Routine Sperm Parameters Research Articles

The role of the intrinsic pathway of apoptosis in human ejaculated sperm damage under a state of scrotal heat stress

PurposeThe study aimed to determine the associations among standard sperm characteristics and oxidative/apoptotic markers in ejaculated sperm of men exposed to prolonged scrotal hyperthermia of either environmental or clinical origin.MethodsThe original study design included four research groups: professional drivers (n = 54), infertile men with varicocele (n = 78), infertile men not exposed to prolonged genital heat stress (n = 37), and fertile individuals serving as the control group (n = 29). Standard semen analysis was performed according to the 5th WHO laboratory manual. The following oxidative and apoptotic parameters of sperm were investigated: mitochondrial superoxide anion generation (MitoSOX Red dye), phosphatidylserine externalization (Annexin V binding assay), mitochondrial membrane potential (JC-1 dye), DNA fragmentation (TUNEL/PI assay), and membrane fluidity (merocyanine 540 dye).ResultsAll the studied groups presented a strong deterioration in routine sperm parameters and a strongly apoptotic phenotype in sperm, characterized by both decreased mitochondrial membrane potential and enhanced DNA fragmentation, regardless of the thermal insult. Significant induction of mitochondrial superoxide anion generation was noted only in the groups exposed to genital heat stress. A positive correlation between the production of superoxide anion in the mitochondrial chain and the level of DNA fragmentation in drivers was also noted.ConclusionLong-term exposure to scrotal hyperthermia in real-life situations is sufficient to reduce sperm quality in humans. The thermal stress directly induces the oxidative stress cascade in ejaculated sperm, affecting the plasma membrane fluidity, mitochondrial homeostasis, and sperm DNA integrity.

ATP5D Is a Potential Biomarker for Male Fertility.

Infertility is a global medical and social problem that affects human health and social development. At present, about 15% of couples of the right age in the world are infertile. As all we know, genetic defects are the most likely underlying cause of the pathology. ATP5D is also known as the delta subunit of mitochondrial ATP synthase. Mitochondria maintain sperm vitality, capacitation, acrosome reaction, and DNA integrity through ATP. Mitochondrial damage can trigger energy synthesis disorders, resulting in decreased sperm quality and function or even disappearance. The specific role of ATP5D in regulation of the male reproductive system remains elusive. In this study, semen from normal and infertile males were collected and their indicators were examined by analysis of routine sperm parameters; ATP5D protein content in semen was examined by ELISA. Singer sequencing was used to detect whether there was a mutated of ATP5D in semen. Meanwhile, ATP5D knockout (KO) and knockin (KI) male mice were selected at 8-12 weeks of age and mated with adult wild-type (WT) female mice for more than two months to assess their fertility and reproductive ability. Morphological changes in tissues such as testes and epididymis were observed by HE staining; spermatozoa were taken from the epididymis of the mice; sperm counts were performed and morphological changes were observed by Diff-Quik staining. The results showed that the expression of ATP5D in infertile males was significantly lower than that in normal males (P < 0.001) and the normal morphology rate of spermatozoa was much lower than that of normal males, and the sequencing results showed no mutations. The animal reproductive experiments showed no significant changes in the number of fertility in KO/KI mice compared with WT mice, but the duration of fertility was significantly longer (P = 0.02). The testicular cells in KO mice were loosely arranged and disorganized, the lumen was larger, the interstitial cells were atrophied, and the number of spermatozoa was reduced and the malformation rate was higher in WT males. This suggests that ATP5D is an essential protein for sperm formation and fertility in male mice and may be used as a biomarker of male fertility. This study found ATP5D correlated with male infertility and the expression levels were significantly reduced in the seminal plasma of all male infertile patients without gene mutations. KO male significantly prolonged fertility time and impaired testicular histomorphology. This suggests that ATP5D may be associated with spermatogenic function and fertility in male mice and may be used as a biomarker for male fertility. Future studies are required to elucidate the potential mechanisms. The trial registration number is KLL-2021-266.

