Sperm Nuclear DNA Research Articles

The Sperm Chromatin Structure Assay (SCSA®) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility

Thirty-five years ago the pioneering paper in Science (240:1131) on the relationship between sperm DNA integrity and pregnancy outcome was featured as the cover issue showing a fluorescence photomicrograph of red and green stained sperm. The flow cytometry data showed a very significant difference in sperm DNA integrity between fertile and subfertile bulls and men. This study utilized heat (100°C, 5min) to denature DNA at sites of DNA strand breaks followed by staining with acridine orange (AO) and measurements of 5000 individual sperm of green double strand (ds) DNA and red single strand (ss) DNA fluorescence. Later, the heat protocol was changed to a low pH protocol to denature the DNA at sites of strand breaks; the heat and acid procedures produced the same results.SCSA data are very advantageously dual parameter with 1024 channels (degrees) of both red and green fluorescence. Hundreds of publications on the use of the SCSA test in animals and humans have validated the SCSA as a highly useful test for determining male breeding soundness. The SCSA test is a rapid, non-biased flow cytometer machine measurement providing robust statistical data with exceptional precision and repeatability. Many genotoxic experiments showed excellent dose response data with very low coefficient of variation that further validated the SCSA as being a highly powerful assay for sperm DNA integrity.Twelve years following the introduction of the SCSA test, the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) test (1993) for sperm was introduced as the only other flow cytometric assay for sperm DNA fragmentation. However, the TUNEL test can also be done by light microscopy with much less statistical robustness.The COMET (1998) and Sperm Chromatin Dispersion (SCD; HALO) (2003) tests were introduced as light microscope tests that don’t require a flow cytometer. Since these tests measure only 50–200 sperm per sample, they suffer from the lack of the statistical robustness of flow cytometric measurements. Only the SCSA test has an exact standardization of a fixed protocol. The many variations of the other tests make it very difficult to compare data and thresholds for risk of male factor infertility. Data from these four sperm DNA fragmentation tests plus the light microscope acridine orange test (AOT) are correlated to various degrees.

Summary evidence on the effects of varicocele treatment to improve natural fertility in subfertile men.

The objective of this review was to summarize the evidence concerning the benefit of varicocele treatment to improve natural fertility in subfertile males. We also analyzed the effect of varicocele treatment on conventional semen parameters and sperm functional tests. An electronic search to collect the data was performed using the PubMed/MEDLINE databases until July 2015. Data pooled from a variety of study designs indicate that varicocelectomy improves semen parameters in the majority of the treated men with clinical varicocele and abnormal semen parameters regardless of the chosen surgical method. Surgical varicocele repair was beneficial not only for alleviating oxidative stress-associated infertility but also to improve sperm nuclear DNA integrity. However, given the low magnitude of the effect size in sperm DNA integrity, further research is needed to elucidate its clinical significance. Conflicting results on the effect of varicocele treatment on natural fertility seem to be due to heterogeneous study designs and, more importantly, patient selection criteria. When these issues are controlled, current evidence indicates that treatment of subclinical varicocele is not warranted, as it does not seem to improve fertility. On the contrary, fair evidence indicates that varicocele treatment should be offered to infertile patients with palpable varicocele and abnormal semen parameters. This evidence supports the current guidelines issued by the American Urological Association and European Association of Urology, which state that varicocele treatment should be offered to male partners of infertile couples presenting for evaluation with clinical varicocele and semen parameters alterations.

Sperm DNA fragmentation to guide towards the appropriate insemination method

Sperm DNA fragmentation to guide towards the appropriate insemination method

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.

Improvement in Sperm DNA Quality Following Simple Life Style Intervention: A Study in Fathers of Children with Non-Familial Sporadic Heritable Retinoblastoma

Introduction and objectives: Genomic integrity of gametes is important for birth of healthy offspring. As compared to ova the sperm is more susceptible to mutation due to limited DNA damage detection and repair mechanism. Life style habits adopted by father before conception may adversely affect genomic DNA integrity in male gametes. Oxidative stress damages sperm nuclear and mitochondrial DNA. Complementary and alternative medical therapies such as yoga/meditation are being increasingly used as adjuncts to modern medicine in treatment of several clinical conditions. Therefore, in this pilot study, we analyzed the effects of yoga and meditation on seminal oxidative stress and sperm DNA integrity in fathers of children with non-familial sporadic heritable retinoblastoma (Rb) after intervention (3 and 6 months). Methods: A total of 10 men (father of children with sporadic non-familial Rb) were recruited in this study. Semen samples were collected at base line (day 0), 3 months and after 6 months of yoga practice. Reactive oxygen species (ROS), DNA fragmentation index (DFI) and 8-hydroxy-2’-deoxyguanosine (8-OHdG) levels were estimated at day 0.3 months and 6 months interval. Results: There was reduction in mean DFI levels [p>0.05] at 3 months and 6 months [p 0.05] 6 months [p<0.05] with respect to the base line levels (day 0). Conclusions: Yoga/meditation significantly lowers oxidative stress and oxidative nuclear DNA damage, and levels of the mutagenic base 8OHdG. Thus yoga and meditation are simple life style interventions which are therapeutic for maintaining/restoring sperm DNA integrity

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.

