DBD-FISH Using Specific Chromosomal Region Probes for the Study of Cervical Carcinoma Progression.

Genomic instability is an important biomarker in the progression of cervical carcinoma. DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) is a sensitive method that detects strand breaks, alkali-labile sites, and incomplete DNA excision repair in cells of the cervical epithelium. This technique integrates the microgel immersion of cells from a vaginal lesion scraping and the DNA unwinding treatment with the capacity of FISH integrated into digital image analysis. Cells captured within an agarose matrix are lysed and submerged in an alkaline unwinding solution that generates single-stranded DNA motifs at the ends of internal DNA strand breaks. After neutralization, the microgel is dehydrated and the cells are incubated with DNA-labeled probes. The quantity of a hybridized probe at a target sequence corresponds to the measure of the single-stranded DNA produced during the unwinding step, which is equivalent to the degree of local DNA breakage. DNA damage does not show uniformly throughout the entire DNA of a cell; rather, it is confined to specific chromosomal sites. In this chapter, an overview of the technique is supplied, focusing on its ability for assessing the association between DNA damage in specific sequences and in the progressive stages of cervical carcinoma.

Role of DNase Activity in Human Sperm DNA Fragmentation.

In this clinical era of intracytoplasmic sperm injection (ICSI), where a single spermatozoon is chosen for fertilization, the diagnostic functionality of the classical parameters typically associated with fertilization, such as sperm concentration, sperm motility, acrosome integrity, and mitochondria, is perhaps becoming less critical. In contrast, the contribution of sperm DNA quality to our understanding of the impact of male fertility within the context of ICSI is gaining increasing interest and importance. Even with respect to natural conception, high levels of sperm DNA fragmentation (SDF) in the ejaculate can adversely affect reproductive outcomes. However, the precise origin of SDF pathology in sperm cells is often ambiguous and most likely to be multifactorial. Hence, the genetic makeup of an individual, unbalanced REDOX processes, enzymatic activity, environmental and lifestyle factors, and even damage during sperm handling in the laboratory all operate in a unique and often synergistic manner to produce or induce sperm DNA damage. Surprisingly, the contribution of active enzymes as potential agents of SDF has received much less attention and, therefore, is likely to be underrated. This review highlights the roles of different enzymes related to the degradation of sperm DNA as possible effectors of DNA molecules in spermatozoa.

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.

Addendum to: The relationship between sperm nuclear DNA fragmentation, mitochondrial DNA fragmentation and copy number in normal and abnormal human ejaculates.

BACKGROUND: While the kinetics of human sperm nuclear DNA fragmentation (SDF-nDNA) following ejaculation have been described, the dynamics and relationships of mitochondrial DNA copy number per spermatozoon (mtDNAcn) and fragmentation (SDF-mtDNA) remain unexplored. OBJECTIVES: To compare post-ejaculatory kinetics of mtDNAcn, SDF-mtDNA and SDF-nDNA, global, single-strand DNA breaks (SDF-SSBs) and double-strand DNA breaks (SDF-DSBs) in normozoospermic and non-normozoospermic samples. MATERIALS AND METHODS: 28 normozoospermic and 43 non-normozoospermic ejaculates were evaluated at 0, 6, 24 and 48 h of incubation in vitro. SDF-nDNA was determined by sperm chromatin dispersion (SCD) assays. mtDNAcn and SDF-mtDNA were analysed by dPCR. RESULTS: SDF-nDNA-global values increased as a consequence of quadratic SDF-SSBs and linear SDF-DSBs kinetics. Non-normozoospermic samples showed a slower SDF-global rate between 6-24 h, due to lesser SSBs production. Regarding SDF-DSBs, non-normozoospermic samples exhibited a faster initial increase rate, followed by a slower final increment. The mtDNAcn median value decreased linearly, being 3.2× higher in non-normozoospermics at all time points; mtDNAcn in both cohorts reduced to half of the baseline by 48 h. mtDNAcn was identified as a risk factor for discriminating non-normozoospermia, a finding that was further strengthen when combined with SDF-Global or SDF-DSBs values. SDF-mtDNA frequencies were identical, increasing over time correspondingly in both cohorts. The mtDNA fragmentation rate was initially fast, decreasing progressively with time for both cohorts; half of the initially unfragmented copies were fragmented after 48 h. Rates of mtDNAcn loss and SDF-mtDNA increase were only marginally correlated with the rates of nuclear fragmentation. CONCLUSION: mtDNA fragmentation and loss occur post ejaculation. Their dynamics are likely to be associated with different and/or uncoupled mechanisms to that which cause nuclear DNA fragmentation. Our results indicate that while mtDNA fragmentation is not influenced by the sperm quality, the number of copies of sperm mtDNAcn can potentially serve as a risk factor for predicting non-normozoospermia.

