Sperm DNA fragmentation does not affect the clinical outcomes in the cumulative transfers of an ICSI cycle along with blastocyst transfers in couples with normozoospermic male patients

Objective:To know whether sperm DNA fragmentation(SDF)affects the clinical outcomes in the cumulative transfers of an intracytoplasmic sperm injection(ICSI)cycle along with blastocyst transfers in couples with normozoospermic males.Methods:The study included 252 couples who underwent their first ICSI cycles along with blastocyst transfer and whose male partner semen samples were normozoospermic according to the World Health Organization 2010 criteria.All the couples were classified into two groups based on the SDF:the low SDF group(SDF≤30%,n=162)and the high SDF group(SDF>30%,n=90).Clinical as well as laboratory outcomes were correlated between the two groups.Sperm DNA fragmentation was assessed on the post-wash semen samples by acridine orange test.The main outcome measures were the live birth rate and miscarriage rate.Results:A significant decrease in the live birth rates was observed in the high SDF group compared to the low SDF group in fresh embryo transfer cycles(P<0.05).However,no significant difference was observed in the clinical outcomes either in the frozen embryo transfer cycles or in the overall cumulative transfer cycles(P>0.05).No significant difference was observed in the laboratory outcomes between the two SDF groups.A remarkable decrease in sperm motility was observed in the high SDF group compared to the low SDF group(P<0.05).Conclusions:Sperm DNA fragmentation does not affect the clinical outcomes in the cumulative transfers of an ICSI cycle along with blastocyst transfers in couples with normozoospermic males.

New approach to assess sperm DNA fragmentation dynamics： Fine-tuning mathematical models

Background： Sperm DNA fragmentation（sDF） has been proved to be an important parameter in order to predict in vitro the potential fertility of a semen sample. Colloid centrifugation could be a suitable technique to select those donkey sperm more resistant to DNA fragmentation after thawing. Previous studies have shown that to elucidate the latent damage of the DNA molecule, sDF should be assessed dynamically, where the rate of fragmentation between treatments indicates how resistant the DNA is to iatrogenic damage. The rate of fragmentation is calculated using the slope of a linear regression equation. However, it has not been studied if s DF dynamics fit this model. The objectives of this study were to evaluate the effect of different after-thawing centrifugation protocols on sperm DNA fragmentation and elucidate the most accurate mathematical model（linear regression, exponential or polynomial） for DNA fragmentation over time in frozen-thawed donkey semen.Results： After submitting post-thaw semen samples to no centrifugation（UDC）, sperm washing（SW） or single layer centrifugation（SLC） protocols, sD F values after 6 h of incubation were significantly lower in SLC samples than in SW or UDC.Coefficient of determination（R-2） values were significantly higher for a second order polynomial model than for linear or exponential. The highest values for acceleration of fragmentation（aSDF） were obtained for SW, fol owed by SLC and UDC.Conclusion： SLC after thawing seems to preserve longer DNA longevity in comparison to UDC and SW. Moreover,the fine-tuning of models has shown that sDF dynamics in frozen-thawed donkey semen fit a second order polynomial model, which implies that fragmentation rate is not constant and fragmentation acceleration must be taken into account to elucidate hidden damage in the DNA molecule.

The relationship between DNA fragmentation and the intensity of morphologically abnormal human spermatozoa

Objective:To determine the relationship between teratozoospermia and sperm DNA fragmentation(SDF)in the human ejaculate.Methods:This retrospective study included 100 normozoospermic men as a control cohort(abnormal forms>14%),210 patients with a high level of abnormal forms(≤4%)and 65 patients presenting with a moderate level of abnormal forms(>4%to≤14%)based on the World Health Organization definitions.Sperm morphology was assessed using bright field microscopy.Sperm DNA fragmentation was assessed using the sperm chromatin dispersion assay.Non-parametric analyses were conducted to examine the relationship between abnormal sperm morphology and sperm DNA fragmentation;receiver operating characteristic(ROC)analyses were conducted to assess sensitivity and specificity of this relationship.Results:A correlation analysis revealed that the higher the proportion of abnormal spermatozoa in the ejaculate,the higher the level of SDF(Spearman's Rho=-0.230;P<0.001).Significant differences in the proportion of SDF were found when all cohorts were compared(P<0.001);these significant differences were also retained when the different cohorts were compared pairwise.ROC analysis showed a moderate but significant predictive value for SDF to differentiate patients with different levels of teratozoospemia.Conclusions:Although analysis of a more continuous range of values for teratozoospermia would help further clarify any causal relationship with SDF,there is clearly a synergistic or coincident affiliation between these variables that needs to be acknowledged by the clinician when interpreting the spermiogram.