Cytotoxic Effects of Benzene Metabolites on Human Sperm Function: An In Vitro Study

In recent years, individuals are rampantly exposed to vapours of benzene, through paint, plastic, petroleum industries, fuel exhaust, and tobacco smoke. Hence the present investigation was directed towards determining the effect of benzene metabolites, namely, phenol-hydroquinone and catechol, on the motility, viability, and nuclear integrity of the human spermatozoa. From the results obtained it was clear that exposure to phenol-hydroquinone caused a significant decline in both, sperm motility and viability. Exposure to a phenol-hydroquinone (Phase I) microenvironment may therefore inhibit metabolically active enzymes, thus impeding ATP production, and in turn lowers sperm motility and viability. In addition, the present study also revealed that both metabolites of benzene caused significant denaturation of sperm nuclear DNA. Hence, exposure to phenol-hydroquinone in vitro could have resulted in generation of free radicals and altered membrane function, which is reflected by a decline in the motility, viability, and loss of sperm nuclear DNA integrity. In Phase II, the exposure of human sperm in vitro to varied concentrations of catechol caused only insignificant changes in sperm motility and viability as compared to those observed on exposure to phenol-hydroquinone. Hence, exposure to catechol appeared to have less toxic effects than those of phenol-hydroquinone.

Raman microspectroscopy: shining a new light on reproductive medicine

BACKGROUND The last 20 years have seen an enormous upsurge in the number of publications reporting findings obtained by Raman spectroscopy, a non-invasive, non-destructive method which uses the inelastic scattering of light to provide a 'fingerprint' of the sample's chemical composition and constituents. Long neglected because of practical difficulties, the technique has been transformed by recent technological advances into a powerful analytical tool capable of opening avenues of investigation that were previously out of the reach of biomedical scientists. Beyond introducing the approach and describing its relative merits and weaknesses, the aim of this review is to provide a spur for discussion of what may become an invaluable tool for biomedical investigations. METHODS A comprehensive review of the literature was conducted searching PubMed and Ovid databases using numerous MeSH terms associated with reproductive medicine. Furthermore, the reference lists of all reported literature were explored. The searches were restricted to English language articles published in the last 50 years. RESULTS Beginning with simple characterizations of biologically and medically important substances, aided by increasing technological sophistication, the use of Raman spectroscopy in biomedicine has quickly expanded to the investigation of complex biochemical interactions, the assessment of organelles and now the evaluation of living cells and tissue. The first Raman investigations of reproductive organs were primarily oncological in nature; however, the past few years have seen an increase in the application of the technique for the assessment and evaluation of both male and female gametes. In particular, progress has been made in the characterization, identification and localization of sperm nuclear DNA damage. CONCLUSIONS The use of Raman spectroscopy has already provided many tantalizing glimpses into the potential that the technique has to answer many of the unresolved issues in investigative and therapeutic reproductive medicine. However, without stringent assessment and the clear representation of the methods' findings, their true meaning cannot be revealed nor should any conclusions be hastily derived. For the potential of Raman microspectroscopy to be truly realized, the dependability and reliability of the technique and its results can only be ascertained by multidisciplinary collaborations that undertake carefully conducted, controlled and analysed studies.

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.

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

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 study of spermatic DNA fragmentation and sperm motility in infertile subjects

Although the pathophysiology of the testicular damage associated with varicocele remains unclear, sperm DNA damage has been identified as a potential explanation for this cause of male infertility. The current study was designed to determine the extent of sperm nuclear DNA damage in patients with varicocele, and to examine its relationship with parameters of seminal motility. Semen samples from 60 patients with clinical varicocele and 90 infertile men without varicocele were examined. Varicocele sperm samples were classified as normal or pathological according to the 1999 World Health Organizzation guidelines. Sperm DNA damage was evalutated using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. The DNA fragmentation index (DFI: percentage of sperm with denatured nuclei) values was significantly higher in patients with varicocele, either with normal or abnormal (DFI 25.8 ± 3.2 vs 17.4 ± 2.8 - P < 0,01) semen profiles. In addition, an inverse correlation was found between spermatic motility and the degree of spermatic DNA fragmentation in patients with clinical varicocele. Varicocele is associated with high levels of DNA-damage in spermatozoa. In addition, in subjects with varicocele, abnormal spermatozoa motility is associated with higher levels of sperm DNA fragmentation. DNA fragmentation may therefore be an essential additional diagnostic test that should be recommended for patients with clinical varicocele.

