an increase in HPV infection in infertile men with respect to fertile men. However, the sperm DNA fragmentation index was not increased in semen containing these viruses.     

Comparison of cryopreserved human sperm from solid surface vitrification and standard vapor freezing method: on motility, morphology, vitality and DNA integrity

Solid surface vitrificaition (SSV) is a cryoperservative method that has been used in the cryopreservation of oocytes, and embryos. Here, we report an application of the SSV in the cryopreservation of human spermatozoa. We compared the SSV with a standard freezing method in terms of sperm motility, morphology, vitality and DNA integrity. Sperm motility was determined by computer assisted semen analysis, morphology and vitality were determined by eosin-methylene blue staining, and DNA integrity was determined by a TUNEL assay. We found that while both cryopreservative methods produced spermatozoa with comparable vitality and motility, the SSV gave slightly, but significantly fewer sperm with DNA damage, and loose tail. We concluded that, a cryopreservation of human spermatozoa by SSV is feasible and provides a quick and practical way to preserve human spermatozoa with a comparable, if not better, quality of the preserved spermatozoa to the standard freezing method.