Sperm nuclear DNA fragmentation and its association with semen quality in Greek men.

Conventional semen parameters and DNA fragmentation in relation to fertility status in a Greek population

Sperm nuclear apoptotic DNA fragmentation in men with spinal cord injury

Background: The sperm DNA fragmentation index (DFI) is regarded as an important tool but its role in evaluating male infertility still remains controversial. The aim of this study is to evaluate the correlation between sperm DNA fragmentation (SDF) with semen parameters and lifestyle patterns. Methods: In our retrospective study which was conducted at the Institute of Reproductive medicine and women health, Madras medical mission, Chennai from 2019-2023, 120 males with subfertility undergoing treatment were included. DFI was measured using the sperm chromatin dispersion assay (SCD) and based on the results they were divided into 3 groups: Low-DFI ≤15%, medium DFI-15%< DFI < 30% and high DFI ≥30%. The correlation between DFI and semen parameters were analysed using Spearman's rank correlation coefficient. Results: In the correlation analysis, DFI showed significant positive correlation with age (p=0.001) and negative correlation with semen parameters like semen volume (p=.0.010), rapid progressive motility (p=0.002) and total motile sperm count (p=0.004). Lifestyle characteristics like body mass index, smoking, consumption of alcohol, diabetes and hypertension showed no significant correlation with DFI. Conclusions: The results indicate a significant relationship between SDF and routine semen parameters, suggesting the inclusion of DNA fragmentation index assessments in male infertilityevaluations.

Male infertility is a complex issue influenced by multiple environmental and pathological factors. In this context, the impact of Human papillomavirus (HPV) infection on male fertility remains controversial. The introduction of new WHO 2021 evaluation criteria, included in the 6th ed. of Laboratory Manual for the examination and processing of human semen, i.e. DNA fragmentation index (DFI), slow and rapid progressive motility, could provide additional information about this correlation. 121 semen samples of male partners of HPV-positive women attending In Vitro Fertilization (IVF) were evaluated following WHO 2021 and HPV-DNA test. Comparing HPV-negative and positive samples for rapid and slow progressive motility showed significantly different results (p = 0.0018, p = 0.0004), contrary to what was observed for total progressive motility. Regarding sperm DFI, only high-risk HPV infections affected DNA integrity. In addition, the correlation between the different semen parameters revealed a significant correlation between midpiece morphological defects and rapid progressive motility in the HPV-positive group (rho = 0.43, p = 0.0006). In conclusion, WHO 2021 provides additional information regarding HPV’s impact on seminal parameters. The correlation between HPV positivity, midpiece defects and a higher rapid progressive motility opens new research perspectives that may help unravel the issues surrounding the role of HPV in compromising sperm quality.

Soy lecithin replaces egg yolk for cryopreservation of human sperm without adversely affecting postthaw motility, morphology, sperm DNA integrity, or sperm binding to hyaluronate

Reliability of the sperm chromatin dispersion assay to evaluate sperm deoxyribonucleic acid damage in men with infertility

Sperm DNA fragmentation (SDF) is a functional sperm abnormality that can impact reproductive potential, for which four assays have been described in the recently published sixth edition of the WHO laboratory manual for the examination and processing of human semen. The purpose of this study was to examine the global practices related to the use of SDF assays and investigate the barriers and limitations that clinicians face in incorporating these tests into their practice. Clinicians managing male infertility were invited to complete an online survey on practices related to SDF diagnostic and treatment approaches. Their responses related to the technical aspects of SDF testing, current professional society guidelines, and the literature were used to generate expert recommendations via the Delphi method. Finally, challenges related to SDF that the clinicians encounter in their daily practice were captured. The survey was completed by 436 reproductive clinicians. Overall, terminal deoxynucleotidyl transferase deoxyuridine triphosphate Nick-End Labeling (TUNEL) is the most commonly used assay chosen by 28.6%, followed by the sperm chromatin structure assay (24.1%), and the sperm chromatin dispersion (19.1%). The choice of the assay was largely influenced by availability (70% of respondents). A threshold of 30% was the most selected cut-off value for elevated SDF by 33.7% of clinicians. Of respondents, 53.6% recommend SDF testing after 3 to 5 days of abstinence. Although 75.3% believe SDF testing can provide an explanation for many unknown causes of infertility, the main limiting factors selected by respondents are a lack of professional society guideline recommendations (62.7%) and an absence of globally accepted references for SDF interpretation (50.3%). This study represents the largest global survey on the technical aspects of SDF testing as well as the barriers encountered by clinicians. Unified global recommendations regarding clinician implementation and standard laboratory interpretation of SDF testing are crucial.

