Sperm Chromatin Dispersion Test Research Articles

Application of Autologous Platelet-rich Plasma Exerts Cryoprotective Effects on Biological Characteristics of Human Oligoasthenoteratospermia Samples after Freezing and Thawing Procedures.

Platelet-rich plasma (PRP) is enriched with active biological components which showed proliferative and cytoprotective properties in healing different injuries in medicinal fields. This study was designed to assess cryoprotective effects of autologous PRP on quality of oligoasthenoteratospermia (OAT) samples during freezing and thawing procedure. The present study is an experimental research. Twenty OAT semen samples were obtained from individuals and prepared by discontinuous density - gradients technique. Control group is sperm samples after DGC. After the procedure, the specimen divided into four groups. Freeze group which has no additive and other three groups were cryopreserved with different concentrations of PRP (1×105/µL, 0.5×105/µL and 0.25×105/µL). Autologous PRP was provided by each participant. After thawing, sperm parameters, DNA fragmentation by sperm chromatin dispersion test (SCD), protamine deficiency by (Chromomycin A3) CMA3 staining, acrosome integrity and malondialdehyde (MDA) level were evaluated. Cryopreservation resulted in significant decreased in all factors compared to the control group. There were no significant changes on sperm count, morphology, non-progressive motility and acrosome reaction by adding PRP as cryoprotectant in comparison with freeze group. PRP at all three concentrations showed significant increase in progressive motility (3.05±2.01 vs. 14.05±4.13, 12.35±4.90 and 12.15±9.65, P<0.001) and viability (36.85±10.25 vs. 47.85±5.86, 51.30±5.54 and 50.05±5.67, P<0.001) compared to the sperm samples without PRP. The percentage of immotile sperms decreased at all PRP concentrations compared to the freeze group. Moreover, PRP at 1×105/µL concentration showed cryoprotective effects on DNA fragmentation, protamine deficiency and MDA level compared to the other three concenterations. Cryopreservation and thawing procedures may exert adverse effects on biological factors of sperm samples. Therefore, adding PRP as cryoprotectant at all three concentrations especially 1×105/µL can be promising strategy to reduce adverse effects of cryopreservation on OAT samples.

Sperm Chromatin Dispersion Test Detects Sperm DNA Fragmentation Mainly Associated with Unviable Spermatozoa and Underestimates the Values with Respect to TUNEL Assay.

Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes.

Evaluation of Sperm DNA Fragmentation Using Halosperm Technique after the Freezing–Thawing Process in Men: A Study on the Validation of the SCD Protocol

Background: DNA fragmentation index (DFI) enhances routine semen analysis by providing valuable insights into male reproductive potential. Utilizing Halosperm test, a sperm chromatin dispersion (SCD) assay based on induced condensation. The purpose of this study was to assess sperm DNA damage both before and after freezing. By following the specified kit instructions, an attempt was made to validate the SCD test protocol, with a particular emphasis on the implications of sperm freezing on its DNA integrity.&#x0D; Methods: In total, 380 fresh human semen samples from normozoospermic patients were frozen at -20°C for 10 days, using SCD cryopreservation reagent. Routine semen analysis and DNA fragmentation index (DFI) were determined for each sample before freezing and after thawing. Semen morphology and sperm DFI were compared before and after freezing/thawing process.&#x0D; Results: There was a significant decrease in sperm normal morphology after thawing (9.31±2.42% vs. 7.1±1.53%, p&lt;0.05, respectively). The sperm head, midpiece, and tail defect rate increased after freezing at -20°C. Moreover, DFI was significantly higher after thawing compared to before freezing (20.71±1.61% before freezing vs. 29.1±0.21% after thawing with p&lt;0.001).&#x0D; Conclusion: Cryoconservation of semen samples at -20°C for 10 days using SCD cryopreservation reagent seems to damage sperm morphology, resulting in a reduction in sperm DNA integrity. The measurement of DFI on a fresh sample remains the most reliable technique for obtaining accurate results.

