Sperm Nuclear DNA Fragmentation Research Articles

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

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. 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. 28 normozoospermic and 43 non-normozoospermic ejaculates were evaluated at 0, 6, 24 and 48h of incubation in vitro. SDF-nDNA was determined by sperm chromatin dispersion (SCD) assays. mtDNAcn and SDF-mtDNA were analysed by dPCR. 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 6-24h, 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 48h. 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 48h. Rates of mtDNAcn loss and SDF-mtDNA increase were only marginally correlated with the rates of nuclear fragmentation. 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.

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.

Nuclear DNA Fragmentation in Boar Spermatozoa: Measurement Methods and Reproductive Performance Implications

The aim of this research was to compare the different techniques to measure sperm nuclear DNA fragmentation (sDF) and to check its relations to boar reproductive value, classical spermiogram parameters, and reproductive results of the doses in sows. Sperm chromatin stability assay (SCSA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and sperm chromatin dispersion test (SCD, Halomax®) results were compared, finding a statistically significant correlation only between SCSA and TUNEL results. The fertility direct boar effect (DBE) index, calculated from the whole productive life of the boar, was not correlated (p > 0.05) with sDF (measured by any technique). Total or progressive sperm motility was not correlated with sDF, while it found a positive correlation between TUNEL measure and abnormal acrosomes (%) and between SCD measure and total sperm morphological abnormalities (%). No significant correlations were obtained between fertility or prolificacy results and sDF results with the different techniques. However, in the case of total born and SCSA measure, the correlation was close to significance (r partial = −0.095; p = 0.066), appointing to a tendency; as SCSA increases, the number of total piglets born decreases. In conclusion, although the different techniques for the sDF seem not to target exactly the same DNA events and the relationship between their values and the reproductive results and the classical spermiogram results is still to be elucidated, the studied sDF techniques may offer extra information that could be useful for the management of AI studs.

Can leukocytospermia predict prostate cancer via its effects on mitochondrial DNA?

Leukocytospermia was previously reported to affect sperm quality by the production of reactive oxygen species (ROS) leading to oxidative stress (OS). In turn, OS decreases sperm functional integrity, increases sperm DNA damage and ultimately alters fertility status. To elucidate the impact of leukocytospermia on sperm nuclear DNA integrity and mitochondrial DNA (mtDNA) structure, we conducted a study including 67 samples from infertile patients with low level of leucocytes (Group 1: n=20) and with leukocytospermia (Group 2: n=47). In addition to standard sperm parameters' assessment, we measured the levels of inflammation biomarkers [interleukin-6 (IL-6) and interleukin-8 (IL-8)] and evaluated the oxidative status [malondialdehyde (MDA) and enzymatic and non-enzymatic antioxidants]. In addition, we evaluated the level of sperm nuclear DNA fragmentation and analysed mitochondrial DNA (mtDNA) of sperm cells by sequencing of 5 genes [cytochrome oxidase I (COXI), cytochrome oxidase II (COXII), cytochrome oxidase III (COXIII), adenosine triphosphate synthase 6 (ATPase 6) and adenosine triphosphate synthase 8 (ATPase 8)]. As expected, patients with leukocytospermia had significantly higher MDA levels (32.56±24.30 nmole/ml) than patients without leukocytospermia (17.59±9.60nmole/ml) (p<.018). Also, sperm DNA fragmentation index (DFI) was significantly higher in Group 2 (33.05±18.14%) as compared to Group 1 (14.19±9.50%) (p<.001). The sequencing of mtDNA revealed a high number of substitutions in Group 2 (n=102) compared to Group 1 (n=5). These substitutions were observed mainly in COXI. Among COXI substitutions found in Group 2, twelve changes were previously described in patients with prostate cancer and six of them were shown associated with this pathology. These findings suggest that leukocytospermia may predispose to the manifestation of prostate cancer through modification of mitochondrial DNA and this may be promoted by OS.

Seasonal Changes of Nuclear DNA Fragmentation in Boar Spermatozoa in Spain.