The aim of our study was to investigate the effect of vitrification with sucrose on swim-up-prepared human spermatozoa in comparison with standard, conventional manual freezing with permeable cryoprotectants. After informed consent, 35 ejaculates were obtained from 35 patients with normozoospermia who were patients of a fertility clinic. All specimens used for this study had fulfilled the following quality criteria for spermatozoa concentration and motility on IVOS (Hamilton Thorne). Semen analysis was performed according to published guidelines of the World Health Organization (WHO Laboratory Manual for the Examination and Processing of Human Semen, 2010). After swim-up, each sample was centrifuged, resuspended with the basic medium (human tubal fluid + 1% human serum albumin) to achieve a concentration of 5×106 spermatozoa/mL, and finally aliquoted into two equal subsamples. Each of these aliquots was assigned to one of two groups: group 1 included conventionally cryopreserved spermatozoa and group 2 included spermatozoa that were vitrified. For conventional cryopreservation, freezing media (15% (vol/vol) glycerol, 20% (vol/vol) egg yolk) and citrate was added to the washed spermatozoa in a 1:2 ratio. The sperm suspension was aspirated into 0.5-mL straws (CryoBioSystem). Subsequent to the room-temperature incubation for 10min, straws were placed horizontally in the vapour phase for 15min and then submerged into liquid nitrogen. For thawing, cryopreserved straws were immersed in water (23°C) for 5min. For preparation of vitrification solution, the basic medium (human tubal fluid + 1% human serum albumin) was diluted 1:1 with 0.5M sucrose. Immediately after processing, the sperm suspension was diluted in a 1:1 ratio with the vitrification solution to reach a final sucrose concentration of 0.25M. The vitrification and sperm solution (300μL) were aspirated into the straws 0.5mL. Straws were then left at room temperature (20-21°C) for 10min and subsequently submerged horizontally into the liquid nitrogen (Isachenko et al. 2012 J. Androl. 33, 462-468; https://doi.org/10.2164/jandrol.111.013789) and stored similarly to the conventionally cryopreserved straws. To thaw, vitrified straws were immersed in a water bath (42°C) for 20s. The DNA fragmentation was analysed using the APO DIRECT kit (BD PharmingenTM). The cells were stained according to the manufacturer's protocol, followed by flow cytometry analysis CyFlow (Sysmex-Partec). An analysis of variance with a significance of 0.05 for nonparametric statistical analysis to establish differences between groups was used. In our study, no statistically significant differences were observed in the total motility, progressive motility, or velocity parameters of spermatozoa (P&amp;gt;0.05) post-thawing. Also, higher percentages of DNA fragmentation (35.1±8.1% vs. 20.1±6.8%; P&amp;lt;0.05) were found in spermatozoa cryopreserved by means of vitrification with sucrose compared with conventional cryopreservation. Therefore, these methods are comparable and either can be implemented for the storage of spermatozoa to be used for future assisted-reproduction-technology procedures. Vitrification of human spermatozoa provides a simpler, faster, more cost-effective alternative to conventional cryopreservation methods.

Although the effect of maternal age on fertility is well known, it is unclear whether paternal age also affects fertility. The aim of this retrospective study was to establish an association between the age of the individuals from Medellin, Colombia with semen volume, rapid progressive motility (a), total progressive motility (a + b) and concentration. We evaluated semen volume using a graduated tube, progressive motility using light microscopy (40×) and sperm concentration using a Makler Chamber. Semen samples were grouped according to age into three arbitrary groups (≤ to 30 years; between 31 and 39 years; and ≥ to 40 years). The semen volume, rapid progressive motility (a) and total progressive motility (a + b), concentration and total sperm count were found to be inversely related to age (p < 0.05). The reduction in semen parameters of 1364 men attending an andrology center was associated with increasing age of the individuals.