The relationship between sperm nuclear DNA fragmentation, mitochondrial DNA fragmentation, and copy number in normal and abnormal human ejaculates.

BACKGROUND: 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. OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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 25.00 vs. 13.67). mtDNA copy number per spermatozoon was 3× higher in non-normozoospermic ejaculates (median 16.06 vs. 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). CONCLUSION: 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.

Intra-individual variation of sperm DNA fragmentation in the Human ejaculate.

This retrospective study assessed the biological intra-individual variability of the percentage of sperm with DNA damage (SDF) observed in subsequent ejaculates of the same individual. Variation in SDF was analyzed using the Mean Signed Difference (MSD) statistic based on 131 individuals, comprising 333 ejaculates. Either two, three or four ejaculates were collected from each individual. With this cohort of individuals two main questions were addressed; (1) does the number of ejaculates analyzed influence the variability in the level of SDF associated with each individual? and (2) is the variability observed in SDF similar when individuals are ranked according to their level of SDF? Results showed that the variation observed in mean SDF was not different when 2, 3 or 4 ejaculates were analyzed; consequently, we suggest that the assessment of SDF based on two ejaculates is likely to be representative of the mean SDF expected for the individual. In parallel, it was determined that the variation in SDF increased as SDF increased; in individuals presenting with an SDF value of lower than 30% (potentially fertile), only 5% possessed levels of MSD that could be considered as variable as that presented by individuals presenting with a recurrent high SDF. Finally, we showed that a single assessment of SDF in individuals with medium SDF (20-30%) was less likely to be predictive of the SDF value in the next ejaculate, and therefore, less informative of the patient's SDF status.

Chromatin Dispersion Test to Asses DNA Damage in Cervical Epithelial Cells.

The chromatin dispersion test (CDT) is based on the removal of nuclear proteins under the assumption that cells with fragmented DNA produce a typical halo of circular DNA loops, which is absent in cells with non-fragmented DNA. This method represents a simple, rapid, accurate, highly reproducible, and inexpensive technique to assess nuclear DNA damage in somatic cells. The visualization of DNA damage and the capacity of the test to provide a threshold value to discriminate between high and low levels of cervical lesions would aid in determining the malignant transformation. All of these advantages associated with the CDT protocol could promote this technique as a tool for the quick and reliable diagnosis of cervical epithelial disorders, even at primary-care centers.

Bacterial DNase activity as a putative inductor of sperm DNA fragmentation in infected bull frozen-thawed semen samples.

The aim of this study was to investigate the relationship between DNase activity associated with bacterial contamination of incubated bovine frozen-thawed spermatozoa and elevated sperm DNA fragmentation. Electrophoresis analysis of plasmid PBR322 incubated for 30 min at 37 °C with the supernatant of the diluent of frozen-thawed centrifuged bovine semen straws infected with bacteria showed clear evidence of DNase activity when compared to plasmid incubated in similarly prepared non-infected bovine diluent supernatant (Experiment 1). This DNase activity was subsequently found to be time dependent (0-60 min) and its activity prevented in the presence of EDTA (10 and 20 mM; Experiment 2). Semen straws infected (n = 10) and not infected (n = 10) with bacteria where incubated at 37 °C for up to 48h post-thaw. Semen infected with bacteria showed an exponential increase in bacterial growth and a corresponding increase in sperm DNA fragmentation. Non-infected semen samples showed no change in the incidence of sperm DNA fragmentation over the same period of incubation (Experiment 3). Our experiments reinforce the idea that exogenous DNases present in the semen should be considered as one of the primary contributing causes of sperm DNA fragmentation post ejaculation. In the case of the bull, post-thaw incubation of commercial straws contaminated with bacteria, resulted in increased levels of sperm DNA fragmentation, most likely associated with DNase activity (potentially restriction endonucleases) derived from the bacteria. Such adverse changes in sperm DNA fragmentation, as described here in vitro, may be also operative after insemination in the female reproductive tract (in vivo) and highlight the importance of implementing high levels of hygiene practice during semen processing, especially in light of future trends of bacterial resistance to the common antibiotics used in semen diluents.