Novel insights into the pathophysiology of varicocele and its association with reactive oxygen species and sperm DNA fragmentation

Varicocele has been associated with reduced male reproductive potential. With the advances in biomolecular techniques, it has been possible to better understand the mechanisms involved in testicular damage provoked by varicocele. Current evidence suggests the central role of reactive oxygen species （ROS） and the resultant oxidative stress （OS） in the pathogenesis of varicocele-associated male subfertility although the mechanisms have not yet been fully described and it is likely to be multifactorial. Excessive ROS is associated with sperm DNA fragmentation, which may mediate the clinical manifestation of poor sperm function and fertilization outcome related to varicocele. Testing of ROS/OS and DNA fragmentation has the potential to provide additional diagnostic and prognostic information compared to conventional semen analysis and may guide therapeutic management strategies in individual patient.

Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation： a SWOT analysis

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation （SDF） in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology （ART）, an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection （Testi-ICSI）. In this article, we used a SWOT （strengths, weaknesses, opportunities, and threats） analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval.

Use of testicular sperm in couples with SCSA-defined high sperm DNA fragmentation and failed intracytoplasmic sperm injection using ejaculated sperm

Sperm DNA fragmentation(SDF)has been linked with male infertility,and previous studies suggest that SDF can have negative influence on pregnancy outcomes with assisted reproduction.We performed a retrospective review of consecutive couples with a high SDF level that had intracytoplasmic sperm injection(ICSI)using testicular sperm(T-ICSI).We compared the T-ICSI outcomes to that of two control groups:87 couples with failed first ICSI cycle and who had a second ICSI cycle using ejaculated sperm(Ej-ICSI),and 48 consecutive couples with high sperm chromatin structure assay(SCSA)-defined SDF(>15%)that underwent an ICSI cycle using ejaculated sperm after one or more failed ICSI cycles(Ej-ICSI-high SDF).The mean number of oocytes that were retrieved and the total number of embryos were not different among the three groups.The mean number of transferred embryos in the T-ICSI group was higher than the Ej-ICSI group but not significantly different than the Ej-ICSI-high SDF group(1.4,1.2,and 1.3,respectively,P<0.05).Clinical pregnancy rate in the T-ICSI group was not significantly different than the Ej-ICSI and Ej-ICSI-high SDF groups(48.6%,48.2%,and 38.7%,respectively,P>0.05).No significant difference was found in live birth rate when comparing T-ICSI to Ej-ICSI and Ej-ICSI-high SDF groups.The results suggest that pregnancy outcomes and live birth rates with T-ICSI are not significantly superior to Ej-ICSI in patients with an elevated SCSA-defined sperm DNA fragmentation and prior ICSI failure(s).

Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors？

This study compared the potential of assessing sperm DNA fragmentation （SDF） from neat semen and the subsequent swim-up （SU） procedure to predict pregnancy when conducting ICSI of fertile donor oocytes. Infertile females （n=81） were transferred embryos resulting from intracytoplasmic sperm injection （ICSI） of their partner＇s spermatozoa and proven donor oocytes. This model normalized the impact of female factor in putative sperm DNA repair. Semen was blindly assessed for SDF using Halosperm immediately following ejaculation （NS） and after swim-up at the time of ICSI fertilisation. There was a decrease in SDF values of the ejaculated semen sample following the swim-up protocol （P=0.000）. Interestingly, pregnancy could be equally predicted from SDF values derived from either neat or swim-up semen samples. Receiver operator curves and the derived Youden＇s indices determined SDF cutoff values for NS and SU of 24.8% and 17.5%, respectively. Prediction of pregnancy from NS SDF had a sensitivity of 75% and a specificity of 69%, whereas for SU SDF was 78% and 73%, respectively. While increased levels of SDF negatively impact reproductive outcome, we have shown that a reduction in SDF following sperm selection using ICSI with proven donor oocytes is not mandatory for achieving pregnancy. This suggests that a certain level of DNA damage that is not detectable using current technologies could be impacting on the relative success of assisted reproductive technology （ART） procedures. Consequently, we propose a modification of the so called ＇iceberg model＇ as a possible rationale for understanding the role of SDF in reproductive outcome.