Simple SummaryArtificial insemination is widely used in pig production and currently a boar performs several thousand matings per year. Traditionally sperm quality is focused on the number of spermatozoa, their motility and morphology. However, the quality of sperm DNA, which contains genetic information, is also related to fertility problems. The aim of this research was to study the effect of natural light hours and age of the boar on the status of the sperm DNA. After a powerful statistical analysis, it was found that the percentage of spermatozoa with fragmented DNA decreases within the observed age range as the boar gets older. On the other hand, the amount of spermatozoa with fragmented DNA was the lowest in autumn while it was the highest in summer. This study demonstrates the remaining seasonality of boars in Spain and highlights the importance of controlling the environmental conditions in the farms. Sperm DNA testing provides a basis for improving the selection of AI boars by excluding males with higher DNA fragmentation due to their very young reproductive age that may pose a potential subfertility.There are numerous cases when conventional spermiogram parameters are all within an acceptable range but boar subfertility persists. The total sperm nuclear DNA fragmentation index (tDFI) is a trait related to fertility and prolificacy problems that is not routinely evaluated in commercial AI boars. The aim of this research was to study the effect of the photoperiod, season and reproductive age of the boar on tDFI (measured by SCSA) of 1279 ejaculates from 372 different boars belonging to 6 different breeds located in 6 AI studs in Spain. tDFI data ranged from 0.018% to 20.1%. Although there was a significant single boar effect in the tDFI occurrence, a negative correlation between the tDFI and the age of the boar was found (p < 0.001). tDFI would decrease due to aging of the boar 0.66% each year old within the observed age range. After including age as a covariate in the ANCOVA, no differences were found in tDFI between photoperiods when the sperm collection date was evaluated. However, when the date of the production of semen in the testis was evaluated, the total percentage of spermatozoa with fragmented nuclear DNA was 1.46% higher in the increasing photoperiod in comparison to the decreasing photoperiod (p < 0.0001). On the other hand, for both dates, the lowest tDFI values corresponded to minimum day length for decreasing photoperiod phase (autumn), while the highest tDFI values were found in summer (maximum day length for decreasing photoperiod phase).

Correlation between mitochondrial dysfunction of spermatozoa and their biological adequacy

One of the most significant indicators affecting male fertility is the sperm nuclear and mitochondrial DNA fragmentation index (DFI). DNA damage depends on biotic and abiotic factors, leading to oxidative stress (O.S.). This research aimed to investigate the relationship between mitochondrial dysfunction of spermatozoa and their biological adequacy. The research material was frozen-thawed sperm samples from the Ayrshire, Russian Black Pied Holstein, Russian Red Pied Holstein, Limousin, and Polled Russian breeding bulls. Assessments of mobility, morphology, and fragmentation index were performed using computer-assisted sperm analysis (CASA). It was found that there is a negative correlation between sperm activity and mitochondrial dysfunction with the correlation coefficient r = -0.24. The incidence of abnormal spermatozoa correlated with sperm dysfunction r = 0.77. The nDNA fragmentation index in chromatin varied from 0 to 25%.

Relationship between sperm morphology and sperm DNA dispersion.

BackgroundThe pathogenesis of teratozoospermia (<4% morphologically normal sperm cells) and the relationship between sperm morphological abnormalities and abnormal sperm nuclear DNA fragmentation, which are considered indicators of male fertility, have not been elucidated. Our research was designed to determine the prevalence of different sperm DNA fragmentation (SDF) levels in men with teratozoospermia and to establish a discriminating threshold value for SDF in assessing sperm morphology.MethodsBasic semen characteristics and detailed sperm morphological analysis (head, neck, midpiece, and tail defects and excess residual cytoplasm) (WHO, 2010), and the nuclear sperm DNA dispersion test were performed on semen samples obtained from 523 men with teratozoospermia (n=296) and those without teratozoospermia (n=227).ResultsSubjects with abnormal sperm morphology had not only lower results for standard sperm characteristics, including detailed sperm morphological abnormalities, but also a higher proportion of sperm cells with SDF vs. men with normal sperm morphology. Moreover, significantly fewer subjects with low SDF levels (≤15%), more subjects with high SDF levels (>30%) and a higher odds ratio (OR) for having high SDF levels were found in the group of men with teratozoospermia vs. men without teratozoospermia. However, the receiving operating characteristic (ROC) curve analysis indicated that a SDF >18% was a significant negative predictive value to distinguish between men with normal sperm morphology or men with abnormal sperm morphology. The optimal area under the ROC curve (AUC) was 0.746. In the group of men with teratozoospermia, a higher incidence of men with >18% SDF and a higher OR for having >18% SDF were observed. SDF negatively correlated with sperm number, morphologically normal sperm cells, sperm motility and sperm vitality but positively correlated with the teratozoospermia index (TZI) and detailed sperm morphological abnormalities.ConclusionsThe obtained findings demonstrated that: (I) detailed sperm structural defects coexist with abnormal nuclear sperm DNA dispersion, (II) men with teratozoospermia may have a higher risk for sperm DNA damage, (III) the calculated optimal SDF value of 18% measured by the DNA sperm dispersion test is the best criterion to predict normal and abnormal sperm morphology.