The efficacy of novel centrifugation-free sperm selection (Io-Lix) on sperm parameters and ICSI reproductive outcomes.

RESEARCH QUESTION: What is the effect of a novel non-centrifugation method (Io-Lix) of sperm selection on sperm parameters and intracytoplasmic sperm injection (ICSI) reproductive outcomes? DESIGN: This pilot study elevated the capacity of the Io-Lix sperm selection protocol to improve sperm parameters (concentration, motility and sperm DNA fragmentation) of the neat ejaculate. Once established, the reproductive outcomes of Io-Lix selected spermatozoa were used for autologous and donor oocyte ICSI programmes and their efficacy compared with those using conventional swim-up. RESULTS: Io-Lix sperm selection resulted in lower sperm concentration yield (P < 0.001) and sperm DNA fragmentation (P < 0.001) but higher sperm motility (P < 0.001) when compared with spermatozoa in the neat ejaculate. When compared with swim-up sperm selection the Io-Lix protocol resulted in a 14.7% (P = 0.028) increase in pregnancy rate and 16.3% (P = 0.047) reduction in miscarriages in the autologous ICSI programme. A similar comparison of sperm selection procedures employed for a donor oocyte ICSI programme showed no difference in terms of their respective reproductive outcomes. CONCLUSIONS: The Io-Lix sperm selection protocol resulted in improved pregnancy rate and reduction in miscarriage when applied to autologous ICSI, which was attributed to a reduction in the proportion of spermatozoa with DNA damage post-selection. A similar finding was not apparent in the donor oocyte programme, which may be associated with the capacity of the donor oocyte to repair sperm DNA post-syngamy.

DNA fragmentation of equine cumulus cells from Cumulus-Oocyte complexes submitted to vitrification and its relationship to the developmental competence of the oocyte.

The objectives of this study were to evaluate the effect of vitrification on the DNA fragmentation rate of equine cumulus cells and to assess its relationship to oocyte in vitro maturation (IVM) after vitrification. Cumulus cells (CC) from 14 mares were recovered from COCs, previously submitted to vitrification (VIT) and IVM. The DNA fragmentation rate of the cumulus cells (CC-DF) was assessed using a chromatin dispersion test. CC-DF rates between vitrified and control COCs were statistically compared by Student's t-test. The rates of CC-DF from control COCs were lower than in vitrified COCs. The percentage of CC-DF was not significantly different (p > .05) between groups of COCs able to reach metaphase II (MII > 0) and those in which oocyte maturation was not achieved (MII = 0). In conclusion, vitrification has a deleterious effect on the DNA fragmentation of equine cumulus cells; however, this parameter cannot be used as a predictor for IVM success after COCs vitrification.

Sperm DNA damage in men with spinal cord injury: the relative incidence of single- and double-strand DNA breaks.