Seminal Oxidative Stress and Sperm DNA Fragmentation in Men from Couples with Infertility or Unexplained Recurrent Pregnancy Loss

(1) Background: This case–control study examined whether men from couples with unexplained recurrent pregnancy loss (RPL) or infertility exhibited higher seminal oxidative stress (OS) and sperm DNA fragmentation (SDF) compared to fertile controls. (2) Methods: The study included 30 participants from each group: unexplained RPL, unexplained infertility, and proven fertility. Data were collected at Aalborg University Hospital tertiary RPL and fertility treatment clinics (Aalborg, Denmark), excluding couples with mixed conditions for homogeneity. Semen samples were analyzed using computer-aided sperm analysis (CASA) for concentration, motility, and morphology. SDF was assessed via a CASA-based sperm chromatin dispersion test. OS was measured as static oxidation-reduction potential (sORP). (3) Results: The results showed no significant OS differences between groups. The RPL group had significantly lower SDF levels than the control group. A significant positive correlation between SDF and OS was observed in the infertility group. Overall, this study did not find significant differences in OS levels between men from couples with unexplained RPL or infertility and fertile controls, while SDF levels were lower in the RPL group compared to controls. (4) Conclusion: In conclusion, despite the existing literature suggesting that OS and SDF are negative prognostic factors, our findings suggest they may not be reliable diagnostic markers for RPL and infertility.

Gaussian clustering and quantification of the sperm chromatin dispersion test using convolutional neural networks.

Sperm DNA fragmentation is a sign of sperm nuclear damage. The sperm chromatin dispersion (SCD) test is a reliable and economical method for the evaluation of DNA fragmentation. However, the cut-off value for differentiation of DNA fragmented sperms is fixed at 1/3 with limited statistical justification, making the SCD test a semi-quantitative method that gives user-dependent results. We construct a collection of deep neural networks to automate the evaluation of bright-field images for SCD tests. The model can detect valid sperm nuclei and their locations from the input images captured with a 20× objective and predict the geometric parameters of the halo ring. We construct an annotated dataset consisting of N = 3120 images. The ResNet 18 based network reaches an average precision (AP50) of 91.3%, a true positive rate of 96.67%, and a true negative rate of 96.72%. The distribution of relative halo radii is fit to the multi-peak Gaussian function (p > 0.99). DNA fragmentation is regarded as those with a relative halo radius 1.6 standard deviations smaller than the mean of a normal cluster. In conclusion, we have established a deep neural network based model for the automation and quantification of the SCD test that is ready for clinical application. The DNA fragmentation index is determined using Gaussian clustering, reflecting the natural distribution of halo geometry and is more tolerable to disturbances and sample conditions, which we believe will greatly improve the clinical significance of the SCD test.

Urinary concentrations of bisphenol A, parabens and benzophenone-type ultra violet light filters in relation to sperm DNA fragmentation in young men: A chemical mixtures approach

People are daily exposed to multiple endocrine disruptor compounds (EDCs) that may interfere with different molecular and cellular processes, promoting a potential estrogenic, androgenic, or anti-androgenic state. However, most epidemiological studies attempting to establish relationships between EDCs exposure and health effects are still considering individual compounds. A few studies have shown associations between exposure to individual non-persistent EDCs and sperm DNA fragmentation (SDF) in different male populations. Thus, the aim of this study was to investigate associations between combined exposure to non-persistent EDCs and SDF index in young men. A cross-sectional study was conducted with 158 healthy university students from Southeaster Spain. The participants provided spot urine and semen samples on the same day. The concentrations of urinary bisphenol A (BPA), benzophenones [2,4-dihydroxybenzophenone (BP-1); 2,2′,4,4′-tetrahydroxybenzophenone (BP-2), 2-hydroxy-4-methoxybenzophenone (BP-3), 2,2′-dihydroxy-4-methoxybenzophenone (BP-8), 4-hydroxybenzophenone (4OHBP)], and parabens (methylparaben, ethylparaben, propylparaben, butylparaben) were measured by dispersive liquid-liquid microextraction and ultrahigh-performance liquid chromatography with tandem mass spectrometry detection. SDF was analysed using a Sperm Chromatin Dispersion test. Statistical analyses were carried out using Bayesian Kernel Machine Regression models to evaluate associations between combined exposure to these compounds and SDF index while adjusting by relevant covariates. The increase in urinary concentration of 4OHBP was found to be the most important contributor to the negative association between urinary EDCs concentrations and SDF index, being of −5.5 % [95 % CI: −10.7, −0.3] for those in percentile 50, and − 5.4 % [95 % CI: −10.8, −0.1] for those in percentile 75. No significant associations were observed between other EDCs and SDF index. Our findings show that urinary 4OHBP levels may be associated with a decrease in the SDF index. Nonetheless, the effects we observed were likely to be small and of uncertain clinical significance. Further research is needed to replicate our findings in other male populations.

Low human sperm motility coexists with sperm nuclear DNA damage and oxidative stress in semen.