Reply of the Authors

Dear Editor, We are very glad to see that the Asian Journal of Andrology published a Special Issue on Semen Analysis in 21st Century Medicine, which well revealed some behind-the-scene controversies of the 5th edition of the WHO laboratory manual for the examination and processing of human semen 1. Three articles from the special issue, two on the reference values of semen parameters 2, 3 and another presenting the investigation results of 118 laboratories performing semen analysis in Mainland China 4, are very thought-provoking and we would like to share some of our views on these topics. First, we noticed that the lower limit of the reference values of semen parameters in the 5th edition of the manual is reduced. Can this reduction reflect a genuine decline in semen quality? We are doubtful on this point. The reasons for the reduction vary from the variations in population selection, and the biases in methodologies of measurement and data processing to the recommended application of 'stricter criteria'. It has been a long time since the change in semen quality of healthy men become a controversial issue throughout the world. There is no definite conclusion on the trend of overall semen quality, owing to the differences in study design, definition of normal men, specimen collection methods, methods used for measurement of sperm and for statistical analysis, selection biases, measurement errors, regional differences, and other factors such as age, years spent in semen collection, the period of abstinence, etc. In addition, researches on change of semen quality are only retrospective analyses. Various insurmountable biases and confounding factors also make the conclusion poorly reliable. Although careful efforts were made to reduce the biases in some studies, discrepancies in results still remained. With the publication of the 5th edition of the WHO manual, more concerns and debates would be aroused. So far, it is not yet known whether the reduced lower limit of the reference values can indicate the decline in semen quality, but strictly evidence-based study design, standardization of measurement criteria and quality control (QC) of semen analysis would explain the reduction in the lower limit. Second, we think it is necessary to establish the reference intervals for values of semen parameters based on a fertile population using the time-to-pregnancy method in China. Semen quality is commonly taken as a surrogate indicator of male fertility, reproductive toxicology, epidemiology and pregnancy risk assessments. Reference intervals for values of semen parameters from a fertile population and the standardization of procedures for the examination of human semen given in the 5th edition of the manual provide a realistic basis for medical professionals all over the world, especially in China, where more than one-fifth of the world population inhabits. China is in urgent need of reference values of semen parameters derived from fertile Chinese men, which calls for a prospective, multi-center and nation-wide population-based study. Such a large-scale study will provide evidence for the prognosis of fertility, diagnosis of infertility and impact of environmental factors on human existence. It will also be helpful for the State Food and Drug Administration of China to establish medical guidelines for male fertility regulation. Third, internal and external QC of semen analysis need to be further improved. Semen analysis is used widely and has a significant role in the assessment of male fertility. There have been reports on the absence of standardization on semen analysis 5, 6, 7, 8 and obvious differences among laboratories 9, 10, 11, and some researchers have called for a comprehensive QC 12, 13, 14, 15, 16. The lack of consistency and standardization 17 incurs difficulties for clinicians to interpret or compare the results of semen analysis from different laboratories. Therefore, improvements in the following aspects would help improve the quality of semen analysis and ensuring the reliability of the results: using accredited and standard measurement, implementing internal QC and external QC. Finally, the criterion for determining an eligible donor in China should be revised accordingly to elevate the present poor eligibility after comprehensive investigations. The current quality requirements for an eligible donor's semen as per the Essential Criterion and Technical Specification of the Human Sperm Bank, issued by the Ministry of Health of China, are based on the 4th edition manual. With the reduction in certain semen parameters such as semen concentration, motility and semen volume in the 5th edition, we believe that studies on the reference intervals for values of semen parameters from fertile men must be carried out before any modification of the criteria is made in China. Such expanded studies would lead experts to conduct extensive discussions and would thus provide informative suggestions for policy makers before making any modification in the criteria of the human sperm bank in China.