BACKGROUND: Men with spinal cord injury (SCI) show a high proportion of sperm DNA damage in their ejaculate but the underlying pathology remains elusive. OBJECTIVE: To investigate the relative incidence of single (SSBs) and double-strand DNA breaks (DSBs) and DNase activity in men with SCI. MATERIALS AND METHODS: This study included ejaculates of 20 men with SCI and 27 normozoospermic (sperm donors). A TwoTails comet assay (TTComet) allowed visualization of three categories of sperm DNA damage corresponding to SSBs, DSBs and those with a combination of SSBs and DSBs, facilitating accurate calculation of the total proportion of SSBs and DSBs. A subset of 15 individuals (sperm donors and SCI patients) was used to test for DNase activity in the seminal plasma. RESULTS: While the proportion of DSBs in men with SCI (median-57.5%) was higher (P = 0.000) than normozoospermic samples (median-4.6%), the proportion of SSBs was higher (P = 0.022) in the normozoospermic ejaculates (median-6.0%) compared to men with SCI (median-2.5%). The relative proportion of the total DSBs with respect to the total SSBs was 3.3× in men with SCI but 0.9× in normozoospermic samples. We further confirmed the high DNase activity in the seminal plasma of men with SCI. DISCUSSION: The TTComet assay provided new insights to the pathology of sperm DNA in men with SCI and may have diagnostic value in developing sperm selection methodologies to reduce DSBs prior to ART. CONCLUSION: Men with SCI are characterized by a high proportion of sperm with DSBs and high levels of DNase activity in the seminal plasma compared to normozoospermic men.

Cumulus Cell DNA Damage as an Index of Human Oocyte Competence.

The determination of oocyte quality is crucial for achieving effective syngamy post-sperm injection and embryonic development. Cumulus cells (CCs) have been proposed as biomarkers of oocyte quality because of their close bio-dynamic relationship with the oocyte. To determine the quality of the oocyte, CCs were sampled during oocyte preparation for ICSI to determine a CC DNA fragmentation index (CCDFI) of each individual oocyte using a variant of the chromatin dispersion test. One hundred and thirty oocytes were selected and studied from two Spanish fertility clinics, 90 of which were fertilized and developed to embryos. Significant differences were found between the CCDFI of unfertilized and fertilized oocytes (p < .001) and between the CCDFI of embryos that were discarded and those that developed suitable for transfer or cryopreservation (p < .001). Oocyte quality was negatively correlated with CCDFI (Spearman's rho = - 0.45; p < .001). Receiver operator characteristics curves (ROC) suggested that a cut-off value of 24% CCDFI was able to discriminate the capacity of the gametes to result in syngamy with a sensitivity and specificity of 75.6% and 65%, respectively. This cut-off supports the application of CCDFI as potential index for the evaluation of the reproductive potential of oocytes prior to fertilization.

Investigation of pathology associated with Chlamydia pecorum infection in the male reproductive tract, and the effect on spermatogenesis and semen quality in the koala (Phascolarctos cinereus).

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morphology, suggestive of chronic exposure to C. pecorum infection and/or inflammation in the testis and epididymis, with residual pathology and lesions disrupting spermatogenesis and maturation of spermatozoa. This study specifically aimed to determine whether pathology associated with chlamydial infection in different regions of the male koala reproductive tract had an adverse effect on classical seminal parameters, sperm DNA quality and endocrine function (testosterone secretion) of naturally infected males. Semen from 58 sexually mature male koalas deemed not suitable for rehabilitation or treatment was assessed, in addition to undertaking a GnRH challenge to determine the androgenic capacity of the testis. Following euthanasia, tissue samples from testes, epididymis and prostate were evaluated for histopathology and real time polymerase chain reaction (qPCR). A significant difference in sperm concentration was observed between males with unilateral and bilateral testicular atrophy and C. pecorum infection (P = 0.011); and between males with unilateral atrophy and C. pecorum infection in one testis and bilateral normal testes with no C. pecorum infection (P = 0.008). No significant association was found for any other semen parameters when categorised by histopathology and C. pecorum tissue presence within the testes, epididymis and prostate. Plasma testosterone concentrations did not significantly differ between testicular histopathology diagnosis and/or C. pecorum infection status. This study suggests Chlamydia infection and inflammation may not be the predominant reason of disruption to spermatogenesis in the wild koala but rather testicular degeneration and atrophy, irrespective of Chlamydia infection, appears to be the primary reason of decreased sperm concentration.