Opioid replacement therapy with methadone or buprenorphine effects on male mice reproduction.

Opioid use disorders are commonly treated by long-acting agonist opioids including methadone and buprenorphine which could affect various aspects of male reproduction especially spermatogenesis. We aimed to determine whether detoxification with methadone or buprenorphine was associated with reproductive disorders in male mice. We orally induced morphine dependence in NMRI male mice, and then performed detoxification programs using either methadone or buprenorphine. Testis architecture and sperm parameters including sperm nuclear DNA integrity, mitochondrial activity, oxidative stress in seminal plasma, and routine sperm parameters were assessed to find the involved mechanisms. The number of Leydig cells and the thickness of germinal epithelium reduced following morphine use and increased differently after detoxification with methadone or buprenorphine. Morphine dependence and detoxification with methadone and buprenorphine had different effects on sperm parameters. Morphine altered chromatin integrity, mitochondrial activity, and oxidative stress in sperm. Detoxification with methadone improved mitochondrial activity but worsened chromatin integrity, whereas detoxification with buprenorphine improved neither chromatin integrity nor mitochondrial activity. Seminal plasma oxidative stress was higher in the treated groups compared to control groups but was comparable among treatment groups. Our study revealed that long-term morphine use followed by detoxification with methadone or buprenorphine impairs testis structure and sperm parameters. Detoxification from morphine use with methadone and buprenorphine led to different preclinical outcomes in semen quality parameters, including chromatin integrity. Therefore, clinical detoxification protocols should be performed more cautiously, considering the desire of the individuals to reproduce.

Vitamin D levels and human sperm DNA fragmentation: a prospective, cohort study

BackgroundIntracytoplasmic sperm injection (ICSI) has revolutionized the treatment of couples with male factor infertility but results remain suboptimal and suggest the need for further investigation into the molecular biology of spermatozoa. Vitamin D has been implicated in spermatogenesis and sperm function. Hypovitaminosis D has been associated with abnormal testicular function, including elevated sperm DNA fragmentation in a murine model. This study’s objective was to evaluate if there is a correlation between Vitamin D sufficiency and human spermatozoa DNA fragmentation index % (DFI%) in infertile couples.ResultsA prospective cohort study using a consecutive, convenience sample of subjects with infertility. The primary endpoint was the effect of Vitamin D sufficiency on human spermatozoa DFI%, and secondary outcomes included Vitamin D’s effect on moderate DFI%, high DFI%, High DNA stainability % (HDS%), sperm density (million/mL), sperm total motility (% total) and sperm strict morphology (% total). Of the 111 participating, 9 were excluded, leaving 102subjects. The subjects were stratified by vitamin D levels: deficient (< 20 ng/mL; n = 24), insufficient (20–30 ng/mL; n = 43),, and sufficient (> 30 ng/mL; n = 35). There were no statistical difference between the categories of serum vitamin D levels and sperm DFI% as well as the secondary outcomes. An increased BMI was associated with low serum vitamin D levels (p = 0.0012).ConclusionVitamin D deficiency was not associated with sperm DFI% or routine sperm parameters. Previous animal and human studies have demonstrated conflicting results between sperm parameters and Vitamin D levels. Redundant pathways in Vitamin D and calcium homeostasis in the human male reproductive tract may maintain essential reproductive processes during Vitamin D insufficiency or deficiency.Trial registrationTrial Registration Number: MOD00002311 (ClinicalTrials.gov).

Evaluation of DNA fragmentation in dog sperm using the sperm chromatin dispersion test.

