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.

Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation

Aim： To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm quality. Methods： Two early apoptotic changes in the semen of 56 men were assessed using Annexin V （AN）/propidium iodide （PI） staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential （MMP）. The results were compared with conventional semen parameters and DNA fragmentation identified using the TUNEL assay. Results： The different labeling patterns in the bivariate Annexin V/PI analysis identified four distinctive spermatozoa populations. The percentage of AN^-/PI^- spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL （＋） spermatozoa. As for the AN^-/PI^＋ fraction, we found an opposite result in comparison to AN^-/PI^- spermatozoa. The level of early apoptotic AN^＋/PI^- spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic AN^＋/PI^＋ spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL （＋） spermatozoa. MMP positively correlated with conventional semen parameters, but negatively correlated with TUNEL （＋） spermatozoa. Conclusion： Although early apoptotic AN^＋/PI^- spermatozoa only negatively correlates with sperm motility, the differences in proportion of each subpopulation of spermatozoa （especially, the percentage of AN^-/PI^- spermatozoa）, and decreased MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of in vitro fertilization. （Asian J Androl 2008 Mar; 10： 227-235）

Diagnostic value of sperm DNA fragmentation and sperm high-magnification for predicting outcome of assisted reproduction treatment

Over the last years, major improvements in the field of male infertility diagnosis have been achieved. The aim of this study was to determine the diagnostic usefulness of sperm DNA integrity and sperm vacuolisation for predicting outcome in infertile couples undergoing in vitroferti lisation （IVF） and intracytoplasmic sperm injection （ICSI） treatments. A cohort study from 152 infertile couples undergoing sperm DNA fragmentation and high-magnification tests prior to an assisted reproduction treatment was designed. We found that the most predictive cutoff for pregnancy was 25.5% of DNA fragmentation with a negative predictive value of 72.7% （P=0.02）. For the degree of vacuolisation, the best predictor of pregnancy was 73.5% of vacuolated sperm grades Ⅲ ＋ Ⅳ with a negative predictive value of 39.4% （P=0.09）, which was not statistically significant. In conclusion, sperm DNA fragmentation greater than 25.5% could be associated with higher probability of failure IVF treatment. Regarding the results of the sperm analysis at high magnification, they do not allow us to predict whether or not oatients will become pregnant.

DNA fragmentation in apoptosis

Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

Lethal effect and apoptotic DNA fragmentation in response of D-GalN-treated mice to bacterial LPS can be suppressed by pre-exposure to minute amount of bacterial LPS: Dual role of TNF receptor 1

AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 μg). Both control and experimental groups simultaneously were challenged with LPS plus D-GaIN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GaIN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GaIN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GaIN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.

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.

LDFF,the large molecular weight DNA fragmentation factor,is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts

DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis,the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg2+-dependent and Ca2+-independent, can occur in both acidic and neutral pH conditions and can tolerate 45℃ treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.

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.

Nonextensivity and Tsallis Entropy in DNA Fragmentation Patterns by Ionizing Radiation

Nonextensive statistical mechanics as in Tsallis formalism was used in this study, along with the dynamical Hamiltonian rod-like DNA model and the maximum entropy criteria for Tsallis’ entropy, so as to obtain length distribution of plasmid fragments, after irradiation with very high doses, assuming that the system reaches metaequilibrium. By intensively working out the Grand Canonical Ensemble (used to take into account the variation of the number of base pairs) a simplified expression for Fragment Size Distribution Function (FSDF) was obtained. This expression is dependent on two parameters only, the Tsallis q value and the minimal length of the fragments. Results obtained from fittings to available experimental data were adequate and the characteristic behavior of the shortest fragments was clearly documented and reproduced by the model, a circumstance never verified from theoretical distributions. The results point to the existence of an entropy which characterizes fragmentation processes and depending only on the q entropic index.

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.

Sperm DNA fragmentation in Chinese couples with unexplained recurrent pregnancy loss

We aimed to study the association between sperm DNA fragmentation and recurrent pregnancy loss(RPL)in the Chinese population via a retrospective observational study of Chinese couples who had experienced RPL between May 2013 and August 2018.The study population included 461 men from couples with RPL and 411 men from a control group(couples with clinical pregnancy via in v/tro fertiIization owing to female causes).Routine semen analysis,sperm chromatin analysis,and microscopic(high-power)morphological analysis were performed using semen samples.Semen samples were assessed for volume,sperm count,and motility.The sperm DNA fragmentation index(DFI)was calculated,and the median DFI was obtained.Men were categorized as having normal(37.8%;DFI<15.0%),moderate(33.6%;15.0%<DFI<30.0%),or severe(28.6%;DFI A30.0%)DNA fragmentation levels.The percentage of men with severe DNA fragmentation was significantly higher in the RPL(42.3%)group than that in the control group(13.1%),whereas the percentage of men with normal levels of DNA fragmentation was significantly lower in the RPL group(22.8%)tha n that in the control group(54.7%).Subsequent analysis also dem on strated that the sperm DNA fragmentation rate had a moderate reverse correlation with the sperm progressive motility rate(r=-0.47,P<0.001)and the total motile sperm count(r=-0.31,P<0.001).We found a positive correlation between RPL and sperm DNA fragmentation.The results suggest that increased sperm DNA damage is associated with RPL.