Antibiotic toxicity on human spermatozoa assessed using the sperm DNA fragmentation dynamic assay.

Sperm DNA fragmentation (SDF) dynamic assays were piloted on 4 fresh ejaculates to examine the possible sperm toxicity of three common antibiotics, ciprofloxacin, doxycycline and ampicillin, incubated at a concentration estimated to be reached in semen in vivo, and 100×, for 24 h. SDF was assessed in terms of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs). Low and high concentrations of ciprofloxacin and high concentration of doxycycline significantly increased the SDF rate, due to sperm containing SSBs. Ampicillin did not affect SDF dynamics at any dose. Based on these results, the effect of antibiotics on the global-SDF dynamics was further examined in 21 ejaculates assessed at 0, 4 and 6 h. Ciprofloxacin increased the rate of SDF at the low concentration in 17 from 21 subjects; the high concentration resulted in a stronger effect in all individuals. A significant increase in the rate of SDF in 17 ejaculates was also noted when spermatozoa were incubated with the high concentration of doxycycline. The dynamic SDF assay is a rapid and sensitive tool to evidence sperm toxicity. Ciprofloxacin should be avoided when it is necessary to preserve sperm quality for reproductive purposes and as additive in semen diluents.

Sperm DNA Fragmentation: A Critical Assessment of Clinical Practice Guidelines.

Sperm DNA fragmentation (SDF) is implicated in male infertility and adverse reproductive outcomes. With the publication of many studies regarding the etiologies and contributors to SDF, as well as the effects of SDF, guidelines are necessary to aid clinicians in the application of SDF for male fertility evaluation. Two recent clinical practice guidelines were published by Agarwal et al and Esteves et al. In this article, we have evaluated and compared both guidelines. We have found fairly similar recommendations between the two guidelines and have also highlighted the differences between them. Finally, we have summarized and combined the best practice recommendations from both guidelines.

A Comprehensive Guide to Sperm Recovery in Infertile Men with Retrograde Ejaculation.

Retrograde ejaculation (RE) is a condition defined as the backward flow of the semen during ejaculation, and when present can result in male infertility. RE may be partial or complete, resulting in either low seminal volume or complete absence of the ejaculate (dry ejaculate). RE can result from anatomic, neurological or pharmacological conditions. The treatment approaches outlined are determined by the cause. Alkalinizing urinary pH with oral medications or by adding sperm wash media into the bladder prior to ejaculation may preserve the viability of the sperm. This article provides a step-by-step guide to diagnose RE and the optimal techniques to retrieve sperm.

Relevance of Leukocytospermia and Semen Culture and Its True Place in Diagnosing and Treating Male Infertility.

The current WHO 2010 manual for human semen analysis defines leukocytospermia as the presence of peroxidase-positive leukocytes at a concentration >1×106/mL of semen. Granular leukocytes when activated are capable of generating high levels of reactive oxygen species in semen resulting in oxidative stress. Oxidative stress has been correlated with poor sperm quality, increased level of sperm DNA fragmentation and low fertility potential. The presence of leukocytes and pathogens in the semen may be a sign of infection and/or localized inflammatory response in the male genital tract and the accessory glands. Common uro-pathogens including Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, Mycoplasma hominis, and Escherichia coli can cause epididymitis, epididymo-orchitis, or prostatitis. The relationship between leukocytospermia and infection is unclear. Therefore, we describe the pathogens responsible for male genital tract infections and their association with leukocytospermia. The review also examines the diagnostic tests available to identify seminal leukocytes. The role of leukocytospermia in male infertility and its management is also discussed.

Sperm Morphology Assessment in the Era of Intracytoplasmic Sperm Injection: Reliable Results Require Focus on Standardization, Quality Control, and Training.