The study's objective was to adapt the Sperm Chromatin Dispersion (SCD) protocol to evaluate sperm DNA fragmentation and implement a fragmentation control in dogs. Correlation between DNA status and routine sperm parameters was also analysed. To adapt the SCD, two different mercaptoethanol (ME) concentrations were assayed (2.5% and 5%) in fourteen ejaculates from seven dogs and semen incubation with 0.3M NaOH for 15min at room temperature was assayed as a control for sperm DNA fragmentation. Data were analysed using a Mann-Whitney test and either Pearson's or Spearman's correlation. The selected ME concentration to use in the SCD test was 5%, as it produced the largest DNA dispersion halo while preserving the core nucleus structure. Four DNA halo patterns were identified as follows: large dispersion halos, medium halos, small halos and nuclei without halos. Semen incubated with NaOH showed 100% sperm without halos (damaged DNA). A significant positive correlation was observed between sperm with fragmented DNA and sperm with coiled tails. Thus, it was possible to adapt the SCD protocol to evaluate dog sperm DNA fragmentation in raw semen without using a commercial kit and establish incubation with NaOH as a DNA fragmentation control. Only coiled tails showed correlation with DNA fragmentation.

Effect of Huaji Jianpi Decoction on the semen quality of high-fat diet-induced obese mice.

BackgroundAt present, the incidence of obesity is increasing. Several studies have shown that obesity can reduce male fertility by affecting spermatogenesis and semen quality. Traditional Chinese medicine (TCM) has fewer side effects and stable efficacy in the treatment of related diseases. This study aimed to investigate the effect of Huaji Jianpi Decoction on the semen quality of high-fat diet-induced obese mice.MethodsThe obese male mice model was constructed by using high-fat diet and the Huaji Jianpi Decoction was processed into an aqueous extract. Mice were allocated in the normal group (n=30) and five different treatment groups (n=50). Huaji Jianpi Decoction was applied in low-, medium- and high-dose [17.52 g/(kg·d), 35.04 g/(kg·d) and 70.07 g/(kg·d), respectively]. The body weight, body fat, testis wet weight, testis coefficient, and routine sperm parameters were detected and analyzed. Meanwhile, transmission electron microscope (TEM) was used to observe testis ultrastructure. reverse-transcription quantitative PCR (RT-qPCR) was used to measure the expression of tumour necrosis factor α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1).ResultsCompared with normal mice (ND), the testis wet weight and testis coefficient of mice in the blank group were significantly decreased, while the number of mitochondria was observed to be decreased on testis ultrastructure examination, and apoptotic cells and germ cells in the spermatogenic tubules were shed. After Huaji Jianpi Decoction administration, the body fat and blood lipid levels of obese mice were decreased, the testis wet weight and testis coefficient were increased, and semen parameters were increased. Different doses of Huaji Jianpi Decoction could improve testicular weight, sperm density, sperm motility, forward motility, and total sperm motility. Huaji Jianpi Decoction could also downregulate TNF-α and MCP-1 expression and inhibit germ cell apoptosis to improve semen quality.ConclusionsHuaji Jianpi Decoction can improve the semen quality of high-fat diet-induced obese mice by reducing weight and lipid levels.

Ameliorative effect of hesperidin on streptozotocin‐diabetes mellitus‐induced testicular DNA damage and sperm quality degradation in Sprague–Dawley rats

This study aimed to investigate the effect of hesperidin on reproductive damage caused by diabetes mellitus. A total of 24 adult male rats were divided into four groups: control group, hesperidin group, diabetes mellitus group, and diabetes mellitus+hesperidin group. The study was conducted for 4weeks. At the end of the study, the rats were sacrificed and testicular oxidative stress markers (MDA, GSH, GSH-Px, SOD, and CAT), DNA damage in testes (8-OHdG), and routine sperm parameters were evaluated. According to the results of the study, most of the parameters were similar in the control and hesperidin groups but CAT activity in the hesperidin group was statistically higher than the control group. Also, diabetes mellitus (DM) significantly increased MDA levels and decreased enzymatic antioxidant (GSH-Px, SOD, CAT) activities and nonenzymatic (GSH) antioxidant levels. On the other hand, hesperidin supplementation significantly decreased oxidative stress and increased enzymatic antioxidant activities and nonenzymatic antioxidant levels due to the antioxidant effect. Also, DM increased DNA damage levels in testicular tissue and hesperidin supplementation significantly decreased DNA damage levels in testes of diabetic male rats. Besides, sperm motility significantly decreased while abnormal sperm rate and dead sperm rate were significantly increased in diabetic rats. Hesperidin supplementation significantly reduced these side effects in diabetic rats. In conclusion, hesperidin supplementation could be beneficial for decreasing the side effects on the male reproductive system caused by DM in rats. PRACTICAL APPLICATIONS: Diabetes is an important metabolic disease, affecting quality of life and fertility. Hesperidin has an antioxidant effect and has a potential protective effect on reproductive toxicity in diabetic male rats. Hesperidin decreased oxidative stress, and DNA damage in testis resulted from hyperglycemia and improved sperm quality in diabetic rats. The hesperidin supplementation could be a good strategy to protect male fertility in diabetic patients.