Down-regulation of steroidogenesis-related genes and its accompanying fertility decline in streptozotocin-induced diabetic male rats: ameliorative effect of metformin.

Metformin has long been used for glycemic control in diabetic state. Recently, other benefits of metformin beyond blood glucose regulation have emerged. To investigate the effect of metformin on the expression of testicular steroidogenesis-related genes, spermatogenesis, and fertility of male diabetic rats. Eighteen adult male Sprague Dawley rats were divided into three groups, namely normal control (NC), diabetic control (DC), and metformin-treated (300mg/kg body weight/day) diabetic rats (D+Met). Diabetes was induced using a single intraperitoneal injection of streptozotocin (60mg/kg b.w.), followed by oral treatment with metformin for four weeks. Diabetes decreased serum and intratesticular testosterone levels and increased serum but not intratesticular levels of luteinizing hormone. Sperm count, motility, viability, and normal morphology were decreased, while sperm nuclear DNA fragmentation was increased in DC group, relative to NC group. Testicular mRNA levels of androgen receptor, luteinizing hormone receptor, cytochrome P450 enzyme (CYP11A1), steroidogenic acute regulatory (StAR) protein, 3β-hydroxysteroid dehydrogenase (HSD), and 17β-HSD, as well as the level of StAR protein and activities of CYP11A1, 3β-HSD, and 17β-HSD, were decreased in DC group. Similarly, decreased activities of epididymal antioxidant enzymes and increased lipid peroxidation were observed in DC group. Consequently, decreased litter size, fetal weight, mating and fertility indices, and increased pre- and post-implantation losses were recorded in DC group. Following intervention with metformin, we observed increases in serum and intratesticular testosterone levels, Leydig cell count, improved sperm parameters, and decreased sperm nuclear DNA fragmentation. Furthermore, mRNA levels and activities of steroidogenesis-related enzymes were increased, with improved fertility outcome. Diabetes mellitus is associated with dysregulation of steroidogenesis, abnormal spermatogenesis, and fertility decline. Controlling hyperglycemia is therefore crucial in preserving male reproductive function. Metformin not only regulates blood glucose level, but also preserves male fertility in diabetic state.

Clinical utility of sperm DNA fragmentation testing: a commentary.

The andrological factor is implicated in more than 40% of couples attending infertility clinics. Considering that standard semen analysis only poorly predicts the outcome of fertilization and pregnancy in couples including assisted reproduction attempts as it only examines sperm parameters such as sperm concentration, vitality, sperm morphology and motility, it is therefore quite limited in its clinical discriminatory power (1) in about 40% of men with normal ejaculates (2). Contributing factors to this fact are the high biological variability of these parameters, essentially causing each ejaculate being unique, thus often resulting in the vague clinical diagnosis of idiopathic infertility (3). In addition, the fertilization process itself is a multifactorial process in which not only the different sperm functions such as motility, capacitation, acrosome reaction, chromatin condensation or the integrity of the genetic information, but also female and oocyte factors have to be taken into account (4). Among these conventional sperm parameters, morphology evaluated according to strict criteria exhibited a relatively low degree of variability (4). Yet, although morphology appeared as a better parameter than sperm concentration, the diagnostic value of semen analysis is limited (5). For this reason, conventional semen analysis was complemented by a panel of functional sperm parameters including sperm nuclear DNA fragmentation, which is also regarded a parameter with low biological variation (6).