Sperm DNA fragmentation (SDF) testing was recently added to the sixth edition of the World Health Organization laboratory manual for the examination and processing of human semen. Many conditions and risk factors have been associated with elevated SDF; therefore, it is important to identify the population of infertile men who might benefit from this test. The purpose of this study was to investigate global practices related to indications for SDF testing, compare the relevant professional society guideline recommendations, and provide expert recommendations. Clinicians managing male infertility were invited to take part in a global online survey on SDF clinical practices. This was conducted following the CHERRIES checklist criteria. The responses were compared to professional society guideline recommendations related to SDF and the appropriate available evidence. Expert recommendations on indications for SDF testing were then formulated, and the Delphi method was used to reach consensus. The survey was completed by 436 experts from 55 countries. Almost 75% of respondents test for SDF in all or some men with unexplained or idiopathic infertility, 39% order it routinely in the work-up of recurrent pregnancy loss (RPL), and 62.2% investigate SDF in smokers. While 47% of reproductive urologists test SDF to support the decision for varicocele repair surgery when conventional semen parameters are normal, significantly fewer general urologists (23%; p=0.008) do the same. Nearly 70% would assess SDF before assisted reproductive technologies (ART), either always or for certain conditions. Recurrent ART failure is a common indication for SDF testing. Very few society recommendations were found regarding SDF testing. This article presents the largest global survey on the indications for SDF testing in infertile men, and demonstrates diverse practices. Furthermore, it highlights the paucity of professional society guideline recommendations. Expert recommendations are proposed to help guide clinicians.

Alterations affecting the mitochondrial genome and chromatin integrity of spermatozoa impair male reproductive potential. This study aimed to evaluate the impact of mitochondrial DNA (mtDNA) copy number alterations on sperm motility and on the molecular mechanism regulating the number of mtDNA copies, through analysis of mitochondrial transcription factor A (TFAM) gene expression. It also investigated any correlation between mtDNA copy number and sperm DNA fragmentation (SDF). Sixty-three asthenozoospermic semen samples (Group A) and 63 normokinetic semen samples (Group N) were analysed according to WHO (WHO laboratory manual for the examination and processing of human semen, World Health Organization, Geneva, 2010). Sperm mtDNA copy number and TFAM gene expression were quantified by real time quantitative polymerase chain reaction. SDF was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. The mtDNA copy number was higher in asthenozoospermic semen samples and was negatively correlated with sperm concentration, total sperm number and total motile spermatozoa. The caseload showed a global negative correlation of TFAM gene expression with total motile sperm and a positive correlation with abnormal forms, SDF and mtDNA copy number, but this was not confirmed within each subgroup. SDF was significantly increased in asthenozoospermic samples and correlated with abnormal forms. No correlation was found between SDF and mtDNA copy number. Our results suggest a potential role of mtDNA content as an indicator of semen quality and support the hypothesis that dysregulation of TFAM expression is accompanied by a qualitative impairment of spermatogenesis. Since mtDNA copy number alterations and impaired chromatin integrity could affect reproductive success, these aspects should be evaluated in relation to assisted reproductive techniques.

While it is common to clinically evaluate sperm nuclear DNA fragmentation, less attention has been given to sperm mitochondrial DNA. Recently, a digital PCR assay has allowed accurate estimation of the proportion of fragmented mtDNA molecules and relative copy number. To determine the correlation of classical sperm parameters, average mtDNA copies per spermatozoon and the level of mtDNA fragmentation (SDF-mtDNA) to that of nuclear DNA fragmentation (SDF-nDNA), measured as the proportion of global, single-strand DNA (SDF-SSBs) and double-strand DNA breaks (SDF-DSBs). To determine whether the level of nuclear and mitochondrial DNA fragmentation and/or copy number can differentiate normozoospermic from non-normozoospermic samples. Ejaculates from 29 normozoospermic and 43 non-normozoospermic were evaluated. SDF was determined using the sperm chromatin dispersion assay. mtDNA copy number and SDF-mtDNA were analyzed using digital PCR assays. Relative mtDNA copy increased as sperm concentration or motility decreased, or abnormal morphology increased. Unlike SDF-mtDNA, mtDNA copy number was not correlated with SDF-nDNA. SDF-mtDNA increased as the concentration or proportion of non-vital sperm increased; the higher the mtDNA copy number, the lower the level of fragmentation. Non-normozoospermic samples showed double the level of SDF-nDNA compared to normozoospermic (median 25.00vs. 13.67). mtDNA copy number per spermatozoon was 3× higher in non-normozoospermic ejaculates (median 16.06vs. 4.99). Although logistic regression revealed SDF-Global and mtDNA copy number as independent risk factors for non-normozoospermia, when SDF-Global and mtDNA copy number were combined, ROC curve analysis resulted in an even stronger discriminatory ability for predicting the probability of non-normozoospermia (AUC=0.85, 95% CI 0.76-0.94, p<0.001). High-quality ejaculates show lower nuclear SDF and retain less mtDNA copies, with approximately half of them fragmented, so that the absolute number of non-fragmented mtDNA molecules per spermatozoon is extremely low.