Low sperm motility, one of the common causes of male infertility, is associated with abnormal sperm quality. Currently, important sperm/semen biomarkers are sperm chromatin status and oxidation‒reduction potential (ORP) in semen. Because the association between sperm motility and these biomarkers is still not fully clarified, our study was designed to verify the distribution and risk of sperm DNA fragmentation (SDF) and oxidative stress in semen in asthenozoospermic men. This study was carried out on discharged sperm cells of asthenozoospermic men (isolated asthenozoospermia or coexisted with reduced sperm number and/or morphology), nonasthenozoospermic men (reduced total sperm count and/or sperm morphology) (experimental groups) and normozoospermic men (proven and presumed fertility) (control group). Basic semen analysis was evaluated according to the 6th edition of the World Health Organization manual guidelines. SDF was assessed using the sperm chromatin dispersion test, while static(s) ORP in semen was measured by means of a MiOXSYS analyser. The men from the asthenozoospermic group had lower basic semen parameters than those from the control and nonasthenozoospermic groups. In men with poor sperm motility SDF and sORP, prevalence and risk for>20% SDF (high level of DNA damage) and for>1.37 sORP (oxidative stress) were significantly higher than those of control and nonasthenozoospermic subjects. The risk for sperm DNA damage and oxidative stress in asthenozoospermic men was over 10-fold higher and almost 6-fold higher than those in control subjects and almost or over 3-fold higher than those in nonasthenozoospermic men. Poor human sperm motility coexisted with low basic sperm quality. Sperm DNA damage and oxidative stress in semen were much more frequent in asthenozoospermia. These abnormalities can decrease the sperm fertilizing capability under both natural and medically assisted reproduction conditions. Thus, in asthenozoospermia, the evaluation of sperm chromatin status and oxidation-reduction potential in semen is justified and inevitable, and the appropriate antioxidant therapy can be suggested.

Unraveling the Impact of Sperm DNA Fragmentation on Reproductive Outcomes.

In recent years, there has been a growing interest in identifying subcellular causes of male infertility, and sperm DNA fragmentation (SDF) research has been at the forefront of this focus. DNA damage can occur during spermatogenesis due to faulty chromatin compaction or excessive abortive apoptosis. It can also happen as sperm transit through the genital tract, often induced by oxidative stress. There are several methods for SDF testing, with the sperm chromatin structure assay, terminal deoxynucleotidyl transferase d-UTI nick end labeling (TUNEL) assay, comet assay, and sperm chromatin dispersion test being the most commonly used. Numerous studies strongly support the negative impact of SDF on male fertility potential. DNA damage has been linked to various morphological and functional sperm abnormalities, ultimately affecting natural conception and assisted reproductive technology outcomes. This evidence-based review aims to explore how SDF influences male reproduction and provide insights into available therapeutic options to minimize its detrimental impact.

Tip-microVapour Fast Freezing: A novel easy method for cryopreserving severe oligozoospermic samples.

Sperm cryopreservation is an important procedure for oligozoospermic subjects at risk of azoospermia and after surgical recovery of spermatozoa in non-obstructive azoospermic men. Conventional procedures for sperm cryopreservation might be, however, not suitable for samples with a very low sperm number. In this pilot study, we investigated the recoveries of sperm motility and viability in severe oligozoospermic subjects (n=39) after cryopreservation with a tip-microVapour Fast Freezing, a procedure previously developed by our group for men with good semen quality. Sperm DNA fragmentation was also evaluated in a second group of oligozoospermic samples (n=16). We used a Vapour Fast Freezing procedure using 10μL tips as carrier, and Test Yolk Buffer as freezing medium (tip-microVapour Fast Freezing). In a subset of samples (n=22), we compared recovery of motility and viability as obtained with tip-microVapour Fast Freezing and with a Vapour Fast Freezing procedure using 500μL straws. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test. We found a recovery rate (median [interquartile range]) of 0.29 (0.13-0.41) for progressive motility, 0.30 (0.21-0.52) for total motility and 0.48 (0.29-0.60) for viability. Interestingly, we observed that samples with the poorest motility were apparently less damaged by freezing/thawing. In a subset of samples (n=22), we directly compared values of viability, progressive motility and total motility by freezing/thawing with tip-microVapour Fast Freezing and Vapour Fast Freezing conducted with 500μL straws. We found much better values of all sperm parameters in samples after freezing/thawing with tip-microVapour Fast Freezing than with Vapour Fast Freezing in 500μL straws: that is, progressive motility: 7.00 (3.00-8.50)% versus 2.00 (0.00-4.25)%, p<0.001; total motility: 12.00 (8.00-16.25)% versus 6.50 (1.00-9.25)%, p<0.001; viability: 29.75 (23.75-45.25) versus 22.50 (13.75-28.13), p<0.001, respectively. In the second group of oligozoospermic samples, we found that tip-microVapour Fast Freezing produced lower levels of sperm DNA fragmentation than straws (33.00 [19.75-36.00]% vs. 36.00 [22.75-41.87]%, p<0.001). Tip-microVapour Fast Freezing appears to be a very promising method to cryopreserve semen samples from severe oligozoospermic patients.