Age-related changes in human conventional semen parameters and sperm chromatin structure assay-defined sperm DNA/chromatin integrity

Research questionWhat are the correlations between male age, traditional semen parameters, sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) in a sufficiently large sample size? DesignRetrospective cohort study of 18,441 semen samples, with data divided into seven age groups according to male age: ≤25, 26–30, 31–35, 36–40, 41–45, 46–50 and ≥51 years. ResultsAge was negatively correlated with semen volume, total sperm count, motility and HDS, and positively correlated with sperm concentration and DFI (P < 0.001). After 35 years of age, semen volume and total sperm count began to decline. After 30 years of age, motility and HDS decreased consistently. Sperm concentration and DFI increased from 26–30 years of age. DFI was negatively correlated with sperm concentration, total sperm count, motility and normal morphology (P < 0.001) and positively correlated with semen volume and HDS (P < 0.001). HDS was negatively correlated with all parameters (P < 0.001) except semen volume (r = –0.013, P = 0.074) and DFI (r = 0.124, P < 0.001). Patients aged ≥40 years had higher DFI than those aged <40 years in the entire cohort, in the abnormal semen parameters cohort, and in the normal semen parameters cohort (OR 2.145, 2.042, 1.948, respectively, P < 0.001). The ≥40 years age group had a lower HDS than the <40 years age group in the entire cohort and abnormal semen parameters cohort (OR 0.719, 0.677, respectively, P < 0.001). ConclusionsAgeing is a negative effector of sperm quantity and quality, and routine sperm parameters have weak but significant correlations with sperm DNA/chromatin integrity.

Assessment of Dog Testis Perfusion by Colour and Pulsed-Doppler Ultrasonography and Correlation With Sperm Oxidative DNA Damage

The assessment of testicular artery blood flow by colour and pulsed-Doppler ultrasonography is an important diagnostic technique to assess vascular perfusion. Recently, it has been suggested as a good predictor of sperm quality. On the other hand, through the alkaline Comet Assay, it is possible to quantify sperm oxidative DNA damage. The aim of this study was to evaluate the relationship between routine sperm parameters, testicular artery blood flow and oxidative DNA damage in canine sperm. Testicular ultrasonography and sperm collection were performed on 12 male dogs, with the animals being allocated into 2 groups, according to the classification of the ejaculates’ quality, as normozoospermic (N; n = 7) or non-normozoospermic (OAT; n = 5). Seven dogs aged between 1.5 and 8.0 years old were included in group N and 5 dogs, aged between 2.0 and 11.0 years old, were included in group OAT. The sperm-rich fraction of the ejaculates was evaluated for sperm routine parameters and DNA damage by comet assay. Colour and pulsed-Doppler ultrasonography were used to evaluate the blood flow of the supratesticular and marginal arteries of right and left testis. Group OAT presented higher levels of sperm oxidative DNA damage (A.U.) in comparison to group N (N:11.7 ± 9.9; OAT:34.2 ± 6.1; P< .001). The peak of systolic velocity was positively correlated with sperm concentration (r = 0.685; P= .005). The resistive and pulsatility indexes (RI and PI) of the supratesticular artery were negatively correlated with sperm membrane integrity (HOST+) (r = −0.594; P = .042; r = −0.612; P = .035, respectively). The end diastolic velocity (EDV) of the supratesticular artery was positively correlated with sperm concentration (r = 0.748; P = .005) and negatively correlated with sperm oxidative DNA damage (r = −0.766; P = .004). Our results suggest that the assessment of the testicular artery blood flow by colour and pulsed-Doppler ultrasonography could be a good predictor of sperm quality in dogs in terms of sperm concentration, membrane integrity and sperm oxidative DNA damage.