Study question Following 6thWHO (2021), we analysed the correlation between DNA fragmentation index (DFI), Human papillomavirus (HPV), and seminal parameters, highlighting slow and rapid progressive motility alterations. Summary answer DFI rates and seminal parameters correlated with rapid, slow, and progressive motility. However, HPV-positivity caused the loss of association between DFI and slow progressive motility. What is known already HPV detection in semen samples has long opened an investigation into its influence on male infertility. Some studies indicate that HPV can affect sperm quality and DFI, while others have failed to find any correlation. With reference to 2010 WHO guidelines, our latest work highlighted how HPV positivity significantly impairs progressive motility, morphology, and immotile sperm rate. Since the latest 2021 WHO guidelines included the evaluation of slow and rapid progressive motility and DFI, we analysed if these new parameters and the other conventional parameters could be altered by HPV infection. Study design, size, duration From August 2021 to December 2022, 121 semen samples were collected from male partners of HPV-positive women attending in vitro fertilization (IVF). Every specimen underwent DFI evaluation, analysis of seminal parameters, and HPV test. Participants/materials, setting, methods Seminal samples were collected by masturbation after 3-5 days of sexual abstinence. The inclusion criteria were as follows: no other sexually transmitted infections, no genetic diseases, and no inflammatory disorders. Sperm concentration, morphology, non-progressive and immotile sperms, and both slow and rapid progressive motility were evaluated according to WHO 2021 guidelines. DFI analysis was assessed by sperm chromatin dispersion test (SCD), while HPV-DNA detection was performed using InnoLipa HPV Genotyping Extra II (Fujirebio, Tokyo, Japan). Main results and the role of chance Of the 121 semen samples tested, 60 (49.6%) were HPV-positive and 61 (50.4%) were HPV-negative. DFI rates showed a significant negative correlation with rapid progressive motility in both groups and a positive correlation with slow progressive motility in the HPV-negative group. Conversely, the significance of the correlation between DFI and slow progressive motility was completely lost in HPV-positive patients. Sperm concentration, normal forms and immotile spermatozoa percentages were correlated with both motility parameters in the HPV-negative group. Similar results were observed in HPV-positive samples, except for the normal form rate, which was not associated with slow progressive motility. In addition, the same samples displayed a negative correlation between non-progressive motility and rapid progressive motility, absent in HPV-negative samples. Significant associations were found also for the derived parameter of progressive motility, which was correlated with DFI, sperm concentration, immotile sperm, and normal forms rate in both groups. The results suggest how high DFI rates, in the presence or absence of HPV infection, could affect reproductive health through a consistent impairment of spermatozoa motility. In particular, the distinction of slow and rapid progressive motility by WHO 2021 allows a deeper understanding of the possible correlations between DFI, semen parameters and HPV infection. Limitations, reasons for caution This is a preliminary study characterized by a small number of samples. Therefore, confirmation of these findings requires the enlargement of the patient cohort, which is already taking place. Wider implications of the findings Our results highlight how the introduction of the new WHO 2021 evaluation criteria, i.e. DFI, and slow and rapid progressive motility, provides additional information about sperm quality and the impact of HPV infection on it. Trial registration number Not applicable