Effect of season on the dynamics of cat sperm DNA fragmentation

BackgroundFeline species undergo reproductive seasonality; thus, sperm characteristics, such as DNA integrity, can be affected by the photoperiod. This study was conducted to determine the effect of seasonal changes on sperm quality and on the dynamics of sperm DNA fragmentation. Epididymal spermatozoa were collected from 36 tomcats subjected to bilateral orchiectomy during breeding (BS) and non-breeding (NBS) seasons. Sperm samples were obtained by cutting the cauda epididymis and assessed for sperm motility, concentration, acrosome integrity, plasma membrane integrity and sperm morphology. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test after 0, 6, and 24 h of incubation at 37 °C.ResultsThe total sperm motility and plasma membrane integrity values were greater during the BS, while the percentages of abnormal sperm and head defects were lesser (p < 0.05). No significant differences in DNA fragmentation were found between seasons after sperm collection. DNA damage was greater after 24 h of incubation at 37 °C in both seasons, although the percentage of spermatozoa with fragmented DNA was significantly lesser in the BS than in the NBS at 24 h (p < 0.05).ConclusionsThe study suggests seasonal changes in some of the quality parameters of cat sperm. DNA fragmentation dynamics were affected by the time of incubation and reproductive season; therefore, this technique might be used as an additional tool to test the potential fertility of semen samples used in feline-assisted reproduction.

DNA Integrity in Absolute Teratozoospermia Patients and its Impact on Assisted Reproductive Technology Outcome

Numerous studies showed that Sperm DNA Fragmentation (SDF) is inversely correlated to semen parameters and that high SDF is a major contributor to failure of conception. In the present study, the severity of Sperm DNA Fragmentation in absolute teratoozoospermia patients and its impact on ICSI outcome compared to males with normal semen was examined. A prospective study was performed including 36 absolute teratozoospermia and 30 controls. The mean of DNA Fragmentation Index (DFI) using Sperm Chromatin Dispersion (SCD) test in the absolute teratozoospermia was 42.9 ± 13.2, compared to 24.59 ± 6.9 for the controls. The percentage of patients with high DFI (&gt;30) was higher in the absolute teratozoospermia group 78% than in the control group 30%. Moderate DFI (15-30%) was more common in controls 60% than in absolute teratozoospermia 22%. Normal DFI (≤ 15%) was completely absent in absolute teratozoospermia. The pregnancy rate following Intracytoplasmic Sperm Injection (ICSI) in the control group was 66.6% and 50% in the absolute teratozoospermia. The miscarriage rate was 26% in the control group compared to 53% in the absolute teratozoospermia. Live birth was significantly lower in absolute teratozoospermia compared to fertile men; 19% and 52% respectively. High levels of SDF are predominant in absolute teratozoospermia patients consequently affecting the outcome of ICSI.

P-105 The Reliability of Sperm Chromatin Dispersion (SCD) Test to Predict Assisted Reproductive Technology (ART) Treatments Outcomes -A Systematic Review and Meta-Analysis