Random sperm DNA fragmentation index is not associated with clinical outcomes in day-3 frozen embryo transfer

Damage to sperm DNA was proposed to play an important role in embryonic development.Previous studies focused on outcomes after fresh embryo transfer,whereas this study investigated the influence of sperm DNA fragmentation index(DFI)on laboratory and clinical outcomes after frozen embryo transfer(FET).This retrospective study examined 381 couples using cleavage-stage FET.Sperm used for intracytoplasmic sperm injection(ICSI)or in vitro fertilization(IVF)underwent density gradient centrifugation and swim up processing.Sperm DFI had a negative correlation with sperm motility(r=−0.640,P<0.01),sperm concentration(r=−0.289,P<0.01),and fertilization rate of IVF cycles(r=−0.247,P<0.01).Sperm DFI examined before and after density gradient centrifugation/swim up processing was markedly decreased after processing(17.1%vs 2.4%,P<0.01;65 randomly picked couples).Sperm progressive motility was significantly reduced in high DFI group compared with low DFI group for both IVF and ICSI(IVF:46.9%±12.4%vs 38.5%±12.6%,respectively;ICSI:37.6%±14.1%vs 22.3%±17.8%,respectively;both P<0.01).The fertilization rate was significantly lower in high(≥25%)DFI group compared with low(<25%)DFI group using IVF(73.3%±23.9%vs 53.2%±33.6%,respectively;P<0.01)but was equivalent in high and low DFI groups using ICSI.Embryonic development and clinical outcomes after FET were equivalent for low and high DFI groups using ICSI or IVF.In this study,sperm DFI did not provide sufficient information regarding embryo development or clinical outcomes for infertile couples using FET.

Human circBOULE RNAs as potential biomarkers for sperm quality and male infertility

Reliable molecular biomarkers to predict fertility remain scarce.The current study investigated the potential of testis-specific circBOULE RNAs as biomarkers for male infertility and sperm quality.Using reverse transcription-PCR and real-time reverse transcription-PCR assays,we identified seven circular RNAs from the human BOULE gene in human sperm.We observed that the expression level of circEx3-6 was significantly reduced in asthenozoospermia,while the expression levels of both circEx2-6 and circEx2-7 were decreased in terato-zoospermia,compared with the controls.Furthermore,we demonstrated that the expression level of circEx2-6 was negatively correlated with the sperm DNA fragmentation index,and the expression level of circEx2-7 was correlated with both fertilization and cleavage rates in those treated with the assisted reproductive technologies.Further functional analyses in a transgenic fly model supported the roles of circBOULE RNAs in sperm development and human male fertility.Collectively,our findings support that sperm circBOULE RNAs may serve as diagnostic biomarkers for assessing sperm motility and DNA quality.Therefore,clinical application and significance of sperm circBOULE RNAs in the assisted reproductive technologies warrant further investigation.

Resolution of sperm quality impairment following SARS-CoV-2 infection:A prospective study

Objective:To investigate the length of time required to resolve COVID-19 effects on semen quality and DNA integrity.Methods:A prospective cohort study was conducted among 42 men who tested positive for SARS-CoV-2 and underwent semen analysis at baseline and four months’post-recovery.Semen samples were collected and evaluated for macroscopic and microscopic parameters,sperm chromatin maturation,and DNA fragmentation.Results:The mean age of participants was 37(±7)years,and 14%had normozoospermia at baseline.After a four-month recovery from COVID-19,48%of patients had normozoospermia.Sperm count,motility,and morphology increased significantly,while sperm DNA fragmentation and sperm chromatin maturation decreased significantly post-recovery from COVID-19.Conclusions:Sperm parameters improve after a four-month recovery from COVID-19.The findings indicate significant improvements in sperm count,motility,morphology,DNA fragmentation,and chromatin maturation after a four-month recovery period.

Obesity leads to higher risk of sperm DNA damage in infertile patients

There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index （BMI）. A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling （TUNEL） assay, we observed an increased rate of sDerm DNA damage in obese men （odds ratio （95% confidence interval）： 2.5 （1.2-5.1））.