Association Between Sleep Quality and Semen Parameters and Reproductive Hormones: A Cross-Sectional Study in Zhejiang, China.

BackgroundThe effects of sleep duration on semen quality have been documented in many epidemiological studies. However, the association between sleep quality and semen parameters and reproductive hormones is still unclear.Patients Enrollment and MethodsWe conducted a cross-sectional study among 970 outpatients from the Reproductive Medicine Center in Zhejiang, China between October 2017 and July 2019. All participants delivered a semen sample, underwent a physical examination, and answered a questionnaire to provide the following information: demographics, life habits, and sleep habits. Sleep quality was measured using the Pittsburgh Sleep Quality Index (PSQI). We first divided the patients into two groups according to sleep quality (good sleep: PQSI < 5 and poor sleep: PSQI ≥ 5). Then, we analyzed routine sperm parameters (semen volume, sperm total motility, progressive motility, sperm concentration, total sperm number, and normal sperm morphology) and reproductive hormones (follicle-stimulating hormone, luteinizing hormone, estrogen, testosterone, and prolactin) of each group. Finally, we used multivariate linear regression analysis and Spearman correlation coefficients to examine the relationship between sleep quality (discrete variable or dichotomous variable) and sperm parameters, reproductive hormones.ResultsA negative correlation was found between the general PSQI scores and several semen parameters: total motility (r= −0.187979, p< 0.001), progressive motility (r= −0.192902, p< 0.001), concentration (r= −0.167063, p< 0.001), total sperm number (r= −0.160008, p< 0.001), and normal sperm morphology (r= −0.124511, p< 0.001). However, there was no significant correlation between the semen volume, all reproductive hormones and the general PSQI scores. After adjusting for confounders, men with poor sleep had lower total motility (β= −9.287; 95% CI, −12.050, −6.523), progressive motility (β= −8.853; 95% CI, −11.526, −6.180), concentration (log scale, β= −0.131; 95% CI, −0.181, −0.082), total sperm number (log scale, β= −0.137; 95% CI, −0.189, −0.084), and normal sperm morphology (β= −1.195; 95% CI, −1.844, −0.547), but semen volume and all reproductive hormones were not markedly altered.ConclusionPoor sleep quality might be related to impaired semen quality, but we found no evidence that poor sleep quality affects reproductive hormones.

Exposure of environmental Bisphenol A in relation to routine sperm parameters and sperm movement characteristics among fertile men

Although several human studies have examined bisphenol A (BPA) exposure in relation to routine sperm parameters, evidence of BPA’s effects on sperm movement characteristics is limited. We examined associations of BPA exposure with sperm parameters including sperm movement characteristics among fertile men. The cross-sectional study was conducted in Sandu County, Guizhou Province, China. Subjects provided semen samples analyzed by computer-aided sperm analysis (CASA) system and urine samples for BPA assay. They were invited to complete an in-person interview with a structured questionnaire to obtain demographics, lifestyle factors, etc. In final analyses, 500 subjects were included. We used multivariate linear regression analyses to estimate associations between BPA and sperm parameters after adjusting for potential confounders. BPA was detected in 73.6% of urine samples, with a geometric mean of 0.44 μg/gCreatinine. Compared with subjects of undetected BPA, subjects with detected BPA had increased Linearity (LIN, β: 2.19, 95% confidence interval (CI): 0.37, 4.0), Straightness (STR, β: 1.47, 95% CI: 0.19, 2.75), Wobble (WOB, β: 1.75, 95% CI: 0.26, 3.25), reduced Amplitude of lateral head displacement (ALH, β: −0.26, 95% CI: −0.5, −0.02) and Mean angular displacement (MAD, β: −2.17, 95% CI: −4.22, −0.11). Subjects in the highest tertile of creatinine-adjusted BPA group had lower sperm concentration than those with undetected BPA. Dose-response relationships of BPA with LIN, WOB, ALH, MAD and sperm concentration were demonstrated by statistically significant trends across tertiles of creatinine-adjusted BPA concentrations. Similar results were obtained using unadjusted BPA concentrations. Exposure to environmental BPA would decrease sperm concentration and sperm swing characteristics (ALH and MAD), and increase sperm velocity ratios (LIN, STR and WOB), which might mediate further effects on impaired male fecundity.