Abstract Study question Is the sperm chromatin dispersion (SCD) test reliable for predicting reproductive technology (ART) treatment outcomes? Summary answer Assessment of DNA fragmentation using the SCD test failed to predict pregnancy or live births rates. What is known already The detrimental effect of high DNA fragmentation on semen quality, fertility, and poor ART outcomes has been well-studied in recent years. Still, it remains contentious. Several techniques can detect sperm DNA fragmentation, such as TUNEL, comet assay, and SCSA. Although these techniques have been implemented in many andrology laboratories, they remain complex, time-consuming, and expensive. On the other hand, the SCD test indirectly estimates the DNA fragmentation level by manually quantifying the number of halos representing nuclear dispersion after sperm lysis and acid denaturation. While the SCD test is commonly used, it remains subjected to bias and high variability. Study design, size, duration Systematic review and meta-analysis. A PRISMA-guided literature search in the English language was performed in January 2021 using PubMed/MEDLINE, Scopus, and Google scholar. We looked for rates of fertilization, cleavage rate embryos, implantation, clinical pregnancy, miscarriage, and live birth. MedCalc software using the random effects model was used, Sensitivity analysis was conducted to examine heterogeneity and the robustness of the results. Participants/materials, setting, methods After duplicate removal, 179 abstracts were assessed for eligibility, and 55 full-text articles were screened. Forty-one articles were included in the qualitative syntheses, and 16 were included in the quantitative synthesis, representing 6989 participants. Main results and the role of chance No significant difference was identified between the group with high SDF vs. low SDF regarding fertilization (SMD −0.334, 95% CI −0.695, 0.027, p = 0.07), clinical pregnancy (OR 0.901, 95% CI 0.719, 1.130, p = 0.367), miscarriage (OR 0.901, 95% CI 0.719, 1.130, p = 0.367) and live birth (OR 1.105, 95% CI 0.627, 1.946, p = 0.730) rates. The rates of cleavage stage embryos (SMD -1.153, 95% CI -2.113, −0.194, p = 0.019) and implantation (OR 0.472, 95% CI 0.303, 0.737, p = 0.001) were significantly lower in the group with high SDF vs. low SDF. Limitations, reasons for caution A high index of heterogeneity and publication bias was reported regarding fertilization (I2 = 93.23%, Egger’s test: intercept -5.29866, 95% CI -10.41, −0.19, p = 0.02) and cleavage-stage embryos(I2 = 98.49%, Egger’s test: intercept -14.02395, 95% CI -27.47, −0.57, p = 0.02) Wider implications of the findings Assessment of DNA fragmentation using the SCD test failed to predict pregnancy or live births. As the goal of ART is live birth, clinicians should consider the limited yield of this assay to predict the likelihood of live birth. Trial registration number NA

P-038 Correlation between sperm DNA fragmentation index, semen parameters and Human Papillomavirus: an analysis conducted under World Health Organization 2021 guidelines

Abstract 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

P-062 The influence of the ejaculatory abstinence period on semen parameters from the first diagnostic sample – results from analysis of 14000 samples

Abstract Study question Is ejaculatory abstinence period (EAP) associated with semen parameters in males undergoing preliminary fertility investigation? Summary answer Short EAP is associated with increased progressive motility (%), vitality but with reduced volume, concentration, total sperm count. No association was indentified with morphology, DNA fragmentation. What is known already It is known that semen parameters may present important intra-individual variations. At present, the last edition of the WHO Manual recommends ejaculatory abstinence from two to seven days for semen analysis. Nonetheless, this period is not supported by some relevant bodies in the reproductive field guidelines that limit abstinence period to three or four days. Recently published reviews on this matter support that the relationship between the abstinence time and sperm quality is not straightforward. Therefore the aim of this study is to analyze correlation between the ejaculatory abstinence time and sperm parameters in patients having initial work-up in our clinic. Study design, size, duration We conducted a retrospective cross-sectional study on the diagnostic semen samples from individuals having their initial fertility investigation in the Fertility Department of a University Affiliated Hospital between the beginning of 1997 and the end of 2022. Basic semen parameters and sperm deoxyribonucleic acid (DNA) fragmentation were registered in 14067 and 784 samples respectively. Sperm morphology was analysed in 4812 samples. Participants/materials, setting, methods We included exclusively results from the first diagnostic semen sample from males of any age undergoing preliminary infertility work-up. All samples were produced on-site and analysed “ad hoc” for basic semen parameters (according to 6th Edition of WHO Manual) or/and sperm DNA fragmentation (Sperm Chromatin Dispersion test). Samples were divided according to EAP: ≤2(A-group); 2-7(B-group); &amp;gt;7 days (C-group) and compared with ANOVA. Additionally, Spearman Correlation was used to confirm correlation between EAP and sperm parameters. Main results and the role of chance Semen analysis was performed either completely manually (23.4%) or with the use of Computer Assisted Semen Analysis (CASA) system (76.6%) with the exception of morphology which was always assessed manually. Overall, 661 samples were produced after ≤2 days of abstinence, 12734 after 2 to maximum 7 days and 690 after &amp;gt;7 days of which ones 330(50%), 6798(53%) and 336(49%) were normozoospermic, respectively. Mean male age was 39 years (SD 6.5) and EAP 4.5 days (SD 1.9). We found the following parameters positively correlated with EAP in both normozoospermic and abnormal samples: volume (A-2.6ml; B-3.6ml; C-4ml, p &amp;lt; 0.05), concentration (A-72M/ml; B-93M/ml; C-127M/ml, p &amp;lt; 0.05), total sperm count (A-161M; B-285M; C-472M, p &amp;lt; 0.05), total motile count (A-111M; B-186M; C-270M, p &amp;lt; 0.05), straightness coefficient (STR) (A-73.1; B-75.7; C-77.8%, p &amp;lt; 0.05) and Zinc concentration (A-14277; B-17375; C-22538µg/dl, p &amp;lt; 0.05). The following parameters were negatively correlated with EAP in both normozoospermic and abnormal samples: progressive motility (A-46; B-43.6; C-36.6%, p &amp;lt; 0.05), vitality (A-60.5; B-60.3; C-53.7%, p &amp;lt; 0.05). We did not find any correlation with leucocytes, fructose, morphology or DNA fragmentation in unselected patients. However, in the group of abnormal samples there was a negative correlation between EAP and morphology (A-3.7; B-3.1; C-2.8%, p &amp;lt; 0.05) and DNA fragmentation, in generalized additive model (Spearman-Correlation, R2 0.01, p &amp;lt; 0.05). Limitations, reasons for caution This is a cross-sectional study which demonstrates only association and does not allow establishment of a cause-effect relationship between EAP and sperm parameters. Additionally, despite significant correlation between EAP and some sperm parameters, its clinical usefulness remains unclear. Wider implications of the findings To our knowledge this is first study correlating EAP with semen parameters only from diagnostic semen samples produced on-site, which confirms that the abstinence time should be seriously considered when analysing ejaculates, especially when borderline results are obtained. Additionally, different clinical strategies may be considered for normo and non-normozoospermic patients. Trial registration number not applicable