Incidence of sperm nuclear DNA fragmentation in artificial insemination boars measured using the sperm chromatin dispersion test

Incidence of sperm nuclear DNA fragmentation in artificial insemination boars measured using the sperm chromatin dispersion test

The impact of sedentary work on sperm nuclear DNA integrity

Contemporary professional jobs that often enforce a sedentary lifestyle and are frequently associated with testicular overheat, deserve special attention with respect to male fertility potential. Interestingly, the harmful effect of testicular heat stress on sperm characteristics including nuclear DNA integrity was well characterized; however, the influence of sedentary work on sperm chromatin has not yet been documented. Therefore, our research was designed to examine the potential effects of sedentary work not only on conventional semen features but also on sperm nuclear DNA status. The study was carried out on ejaculated sperm cells obtained from men who spent ≥ 50% of their time at work (≥ 17.5 h per week) in a sedentary position (n = 152) and from men who spent < 50% of their time at work in a sedentary position (n = 102). Standard semen characteristics were assessed according to the WHO 2010 recommendations, while sperm nuclear DNA fragmentation (SDF) was evaluated using the Halosperm test. There were no significant differences in the standard semen parameters between the study groups. The groups differed only in SDF parameter. The men who spent at least 50% of their work time in a sedentary position had a higher proportion of SDF than the men who spent < 50% of their time at work in a sedentary position (median value 21.00% vs. 16.50%, respectively). The incidence of low SDF levels (related to 0-15% sperm cells with abnormal DNA dispersion) was significantly lower (27.63% vs. 45.10%), the percentage of men with high SDF levels (related to > 30%) was significantly higher (30.92% vs. 16.67%) in group of men who spent at least 50% of their work time in a sedentary positon. Furthermore, these men were more than twice as likely to have not a low SDF level (OR: 0.4648) and had more than twice the risk of having a high SDF level (OR: 2.2381) than the men in less sedentary occupations. Despite lack of association between sedentary work and conventional semen characteristics our study revealed detrimental effect of seated work on sperm nuclear DNA integrity. A sedentary job doubled the risk of high levels of sperm DNA damage. The pathomechanism could be related to testicular heat stress resulting in sperm chromatin remodelling failure during spermiogenesis. Therefore, it seems reasonable to simultaneously carry out routine seminological analyses and tests assessing sperm chromatin status while diagnosing male infertility.

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

Due to the limitations of conventional semen analysis in predicting a man's fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. This prospective study was undertaken to investigate the associations between conventional seminal parameters and DNA fragmentation in Greek men. A total of 669 subject data were evaluated in two groups, normozoospermic (n = 184) and non-normozoospermic (n = 485), according to the WHO 2010 (WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization), reference limits. For all the subjects, semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were recorded following the WHO 2010 methods and DNA fragmentation was assessed by the sperm chromatin dispersion assay. An inverse correlation was established between DNA fragmentation and all conventional seminal parameters except semen volume in men with seminal profiles below the reference limits, with statistical significance for rapid and total progressive motility. Normozoospermic men exhibited lower levels of DNA fragmentation than their non-normozoospermic counterparts, even though the values were not always below 30%. DNA fragmentation testing and traditional semen analysis should therefore be considered as complementary diagnostic tools in a comprehensive evaluation of male infertility.

Maldi-tof fingerprinting of seminal plasma lipids in the study of human male infertility.

This study proposed lipid fingerprinting of human seminal plasma by mass spectrometry as an analytical method to differentiate biological conditions. For this purpose, we chose infertile men as a model to study specific conditions, namely: high and low seminal plasma lipid peroxidation levels (sub-study 1.1), high and low sperm nuclear DNA fragmentation (sub-study 1.2), and intervention status: before and after subinguinal microsurgical varicocelectomy (study 2). Study 1 included 133 patients, of which 113 were utilized for sub-study 1.1 and 89 for sub-study 1.2. Study 2 included 17 adult men submitted to subinguinal varicocelectomy, before and 90 days after varicocelectomy. Lipids were extracted from seminal plasma and submitted to Matrix-Assisted Laser Desorption Ionization Quadrupole-Time-of-Flight Mass Spectrometry in the positive ionization mode. Spectra were processed using Waters(®) MassLynx, and MetaboAnalyst online software was used for statistical analyses. For sub-studies 1.1 and 1.2, and study 2, univariate analysis revealed 8, 87 and 34 significant ions, respectively. Multivariate analysis was performed through PCA and PLS-DA. PCA generated 56, 32 and 34 components respectively for each study and these were submitted to logistic regression. A ROC curve was plotted and the area under the curve was equal to 97.4, 92.5 and 96.5%. PLS-DA generated a list of 19, 24 and 23 VIP ions for sub-studies 1.1 and 1.2, and study 2, respectively. Therefore, this study established the lipid profile and comparison of patterns altered in response to specific biological conditions.