The ability of sperm selection techniques to remove single- or double-strand DNA damage

A wide variety of techniques for the preparation of sperm are currently available, of which the most commonly employed are densitygradient centrifugation （DGC） and swim-up （SUP）. To date, these methods appear to be effective in selecting functional sperm for assisted reproduction techniques （ART）, but they may have negative effects on sperm DNA. In this study, the ability of these semen processing techniques to eliminate spermatozoa containing single- and double-strand DNA damage was assessed by the two-tailed comet assay and the sperm chromatin dispersion test in 157 semen samples from patients seeking assisted reproduction treatment. Our results indicated that SUP and DGC are equally efficient in eliminating spermatozoa containing double-strand DNA damage and sperm with highly damaged （degraded） DNA, as characterized by the presence of both single- and double-strand DNA breaks. However, DGC is more efficient than SUP in selecting spermatozoa that are free from single-strand DNA damage. Future studies should characterise the importance of the various types of DNA damage and examine the sperm processing protocols used in each laboratory to determine their ability to eliminate DNA damage and hence, prevent the potential transmission of genetic mutations via ART.

Inter-center variation in the efficiency of sperm DNA damage reduction following density gradient centrifugation

This was a prospective multicenter study aiming at comparing the efficiency of sperm selection by density gradient centrifugation (DGC) in reducing sperm DNA fragmentation (SDF) in different ART centers. The study was designed using 290 semen samples collected from 10 different ART centers performing artificial insemination, in vitro fertilization and blind assessment of SDF at the University facilities. The results showed that while there was a significant reduction in the SDF levels in sperm isolated from the gradient centrifuged pellet (DGC) compared to neat semen samples (NSS), there was also significant inter-center variability in the efficiency to reduce SDF values by DGC (78.5% to 29.2%). Surprisingly, for some patients, the level of SDF actually increased following sperm selection. The main conclusions derived from this study were that 1) isolation of sperm from the gradient pellet by DGC must be performed using validated, optimized protocols;2) routine comparison of SDF values in NSS semen and in processed sperm after DGC or swim-up must be recommended as part of the internal quality control (QC) of ART laboratories to test the efficacy of sperm processing;and 3) SDF values in processed spermatozoa should be obtained to compare with the pregnancy rate when insemination or fertilization is about to be performed, otherwise, attempts to predict pregnancy outcome from SDF could be biased or are essentially meaningless.

Can the Prediction of Intrauterine Insemination Results by Used Aniline Blue Stain (ABS) and Sperm Chromatin Dispersion (SCD) Levels?

Introduction: This study aimed to perform routine seminal fluid analysis, sperm DNA fragmentation, and sperm function tests at the chromatin maturation level and evaluate pregnancy in the patients passing intrauterine insemination before starting Intrauterine Insemination (IUI) method. Materials and Methods: In this prospective study, 111 couples who underwent Intrauterine Insemination (IUI) in unexplained infertility patients were admitted to Al-Farah IVF and assisted reproductive center in Baghdad, Iraq between November 2020 and February 2021 were evaluated. Semen fluid analysis was performed based on (WHO 4th) guiding rules. In addition, Sperm Chromatin Dispersion (halo test) and sperm maturation were performed with Aniline Blue Stain (ABS). Results: Sperm Chromatin Dispersion (SCD) groups were compared in terms of pregnancy outcome;the positive pregnancy rate was found to be above in the normal SCD groups (p = 0.0005). In addition, Aniline Blue Stain (ABS) groups were compared in the terms of pregnancy outcome;the positive pregnancy rate was found to be higher in the normal ABS group (p = 0.017). Conclusion: Our study showed that the use of DNA fragmentation (SCD) and sperm maturation tests (ABS) together with routine semen analysis in intrauterine insemination cases will make a significant contribution to the prediction of Intrauterine Insemination (IUI) increased results. So, these results indicate a defect in the effect of DNA fragmentation on the outcome of intrauterine insemination.