P-071 L-proline enhances sperm quality and chromatin condensation of normozoospermic men during in vitro incubation

Abstract Study question Does supplementation of human sperm media with L-Proline improve sperm function and chromatin quality during in Vitro Incubation? Summary answer The inclusion of L-proline as an antioxidant in human sperm media improves sperm function and chromatin integrity probably by modulating oxidative stress What is known already Infertility affects millions of people of reproductive age globally. The quality of gametes is one of the most critical aspects that could affect the success rate of infertility therapy. The storage of spermatozoa at the incubator to achieve capacitation could rise sperm DNA fragmentation as a result of prolonged incubation. Although mild and low levels of Reactive Oxygen Species (ROS) could boost fertilization capacity by improving hyperactivation, and capacitation, oxidative stress is one of the major contributors to low sperm quality. L-Proline plays a pivotal role in many aspects of cell metabolism and physiology, including osmotic protection and oxidative stress Study design, size, duration The study was conducted on thirty-five healthy men attending the Motazedi Hospital, Kermanshah of the Division of Andrology, from December 2020 to June 2021. Each semen sample was equally divided into 4 groups: one group as control and three as treatment groups. The control group contained only sperm media (Hams, F10 medium supplemented with 5 mg/ml HSA). In treatment groups, different concentrations of Proline (1, 2, and 4 mmol/L) were also added. Participants/materials, setting, methods 35 normozoospermic men with healthy lifestyles were included. Sperm parameters including motility, viability, and morphology were evaluated. The levels of ROS, lipid peroxidation (LPO), and total antioxidant capacity (TAC) were determined in sperm media. Sperm chromatin integrity was analyzed by Aniline blue (AB), Toluidine blue (TB), and Chromomycin A3 (CMA3) staining tests. DNA fragmentation was measured by the Sperm chromatin dispersion (SCD) test. All assessments were conducted after 1, 4, and 24 hours of incubation. Main results and the role of chance The inclusion of Proline (2 mmol/L) resulted in noticeably improved maintenance of motility and viability after 24 hr of incubation compared to the control group (P &amp;lt; 0.05). Also, 2 mmol/L of Proline was able to significantly maintain normal morphology compared to other groups (P &amp;lt; 0.01). However, the level of ROS production non-significantly diminished in the L-proline groups compared to the control group (P &amp;gt; 0.05). In terms of lipid peroxidation, the presence of 2 mmol/L of Proline kept the MDA production of spermatozoa at lower levels after 4 hr of incubation compared with the control group (P &amp;lt; 0.05). The supplementation of sperm media with 2 mmol/L of Proline led to improved maintenance of TAC level, compared to any other group (P &amp;lt; 0.01). Additionally, the results of AB and TB tests indicated that chromatin quality after supplementation with 2 mmol/L L-proline significantly improved compared to the control group (P &amp;lt; 0.05). In terms of CMA3 assessment, however, there was a minor improvement between the 2 mmol/L Proline group and the control group. Moreover, 2 mmol/L Proline significantly reduced the level of fragmented DNA by comparison to the control group after 24 hr of incubation (P &amp;lt; 0.05). Limitations, reasons for caution Our team was unable to carry out additional studies to verify the impact of L-proline on mitochondrial activity, membrane potential, and apoptosis and to validate the efficiency and safety of L-proline using additional parameters such as the in vitro embryonic development and live birth rate. Wider implications of the findings While further studies are required to uncover the mechanisms underlying the antioxidant properties of Proline, these findings extend our knowledge of human male fertility and pave the way to improving the human sperm culture and in vitro procedure routinely used in infertility clinics for sperm incubation. Trial registration number IR.KUMS.REC.1399.625