Protective effect of alpha glucosyl hesperidin (G-hesperidin) on chronic vanadium induced testicular toxicity and sperm nuclear DNA damage in male Sprague Dawley rats.

The study was conducted to evaluate the vanadium-induced testicular toxicity and its effect on sperm parameters, sperm nuclear DNA damage and histological alterations in Sprague Dawley rats and to assess the protective effect of G-hesperidin against this damage. Treatment of rats with vanadium at a dose of 1mgkgbw(-1) for 90days resulted in significant reduction in serum testosterone levels, sperm count and motility. Further, a parallel increase in abnormal sperm morphology and adverse histopathological changes in testis was also associated with vanadium administration when compared to normal control. Moreover, sperm chromatin dispersion assay revealed that vanadium induces sperm nuclear DNA fragmentation. A marked increase in testicular malondialdehyde levels and decreased activity of antioxidant enzymes such as superoxide dismutase and catalase indicates vanadium-induced oxidative stress. Co-administration of G-hesperidin at a dose of 25 and 50mgkgbw(-1) significantly attenuated the sperm parameters and histological changes by restoring the antioxidant levels in rat testis. These results suggested that vanadium exposure caused reduced bioavailability of androgens to the tissue and increased free radical formation, thereby causing structural and functional changes in spermatozoa. G-hesperidin exhibited antioxidant effect by protecting the rat testis against vanadium-induced oxidative damage, further ensures antioxidant potential of bioflavonoids.

Seminal plasma lipid fingerprinting and its association to semen oxidative stress and sperm nuclear dna fragmentation

Seminal plasma lipid fingerprinting and its association to semen oxidative stress and sperm nuclear dna fragmentation

Sperm nuclear vacuoles and DNA fragmentation. Is to talk about of the same issue?

The aim of this study was to determine the relationship between the size-number of nuclear vacuoles and DNA damage in spermatozoa from patients with different sperm morphology index. Prospective-comparative study. Forty four men who undertwent ART (2011-2012) were selectec for this study. Accordingly to the WHO manual for semen examination (1999), the inclusion criteria were: sperm concentration ≥20 million/mL, progressive motility ≥30% and strict sperm morphology (Kruger) ≥14% (group 1), 5-13 (group 2) and ≤4% (group 3). After gradient, motile sperm were placed in PVP and selected considering by the number and size of nuclear vacuoles under high magnification (7800X). It was considered the following patterns: A) No vacuoles, B)1 vacuole, size=less than 25% of the nuclear surface, C)2 vacuoles less than 25%, D)1 vacuole greater than 25-50%, E)several vacuoles less than 25% and F)several vacuoles greater than 25%. Selected spermatozoa (by ICSI pipette) were assessed for DNA fragmentation (TUNEL) by immunofluorescence. Statistical analysis was performed by ANOVA. The mean age was: 35±4.5(group 1), 36±3.5 (group 2) and 36±3.0 (group 3). Progressive sperm motility was 59±22 (1), 55±18 (2), 60±18 (3). The average of Kruger was: 16.2±2 (1), 7.5±2.5 (2) and 1.2±1.4 (3). A total of 818 spermatozoa were selected and evaluated for TUNEL. The comparison between sperm populations are shown in table 1.Tabled 1GroupsABCDEFGroup 13.6±2.46.1±4.26.7±3.912.1±3.6ap<0.05.7.3±3.710.8±4.9ap<0.05.Group 25.8±2.26.2±2.97.5±3.113.2±3.4ap<0.05.9.6±2.212.8±3.6ap<0.05.Group 316.8±5.818.2±5.717.4±5.921.4±3.620.4±4.620.6±5.9a p<0.05. Open table in a new tab Sperm DNA fragmentation levels signifantly increases just in spermatozoa with large (D) or several nuclear vacuoles (F) in patients with Kruger between 5-13% or ≥14%, but not in patients with Kruger index ≤4%. This data may be relevant during patients and sperm selection for IMSI. Finally, sperm nuclear vacuoles are not always related with DNA fragmentation.

Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation

Sperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation. Sperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MS(E)). Differentially expressed proteins were used for a functional enrichment study. Two hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells. Sperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.

Sperm FISH analysis of a 44,X,der(Y),t(Y;15)(q12;q10)pat,rob(13;14)(q10;q10)mat complex chromosome rearrangement

Complex chromosome rearrangements (CCR) with two independent chromosome rearrangements are rare. Although CCRs lead to high unbalanced gamete rates, data on meiotic segregation in this context are scarce. A male patient was referred to our clinic as part of a family screening programme prompted by the observation of a 44,X,der(Y),t(Y;15)(q12;q10)pat,rob(13;14)(q10;q10)mat karyotype in his brother. Karyotyping identified the same CCR. Sperm FISH (with locus-specific probes for the segments involved in the translocations and nine chromosomes not involved in both rearrangements) was used to investigate the rearrangements meiotic segregation products and establish whether or not an inter-chromosomal effect was present. Sperm nuclear DNA fragmentation was also evaluated. For rob(13;14) and der(Y), the proportions of unbalanced products were, respectively, 26.4% and 60.6%. Overall, 70.3% of the meiotic segregation products were unbalanced. No evidence of an inter-chromosomal effect was found, and the sperm nuclear DNA fragmentation rate was similar to our laboratory's normal cut-off value. In view of previously published sperm FISH analyses of Robertsonian translocations (and even though the mechanism is still unknown), we hypothesise that cosegregation of der(Y) and rob(13;14) could modify rob(13;14) meiotic segregation.

The oviducal protein, heat-shock 70-kDa protein 8, improves the long-term survival of ram spermatozoa during storage at 17°C in a commercial extender

Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical artificial insemination (AI). Heat-shock 70-kDa protein 8 (HSPA8), found within the oviduct, prolongs boar, ram and bull sperm survival at body temperatures in vitro. Here, we aimed to determine whether supplementing extenders (INRA-96 and RSD-1) with HSPA8 (4 µg mL⁻¹) would improve their performance in maintaining freshly collected ram sperm viability and sperm nuclear DNA integrity during storage over 48 h at 17°C. Sperm function was assessed at 1, 6, 24 and 48h and this experiment was repeated using 25 × 10⁶ and 800 × 10⁶ spermatozoa mL⁻¹. INRA96 supplemented with HSPA8 maintained sperm viability significantly better than INRA96 alone at both sperm concentrations. However, sperm nuclear DNA fragmentation (DF) increased significantly during storage using the higher sperm concentration, irrespective of the extender and the protein treatment used. Increasing levels of sperm nuclear DF over time could explain why poor fertility rates are often observed following cervical AI using stored ram semen. However, further research is required to ascertain whether supplementing the commercially available INRA96 extender with HSPA8 will improve fertility rates following cervical AI using stored ram semen.

Increased receptor for advanced glycation end products in spermatozoa of diabetic men and its association with sperm nuclear DNA fragmentation

Although the majority of patients with diabetes have disorders in sexual function, associations between diabetes mellitus and sperm function at the molecular level are largely unknown. As receptor for advanced glycation end products plays a key role in many diabetic complications, we hypothesised that it may be involved in sperm nuclear DNA fragmentation. RAGE levels were determined using ELISA and western blot analysis in sperm samples from 32 diabetic and 35 nondiabetic men. Sperm DNA fragmentation was assessed using TUNEL assay. Diabetic men had significantly higher mean levels of RAGE protein (P < 0.001) and DNA fragmentation (P < 0.001) in spermatozoa. Sperm RAGE was directly correlated to sperm DNA fragmentation in diabetic men (r = 0.81, P < 0.001). The high positive correlation between RAGE levels and nuclear DNA fragmentation in spermatozoa of diabetic men suggests a central role of RAGE in disturbances in sexual function of diabetic men.