Comparison of sperm parameters and DNA fragmentation index between infertile men with infection and vaccines of COVID-19

Several preventive measures,including vaccination,have been implemented owing to the severe global effect of coronavirus disease 2019(COVID-19),but there is still limited evidence in the effect of this disease and vaccination against it on male fertility.Therefore,this study is to compare sperm parameters of infertile patients with or without COVID-19 infection and the effect of COVID-19 vaccine types on them.Semen samples of infertile patients were collected consecutively at Universitas Indonesia-Cipto Mangunkusumo Hospital(Jakarta,Indonesia).COVID-19 was diagnosed by rapid antigen or polymerase chain reaction(PCR)tests.Vaccination was performed with three types of vaccine,namely inactivated viral vaccine,messenger RNA(mRNA)vaccine,and viral vector vaccine.Spermatozoa were then analyzed on the World Health Organization recommendations,and DNA fragmentation was assayed with the sperm chromatin dispersion kit.The results showed that the COVID-19 group experienced a significant decrease in sperm concentration and progressive motility(both P<0.05),but there was no significant change in morphology or sperm DNA fragmentation index(DFI;both P>0.05).The viral vector vaccine caused a decrease in morphology as well as an increase in DFI compared with the control(both P<0.05),meanwhile results for those who were vaccinated with the inactivated and mRNA types were not significant compared with the control(both P>0.05).We conclude that COVID-19 has negative effects on sperm parametes and sperm DNA fragmentation,and we found that the viral vector vaccines affect sperm parameter values and DNA fragmentation negatively.Further studies with a larger population and longer follow-up are needed to confirm the results.

Study of Sperm Apoptosis and Seminal Oxidant Capacity in Infertile Patients with Genital Tract Infections Evaluation of the Efficacy of Levofloxacin in Two Therapeutic Protocols

Introduction: Antibiotics are the principal treatment of seminal tract infections Our objective is to determine the efficacy of levofloxacin in the eradication of genital tract patogens in infertile patients comparing 28 with 56 days of treatment and to elucidate different seminal parameters availables as markers of antibiotic efficacy in seminal improvement. Materials and Methods: We studied prospectively 50 males patients with seminal tract infections. All patients were treated with levofloxacin 500 mg orally along 28 or 56 days. Two seminal analysis were performed before and after treatment an seminal parameters included TUNEL and nitric oxide levels in seminal plasma were measured. Results: We observed significative differences between both diagrams of treatment. Sperm count and motility increased significatively after treatment. But, sperm morphology do not improve after antibiotic. Necrospermy index but not leucocitospermy had been reduced after levofloxacin. Nitric oxide levels have a direct correlation with sperm count but inverse with motility. The rate of apoptosis determined by TUNEL technique in the study population was 30.46%. There was no correlation between apoptosis parameters and necrozoospermy and there was no correlation between sperm cell apoptosis and NO levels. Conclutions: Bacteriologic cure in seminal infection is higher with a scheme of 56 days than 28 days. Treatment with levofloxacin improves seminal parameters in infertile patients with spermatic tract infection. Nitric oxide but not TUNEL is a good predictive factor of antibiotic efficacy.

Magnetic-activated cell sorting of nonapoptotic spermatozoa with a high DNA fragmentation index improves the live birth rate and decreases transfer cycles of IVF/ICSI

The present study aimed to evaluate the clinical outcomes of magnetic-activated cell sorting(MACS)in sperm preparation for male subjects with a sperm DNA fragmentation index(DFI)≥30%.A total of 86 patients who had undergone their first long-term long protocol were selected.The protocol involved in vitro fertilization(IVF)and intracytoplasmic sperm injection(ICSI)cycles,and the patients were divided into the MACS or control groups.The MACS group included sperm samples analyzed with MACS that were combined with density gradient centrifugation(DGC)and the swim-up(SU)technique(n=39),and the control group included sperm samples prepared using standard techniques(DGC and SU;n=41).No differences were noted with regard to basic clinical characteristics,number of oocytes retrieved,normal fertilization rate,cleavage rate,or transplantable embryo rate between the two groups in IVF/ICSI.In addition,the clinical pregnancy and implantation rates of the first embryo transfer cycles indicated no significant differences between the two groups.However,there was a tendency to improve the live birth rate(LBR)of the first embryo transfer cycle(63.2%vs 53.9%)and the cumulative LBR(79.5%vs 70.7%)in the MACS group compared with the control group.Moreover,the number of transferred embryos(mean±standard deviation[s.d.]:1.7±0.7 vs 2.3±1.6)and the transfer number of each retrieved cycle(mean±s.d.:1.2±0.5 vs 1.6±0.8)were significantly lower in the MACS group than those in the control group.Thus,the selection of nonapoptotic spermatozoa by MACS for higher sperm DFI could improve assisted reproductive clinical outcomes.