Does folate deficiency alter sperm parameters in infertile males?

Introduction: There is not enough evidence regarding the influence of folate deficiency in the etiology of idiopathic male infertility. Objectives: The objective of the current study was to examine whether there is an association between serum levels of folate and sperm parameters among infertile males. Patients and Methods: This cross-sectional study was undertaken on a sample of infertile males referring to an infertility treatment clinic in Isfahan province, Iran. Semen parameters, comprising sperm concentration, progressive/total motility, morphology, semen leukocyte, agglutination, and vitality were assessed based on standard protocols. The sperm DNA fragmentation assay (SDFA) and sperm chromatin maturity assay (SCMA) were also assessed by the sperm chromatin dispersion and aniline-blue tests respectively. Results: In general, 70 infertile males including 46 with sufficient serum folate level and 24 with insufficient serum folate level were examined. Subjects with sufficient serum folate level had a significantly higher mean of sperm concentration, vitality, progressive motility, total motility, SDFA, and SCMA (P&lt;0.05). Higher serum folate level was also associated with lower risk of abnormal sperm agglutination (OR: 0.09, 95% CI: 0.01-0.65), and morphology (OR: 0.12, 95% CI: (0.02-0.60) after adjustment for potential confounders. Conclusion: The present study found a significant association between serum folate level and infertility among Iranian infertile males. However, prospective large-scale population-based research is required to confirm the finding.

Novel sperm chromatin dispersion test with artificial intelligence-aided halo evaluation: A comparison study with existing modalities.

Sperm chromatin dispersion test is a common and inexpensive technique to assess sperm DNA fragmentation, but its subjectivity in assessing a small number of spermatozoa is a disadvantage. To study the efficacy of a new sperm chromatin dispersion test kit (R10) combined with an artificial intelligence-aided halo-evaluation platform (X12) and compare the results to those of existing sperm DNA fragmentation testing methods. Semen samples from normozoospermic donors (n=10) and infertile men with abnormal semen parameters (n=10) were enrolled. DNA fragmentation indices were examined by multiple assays, including R10, Halosperm G2 (G2), sperm chromatin structure assay, and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling. In R10 assay, the DNA fragmentation indices were obtained both manually (manual R10) and by X12 (AI-R10). The obtained DNA fragmentation indices were analyzed by agreement analyses. The DNA fragmentation indices obtained by manual R10 and those obtained by AI-R10 showed a strong significant correlation (r=0.97, p<0.001) and agreement. The number of spermatozoa evaluated by AI-R10 was 2078 (680-5831). The DNA fragmentation indices obtained by manual R10 and AI-R10 both correlated with those of G2 (r=0.90, p<0.001; r=0.88, p<0.001). Between the AI-R10 and G2 results, Passing-Bablok regression showed no systematic or proportional difference, and Bland-Altman plots revealed overall agreement and a mean bias of 6.3% with an SD of 6.9% (95% limit of agreement: -7.2% to 19.9%). AI-R10 and sperm chromatin structure assays showed systematic differences with a mean bias of -1.9%, while AI-R10 and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling revealed proportional differences with a mean bias of -10.7%. The novel sperm chromatin dispersion kit and artificial intelligence-aided platform demonstrated significant correlation and agreement with existing sperm chromatin dispersion methods by assessing greater number of spermatozoa. This technique has the potential to provide a rapid and accurate assessment of sperm DNA fragmentation without technical expertise or flow cytometry.