Sperm DNA and detection of DNA fragmentations in sperm.

The questionable effectiveness of routine sperm parameters in determining male factor infertility problems and increasing the success rates of assisted reproductive techniques have led to the investigation of more detailed sperm parameters that could affect the male fertility and reproduction. Thus, the effects of different sperm parameters such as sperm DNA integrity was started to be investigated thanks to the previously described methods such as single cell gel electrophoresis (COMET) assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), terminal deoxynucleotidyl transferase-mediated deoxyuridine (TdT) triphosphate (dUTP) nick end labeling (TUNEL) assay and sperm chromatin dispersion (SCD) test. However, studying on sperm DNA might be very complex because the sperm DNA differs from the somatic cell DNA with its unique structure. Also, the sperm DNA undergoes many changes during spermatogenesis and it is condensed by being packaged tightly with different types and numbers of protamines in different species. Despite all these difficulties, these methods provide important information about the reasons and consequences of DNA damages in sperm and the effects of these damages on reproduction.

The effect of bacteriospermia and leukocytospermia on conventional and nonconventional semen parameters in healthy young normozoospermic males

Bacterial semen inflammation/infection is an important diagnostic and therapeutic problem in contemporary andrology. The molecular mechanism by which inflammatory mediators compromise the fertilizing potential of germ cells is complex and multifactorial, and it remains unclear. To improve the understanding of the pathophysiology of human subfertility/infertility caused or complicated by reproductive tract inflammation/infection, we simultaneously evaluated a set of conventional (standard semen analysis) and nonconventional sperm parameters, including subcellular changes in sperm membranes (phospholipid scrambling, peroxidative damage, and phosphatidylserine (PS) externalization), mitochondria (mitochondrial transmembrane potential, ΔYm, and oxidoreductive capability), and DNA fragmentation in healthy young normozoospermic males with asymptomatic bacteriospermia and leukocytospermia. Both bacteriospermia and leukocytospermia had a deleterious effect on standard sperm parameters, including sperm concentration, motility and morphology. Bacteriospermia was associated with a simultaneous decrease in mitochondrial transmembrane potential and an increase in PS externalization, and with DNA fragmentation in both live and dead sperm. The highest MDA concentrations in sperm lysates were observed in the presence of leukocytes. This study demonstrates for the first time that bacteriospermia and leukocytospermia compromise sperm quality in healthy young normozoospermic males. Bacteria mainly participate in intrinsic mitochondria-dependent apoptotic cell death mechanisms. Oxidative stress plays a relevant role in decreasing routine sperm parameters during leukocytospermia. The value of these observations may be significant and may support the development of a new diagnostic platform (biomarkers) for infertile males with infections in the reproductive tract.

DNA integrity of canine spermatozoa during chill storage assessed by the sperm chromatin dispersion test using bright-field or fluorescence microscopy