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).

DNA fragmentation in two cytometric sperm populations： relationship with clinical and ultrasound characteristics of the male genital tract

We investigated whether DNA fragmentation in two cytometric sperm populations （PIddimmer and PIbriehter） with different biological characteristics and clinical relevance is related to clinical and color-Doppler ultrasound （CDUS） parameters of the male genital tract. One hundred and sixty males of infertile couples without genetic abnormalities were evaluated for clinical, scrotal, and transrectal CDUS characteristics, presence of prostatitis-like symptoms （with the National Institutes of Health-Chronic Prostatitis Symptom Index） and sperm DNA fragmentation （sDF） in PIdimmer and PIbrighter populations （using TUNEIJPI method coupled with flow cytometry）. Data were adjusted for age （Model 1） along with waistline, testosterone levels, smoking habit, and sexual abstinence （Model 2）. According to the statistical Model 2, PIdi sDF was associated with testicular abnormalities, including lower clinical and ultrasound volume （r = -0.21 and r = -0.20, respectively; P 〈 0.05）, higher FSH levels （r = 0.34, P 〈 0.0001） and occurrence of testicular inhomogeneity （P 〈 0.05） and hypoechogenicity （P 〈 0.05）. PIbrighter sDF was associated with prostate-related symptoms and abnormal signs, including higher NIH-CPSI total and subdomain scores, a higher prevalence of prostatitis-like symptoms and of CDUS alterations such as macro-calcifications, severe echo-texture inhomogeneity, hyperemia （all P 〈 0.05）, and higher arterial peak systolic velocity （r = 0.25, P 〈 0.05）. Our results suggest that DNA fragmentation in PIdimmer sperm, which is related to poor semen quality, mainly originates in the testicles, likely due to apoptosis. Conversely, DNA fragmentation in PIbrighter sperm appears to mainly originate during or after transit through the prostate, increasing with the presence of an inflammatory status of the organ. These results could lead to new perspectives for the identification of therapeutic targets to reduce sDF.

Evaluation of the sperm DNA fragmentation index in infertile Japanese men by in-house flow cytometric analysis

Semen analysis has long been used to evaluate male fertility.Recently,several sperm function tests have been developed.Of those,the sperm DNA fragmentation index(DFI),which describes the status of the sperm DNA,is thought to be a suitable parameter for evaluating male fertility.However,there have been no large-scale studies on the sperm DFI of Japanese men.Therefore,we investigated the feasibility of using an in-house flow cytometry-based sperm DFI analysis based on the sperm DNA fragmentation test of sperm chromatin structure assay(SCSA)to assess male fertility in Japan.This study enrolled 743 infertile and 20 fertile Japanese men.To evaluate reproducibility,inter-and intraobserver precision was analyzed.A receiver operating characteristic curve analysis was used to set a cutoff value for the sperm DFI to identify men who could father children by timed intercourse or intrauterine insemination.The variability of the sperm DFI among fertile volunteers was determined.The relationship between semen parameters and the sperm DFI was assessed by Spearman’s rho test.A precision analysis revealed good reproducibility of the sperm DFI.The cutoff value of sperm DNA fragmentation in infertile men was 24.0%.Semen volume had no relationship with the sperm DFI.Sperm concentration,sperm motility,total motile sperm count,and percentage of normal-shaped sperm were significantly and negatively correlated with the sperm DFI.The median sperm DFI was smaller in fertile volunteers(7.7%)than that in infertile men(19.4%).Sperm DNA fragmentation analysis can be used to assess sperm functions that cannot be evaluated by ordinary semen analysis.

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.

Morphometric comparison by the ISAS CASA-DNAf system of two techniques for the evaluation of DNA fragmentation in human spermatozoa

DNA fragmentation has been shown to be one of the causes of male infertility, particularly related to repeated abortions, and different methods have been developed to analyze it. In the present study, two commercial kits based on the SCD technique （Halosperm~ and SDFA） were evaluated by the use of the DNA fragmentation module of the ISAS vl CASA system. Seven semen samples from volunteers were analyzed. To compare the results between techniques, the Kruskal-Wallis test was used. Data were used for calculation of Principal Components （two PCs were obtained）, and subsequent subpopulations were identified using the Halo, Halo/Core Ratio, and PC data. Results from both kits were significantly different （P 〈 0.001）. In each case, four subpopulations were obtained, independently of the classification method used. The distribution of subpopulations differed depending on the kit used. From the PC data, a discriminant analysis matrix was obtained and a good a posterioriclassification was obtained （97.1% for Halosperm and 96.6% for SDFA）. The present results are the first approach on morphometric evaluation of DNA fragmentation from the SCD technique. This approach could be used for the future definition of a classification matrix surpassing the current subjective evaluation of this important sperm factor.

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.

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.