Semen analysis is the first, and frequently, the only step in the evaluation of male fertility. Although the laboratory procedures are conducted according to the World Health Organization (WHO) guidelines, semen analysis and especially sperm morphology assessment is very difficult to standardize and obtain reproducible results. This is mainly due to the highly subjective nature of their evaluation. ICSI is the choice of treatment when sperm morphology is severely abnormal (teratozoospermic). Hence, the standardization of laboratory protocols for sperm morphology evaluation represents a fundamental step to ensure reliable, accurate and consistent laboratory results that avoid misdiagnoses and inadequate treatment of the infertile patient. This article aims to promote standardized laboratory procedures for an accurate evaluation of sperm morphology, including the establishment of quality control and quality assurance policies. Additionally, the clinical importance of sperm morphology results in assisted reproductive outcomes is discussed, along with the clinical management of teratozoospermic patients.

Determining the effects of sperm activation in anuran cloaca on motility and DNA integrity in Epidalea calamita (Bufonidae).

The effect of time inside the animal's cloaca on sperm quality after hormone-induced spermiation is unknown. However, this knowledge is critical for the development of assisted reproductive biotechnologies in amphibians. Out-of-season spermatozoa were collected from Epidalea calamita for 4h after injection of 10IU g-1 human chorionic gonadotrophin either hourly (Group I (n =10); four samples per male) or every 2h (Group II (n =9); two samples per male). Sperm samples were assessed for motility and DNA integrity using the sperm chromatin dispersion (SCD) test and the sperm chromatin structure assay (SCSA). The collection strategy affected total motility (mean (±s.e.m.) 84.4±9.9% vs 73.6±16.7% in Group I and II respectively; P =0.014) and the sperm motility index (67.6±17.7% vs 57.6±16.3% in Group I and II respectively; P =0.034). There was a significant effect of the male in Group II, but not in Group I. In Group I, the quality of the first samples collected was lower than that of samples collected thereafter (P ≤ 0.032). No significant correlations were found between the results of the SCD test and SCSA, showing that these techniques provide different information in this species. In conclusion, collecting spermatozoa every hour resulted in better sperm quality and may be more efficient. However, the between-male differences were considerable and collection of spermatozoa at just 1h after hormone treatment produced lower-quality spermatozoa.

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

OBJECTIVE: To investigate the intraindividual agreement of the sperm chromatin dispersion (SCD) assay results to assess sperm DNA fragmentation (SDF) in men with infertility. DESIGN: Diagnostic test reliability study. SETTING: Andrology laboratories. PATIENT(S): A total of 219 men with infertility. INTERVENTION(S): Sperm DNA fragmentation assessment in two ejaculates of the same subjects within a 3-month interval, using the SCD assay performed and analyzed by the same observers under similar testing conditions. MAIN OUTCOME MEASURE(S): Cohen's κ statistics to assess the degree of agreement between the pairs of results after converting the nominal SCD values into categorical data, that is, normal (<20%), intermediate (21%-29%), and high (≥30%) SDF rates. We also assessed the pairs of results using reliability measures for numerical variables (intraclass correlation coefficient for consistency using the two-way mixed-effects model and Bland-Altman plots). RESULT(S): The degree of agreement in classifying patients according to normal and pathological SDF classes was overall substantial (κ = 0.632; 95% confidence interval [CI], 0.546-0.718). A total of 76.7% of individuals were classified under the same class using paired ejaculates. The agreement rate was highest (approximately 80%) in ejaculates initially classified as either normal or high and lowest (approximately 60%) among those with intermediate SDF levels. The frequency of intermediate SDF ejaculates downgraded to normal or upgrade to high SDF classes in the second test was similar (approximately 20%). The intraclass correlation coefficient was 0.856 (95% CI, 0.812-0.887), and the mean difference between the pairs of observations was 0.80% (95% CI, -0.72 to 2.23), indicating no systematic difference between paired observations. CONCLUSION(S): Our study indicates a substantial intraindividual agreement of paired SCD assay results to classify men with infertility into three SDF categories: normal, intermediate, and high. The reliability of the SCD assay was adequate and exceeded 0.80 using two ejaculates analyzed within a 3-month interval under similar conditions. Although this evidence overall supports a single SCD test for patient classification using predefined SDF thresholds, particularly when the first test shows normal or high SDF levels, one in four men will have discordant values in paired ejaculates. Clinicians should be judicious when using SDF test results in decision-making.