Effects of microsurgical varicocelectomy on human sperm DNA fragmentation, distribution of nuclear sulfhydryl groups and sperm maturation: a prospective trial

To examine the effect of varicocelectomy on sperm quality, specifically, sperm nuclear chromatin integrity, distribution of nuclear sulfhydryl groups and sperm maturation. A prospective trial. We prospectively evaluated a consecutive series of infertile men (n=29) presenting to Ovo clinic with one year or more history of infertility, a clinically palpable varicocele and abnormal semen parameters. Six sperm donors with normal sperm parameters served as controls. Conventional sperm parameters, sperm nuclear DNA integrity (sperm chromatin structure assay), distribution of nuclear sulfhydryl groups (Iodoacetamide fluorescein stain “5-IAF”) and sperm maturation (Aniline Blue stain “AB”) before and 4 months after microsurgical varicocelectomy. The percentage of spermatozoa with the sperm %DFI, %HDS, positive 5-IAF staining (diffuse and intense head staining), positive AB staining (dark blue) were all significantly lower in the control group compared to infertile men with varicocele (8 vs. 20%, 4.0 vs. 9.6%, 1.7 vs. 16.3%, and 13.5 vs. 2.5% respectively). The percentage of spermatozoa with positive 5-IAF nuclear and positive AB staining decreased significantly after surgery (16.3 to 5.4%, and 13.5% to 5.4% respectively). Similarly, the %HDS and %DFI also decreased significantly after surgery (from 10% to 6% and from 20% to 13%, respectively). The only notable relationships were between aniline blue staining and HDS post varicocelectomy (r=0.57, P<0.05), and both IAF staining and %DFI were inversely correlated with motility (r=-0.44 and -0.43, respectively). The data show that varicocelectomy is associated with a consistent improvement in sperm DNA integrity and chromatin compaction using three different cytochemical assays of sperm chromatin integrity (SCSA, IAF, Aniline Blue).

Dynamic in vivo binding of transcription factors to cis-regulatory modules ofcerandgscin the stepwise formation of the Spemann–Mangold organizer

How multiple developmental cues are integrated on cis-regulatory modules (CRMs) for cell fate decisions remains uncertain. The Spemann-Mangold organizer in Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated by the aforementioned transcription factors through CRMs conserved between X. laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the late blastula stage, and subsequently bound by all five factors at the gastrula stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among others, persists, which corresponds to their co-expression in the prechordal plate. Based on these data, together with detailed expression pattern analysis, we propose a new model of stepwise formation of the organizer, in which (1) maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then (2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the dorsal endodermal and mesodermal regions where all these genes are co-expressed; and (3) these two regions are combined at the gastrula stage to form the organizer. Thus, the in vivo dynamics of multiple transcription factors highlight their roles in the initiation and maintenance of gene expression, and also reveal the stepwise integration of maternal, Nodal and Wnt signaling on CRMs of organizer genes to generate the organizer.

Diabetes Mellitus and Sperm Parameters

Because of the paucity of studies and inconsistencies regarding the impact of diabetes mellitus (DM) on semen quality, this disease is seldom looked for in the infertile patient. Recently, this view has been challenged by findings showing that DM induces subtle molecular changes that are important for sperm quality and function. This brief review shows the main sperm parameters in patients with DM and presents the mechanisms hypothesized to explain the changes observed in these patients. The data available suggest that DM alters conventional sperm parameters. In addition, DM causes histologic damage of the epididymis, with a negative impact on sperm transit. Various mechanisms may explain the sperm damage observed in patients with DM. These include endocrine disorders, neuropathy, and increased oxidative stress. Many authors suggest that DM decreases serum testosterone levels. This is associated with a steroidogenetic defect in Leydig cells. In addition, diabetic neuropathy seems to cause atonia of seminal vesicles, bladder, and urethra. Furthermore, DM is associated with an increased oxidative stress, which damages sperm nuclear and mitochondrial DNA. Finally, spermatogenesis derangement and germ cell apoptosis in type 1 DM may relate to a local autoimmune damage, whereas insulin resistance, obesity, and other related comorbidities may impair sperm parameters and decrease testosterone serum levels in patients with type 2 DM.

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.