The objective of this study was to evaluate the effect of chill storage on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test using bright-field microscopy with Wright solution (sDF-B) or fluorescence microscopy with propidium iodide (sDF-F). The relationship and agreement between the results obtained with both staining methods were analyzed. The values of DNA fragmentation indexes (sDF-F and sDF-B) were compared at each time of chill storage (0, 24, 48, 72, and 96 hours). Additionally, the sperm DNA fragmentation rate (slope) was compared between the methods during chill storage. Good agreement and no significant differences between values obtained with both staining procedures were observed. Finally, the effect of chill storage for up to 96 hours was assessed on sperm motility parameters and DNA fragmentation indexes. Significant differences were found after 48 hours of chill storage, obtaining greater values of fragmented DNA. Progressive sperm motility was lower just after 96 hours of chill storage, and no effect was found in total sperm motility. In conclusion, the Sperm-Halomax kit, developed for canine semen and based on the sperm chromatin dispersion test, can be used accurately under bright-field or fluorescence microscopy to assess the sperm DNA integrity of canine semen during chill storage. The sperm DNA fragmentation index increased after 48 hours of chill storage, thereby detecting sperm damage earlier than other routine sperm parameters, such as sperm motility.

Differential Proteomic Profiling of Spermatozoal Proteins of Infertile Men With Unilateral or Bilateral Varicocele

To compare the sperm protein profile between infertile men with unilateral varicocele and infertile men with bilateral varicocele. This prospective study investigated 50 infertile patients with clinical varicocele (33 unilateral and 17 bilateral) seeking fertility workup between March 2012 and April 2014. Routine sperm parameters, reactive oxygen species, total antioxidant capacity, and sperm deoxyribonucleic acid fragmentation were assessed in their semen. Sperm protein profile was characterized only in pooled samples of 5 unilateral and 3 bilateral varicocele samples, respectively, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Linear Trap Quadrupole-Orbitrap Elite hybrid mass spectrophotometer system. Differences in protein expression were analyzed using gel analysis software, followed by protein identification using mass spectroscopy analysis. Differentially expressed proteins and their abundance were quantified by comparing spectral counts, followed by bioinformatics analysis. Unique expression of 64 proteins in the bilateral group and 31 proteins in the unilateral group was obtained. Core functions of the top protein interaction networks were post-translational modification (∼122 proteins associated with acetylation), protein folding, free-radical scavenging, cell death, and survival. The top molecular and cellular functions were protein degradation, free radical scavenging, and post-translational modifications, whereas the top pathways were protein ubiquitination and mitochondrial dysfunction. Major biological pathways for the 253 differentially expressed proteins were metabolism, apoptosis, and signal transduction. Functional proteomic profiling helps identify the differential processes or pathways that are affected based on the nature of varicocele (bilateral or unilateral) and provide insights into the mechanistic implications of varicocele-associated male infertility.

Beneficial Effect of an Oral Antioxidant Supplementation (Fertimax2) on IVF-ICSI Outcomes: A Preliminary Clinical Study

Background: Considerable evidence points towards a significant role of oxidative stress (OS) in the pathogenesis of sperm dysfunction. OS as a result of an inappropriate balance between oxidants and antioxidants in the semen can cause DNA damage and lipid peroxidation leading to failure of conception, miscarriage or potentially even childhood cancer. The objective of this study was to investigate whether a male antioxidant therapy can improve semen parameters and the results of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Methods: A total of forty eight infertile couples were considered. Male participants were administrated Fertimax2 antioxidant treatment for at least two months prior to their partner’s IVF-ICSI cycle. Sperm parameters (volume, concentration, progressive motility) and the IVF-ICSI outcomes were compared before and after the antioxidant treatment. The primary outcome measures were oocyte fertilization, cleavage and good embryo quality rates; the secondary outcomes were biochemical pregnancies, clinical pregnancies and implantation rates. Results: The principal finding that emerged from this study was that antioxidant therapy resulted in significant improvements in fertilization (p = 00.2), cleavage (p = 0.004) and good-embryo quality (p = 0.002) rates accompanied by a marked increase in clinical pregnancy (18.7% versus 2.5%) and implantation (11.8% versus 1.02%) rates. No significant changes in routine sperm parameters were observed. Conclusion: The Fertimax2 antioxidant therapy appears to influence favorably chances of conception in couples undergoing assisted reproduction treatment (ART).