Decreased AKAP4/PKA signaling pathway in high DFI sperm affects sperm capacitation

The sperm DNA fragmentation index(DFI)is a metric used to assess DNA fragmentation within sperm.During in vitro fertilizationembryo transfer(IVF-ET),high sperm DFI can lead to a low fertilization rate,poor embryo development,early miscarriage,etc.A kinase anchoring protein(AKAP)is a scaffold protein that can bind protein kinase A(PKA)to subcellular sites of specific substrates and protects the biophosphorylation reaction.Sperm protein antigen 17(SPA17)can also bind to AKAP.This study intends to explore the reason for the decreased fertilization rate observed in high sperm DFI(H-DFI)patients during IVF-ET.In addition,the study investigates the expression of AKAP,protein kinase A regulatory subunit(PKARIl),and SPA17 between H-DFI and low sperm DFI(L-DFI)patients.SPA17 at the transcriptional level is abnormal,the translational level increases in H-DFI patients,and the expression of AKAP4/PKARIl protein decreases.H,O,has been used to simulate oxidative stress damage to spermatozoa during the formation of sperm DFI.It indicates that H,O,increases the expression of sperm SPA17 protein and suppresses AKAP4/PKARIl protein expression.These processes inhibit sperm capacitation and reduce acrosomal reactions.Embryo culture data and IVF outcomes have been documented.The H-DFI group has a lower fertilization rate.Therefore,the results indicate that the possible causes for the decreased fertilization rate in the H-DFI patients have included loss of sperm AKAP4/PKARIl proteins,blocked sperm capacitation,and reduced occurrence of acrosome reaction.

Antioxidant therapy in male infertility： fact or fiction？

Infertile men have higher levels of semen reactive oxygen species （ROS） than do fertile men. High levels of semen ROS can cause sperm dysfunction, sperm DNA damage and reduced male reproductive potential. This observation has led clinicians to treat infertile men with antioxidant supplements. The purpose of this article is to discuss the rationale for antioxidant therapy in infertile men and to evaluate the data on the efficacy of dietary and in vitro antioxidant preparations on sperm function and DNA damage. To date, most clinical studies suggest that dietary antioxidant supplements are beneficial in terms of improving sperm function and DNA integrity. However, the exact mechanism of action of dietary antioxidants and the optimal dietary supplement have not been established. Moreover, most of the clinical studies are small and few have evaluated pregnancy rates. A beneficial effect of in vitro antioxidant supplements in protecting spermatozoa from exogenous oxidants has been demonstrated in most studies; however, the effect of these antioxidants in protecting sperm from endogenous ROS, gentle sperm processing and cryopreservation has not been established conclusively.

Green tea extract as a cryoprotectant additive to preserve the motility and DNA integrity of human spermatozoa

Cryopreservation impairs sperm quality and functions,including motility and DNA integrity.Antioxidant additives in sperm freezing media have previously brought improvements in postthawed sperm quality.Green tea extract(GTE)is widely considered as an excellent antioxidant,and its beneficial role has been proven in other human cells.This study aims to evaluate the GTE as a potential additive in cryopreservation media of human spermatozoa.In part one,the semen of 20 normozoospermic men was used to optimize the concentration of GTE that maintains sperm motility and DNA integrity against oxidative stress,induced by hydrogen peroxide(H_(2)O_(2)).Spermatozoa were treated with GTE at different concentrations before incubation with H_(2)O_(2).In part two,the semen of 45 patients was cryopreserved with or without 1.0 ng ml^(-1)GTE.After 2 weeks,the semen was thawed,and the effect on sperm motility and DNA fragmentation was observed.Our data showed that GTE significantly protected sperm motility and DNA integrity against oxidative stress induced by H_(2)O_(2)when added at a final concentration of 1.0 ng ml^(-1).We found that the addition of 1.0 ng ml^(-1)GTE to cryopreservation media significantly increased sperm motility and DNA integrity(both P<0.05).More interestingly,patients with high sperm DNA damage benefited similarly from the GTE supplementation.However,there was no significant change in the reactive oxygen species(ROS)level.In conclusion,supplementing sperm freezing media with GTE has a significant protective effect on human sperm motility and DNA integrity,which may be of clinical interest.