In vitro effects of plasma rich in growth factors on human teratozoospermic semen samples

There is a correlation between teratozoospermia and production of reactive oxygen species leading to poor assisted reproductive techniques outcomes. This study aimed to examine the effect of plasma-rich in growth factors (PRGF) on teratozoospermic samples. Twenty-five teratozoospermic samples were included in this study. After sperm preparation, it was divided into four groups, including 0 (control), 1, 5, and 10% PRGF. Sperm motility, viability (eosin-nigrosin staining), morphology (Papanicolaou staining), DNA fragmentation (sperm chromatin dispersion test), mitochondrial membrane potential (JC-1 staining by flow cytometry), and lipid peroxidation (measurement of malondialdehyde, MDA) were evaluated before and after 1 h of incubation with or without PRGF. Our results showed that after 1 h of incubation, the addition of 1% PRGF improved sperm progressive motility (47.72 ± 13.76%) compared to the control group (17.36 ± 8.50%) (p < 0.001). Also, 1% PRGF preserved the sperm’s total motility (77.50 ± 13.28% vs. 65.63 ± 19.03%, for 1% PRGF and control, respectively) and viability after incubation. The rate of normal sperm morphology was the same between different groups. Higher mitochondrial membrane potential and lower DNA fragmentation were also observed in sperm treated with different concentrations of PRGF compared to the control group, but the differences were non-significant. The MDA levels were significantly decreased in PRGF-treated groups compared to the control group (0.99 ± 0.62, 0.95 ± 0.33, 0.95 ± 0.79, and 1.49 ± 0.27 for 1% PRGF, 5% PRGF, 10% PRGF and control, respectively). Based on our results, it seems that PRGF incubation can improve sperm parameters and especially decrease the level of malondialdehyde as an indicator of oxidative stress, which is one of the main problems of teratozoospermic samples.

Best Washing Method for Oligoasthenospermic Patients by Evaluating DNA Integrity by Using a Sperm Chromatin Dispersion Test

Background: Growing evidence points to sperm DNA fragmentation (DF) as a significant contributor to infertility. One of the most popular techniques to identify sperm DF is the Sperm Chromatin Dispersion test (SCDT), a new assay in semen. The SCDT is based on the idea that after acid denaturation and removal of nuclear proteins, sperm with fragmented DNA are unable to create the distinctive halo of dispersed DNA loops seen in sperm with non-fragmented DNA. As Oligoasthenospermic patients have low sperm concentration, these patients are infertile and opt for fertility treatments for getting a child. There are many steps in the processing of sperm during Assisted Reproductive Technology (ART), and one of the main steps is Sperm Washing. Aim &amp; Objective: The main aim of this research is to determine the best sperm-washing method for Oligoasthenospermic patients by evaluating DNA integrity (DI) by using SCDT. Study Setting: Semen samples were obtained from 40 Oligoasthenospermic patients with progressive motility

Animal board invited review: An update on the methods for semen quality evaluation in swine - from farm to the lab.

Pig breeding is mainly conducted through artificial insemination with liquid-stored semen. It is, therefore, crucial to ensure that sperm quality is over the standard thresholds, as reduced sperm motility, morphology or plasma membrane integrity are associated with reduced farrowing rates and litter sizes. This work aims to summarise the methods utilised in farms and research laboratories to evaluate sperm quality in pigs. The conventional spermiogram consists in the assessment of sperm concentration, motility and morphology, which are the most estimated variables in farms. Yet, while the determination of these sperm parameters is enough for farms to prepare seminal doses, other tests, usually carried out in specialised laboratories, may be required when boar studs exhibit a decreased reproductive performance. These methods include the evaluation of functional sperm parameters, such as plasma membrane integrity and fluidity, intracellular levels of calcium and reactive oxygen species, mitochondrial activity, and acrosome integrity, using fluorescent probes and flow cytometry. Furthermore, sperm chromatin condensation and DNA integrity, despite not being routinely assessed, may also help determine the causes of reduced fertilising capacity. Sperm DNA integrity can be evaluated through direct (Comet, transferase deoxynucleotide nick end labelling (TUNEL) and its in situ nick variant) or indirect tests (Sperm Chromatin Structure Assay, Sperm Chromatin Dispersion Test), whereas chromatin condensation can be determined with Chromomycin A3. Considering the high degree of chromatin packaging in pig sperm, which only have protamine 1, growing evidence suggests that complete decondensation of that chromatin is needed before DNA fragmentation through TUNEL or Comet can be examined.