Relationship of seminal plasma antioxidants and serum male hormones with sperm chromatin status in male factor infertility

We explored the relationship between sperm chromatin integrity, hormone levels, seminal plasma total antioxidant capacity (TAC), and routine sperm parameters in men with male factor (MF, n = 81) and non-male factor (NMF, n = 52) infertility. Semen and blood were collected and examined from men undergoing evaluation for infertility in the Avicenna Infertility Clinic. We have examined each patient for serum hormones (LH, FSH, E2, DHEA), sperm chromatin damage, level of protamination and seminal plasma TAC. Levels of FSH, LH, sperm chromatin damage, and abnormal protamination were significantly higher in MF vs. NMF groups (p < 0.001). Sperm chromatin damage was correlated with percentage of CMA3- positive sperm (r = 0.64, p < 0.001) and with sperm concentration (r = −0.36, p < 0.001), motility (r = −0.21, p < 0.05), and morphologically normal spermatozoa (r = −0.29, p < 0.001). Linear regression showed sperm chromatin damage was related to percentage of CMA3- positive sperm (p < 0.001) in ungrouped patients. It was related to both percentage of CMA3- positive sperm and serum DHEA in the MF group (p < 0.001 and p < 0.05, respectively). Sperm chromatin maturity assessed by CMA3 test was inversely related to sperm chromatin damage assessed by the toludine blue assay. Male factor infertility associated with sperm chromatin damage may be related to sperm protamination and to serum DHEA.

Impact of body mass index on seminal oxidative stress

Male obesity has been linked with a reduction in sperm concentration and motility, an increase in sperm DNA damage and changes in reproductive hormones. Recent large observational studies have linked male obesity with a reduced chance of becoming a father. One of the potential underlying pathological mechanisms behind diminished reproductive performance in obese men is sperm oxidative stress. The primary aim of this study was to determine if sperm oxidative stress was more common in obese/overweight men. A total of 81 men had their body mass index (BMI) correlated with seminal reactive oxygen species (ROS) production (Nitro Blue Tetrazolium assay), sperm DNA damage (TUNEL), markers of semen inflammation (CD45, seminal plasma PMN elastase and neopterin concentration) and routine sperm parameters, together with reproductive hormones. The principal finding from this study was that oxidative stress did increase with an increase in BMI, primarily due to an increase in seminal macrophage activation. However, the magnitude of this increase was small and only of minor clinical significance as there was no associated decline in sperm DNA integrity or sperm motility with increasing ROS production. Increased BMI was also found to be significantly linked with a fall in sperm concentration and serum testosterone, and an increase in serum oestradiol.

Improvement in sperm DNA quality using an oral antioxidant therapy

Oxidative stress is now recognized as a common pathology that affects up to half of all infertile men. One of the principal mechanisms by which oxidative stress produces infertility is by damage to sperm DNA, either through direct oxidation of the DNA by reactive oxygen species (ROS) or by the initiation of apoptosis. The objective of this study was to determine if an oral antioxidant/mineral supplement could improve sperm DNA integrity in men with known oxidative stress. A total of 50 infertile men identified as exhibiting oxidative stress were administered oral antioxidant therapy for a period of 3 months. All participants were assessed at entry and exit for sperm DNA integrity with terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling, apoptosis with annexin V, protamination with chromomycin A(3) and ROS production with nitro blue tetrazolium assay. Sperm concentration, motility and morphology, together with assessment of serum male reproductive hormones (LH, FSH, testosterone, anti-Mullerian hormone), were also monitored. The principal finding that emerged from this study was that antioxidant therapy resulted in significant improvements in sperm DNA integrity (P = 0.002) and protamine packaging (P < 0.001), accompanied by a reduction in seminal ROS production (P = 0.027) and apoptosis (P = 0.004). No significant changes in routine sperm parameters (concentration, motility, morphology) or male reproductive hormones were observed.

ANDROLOGY LAB CORNER*: Utility of Magnetic Cell Separation as a Molecular Sperm Preparation Technique

Assisted reproductive techniques (ARTs) have become the treatment of choice in many cases of infertility; however, the current success rates of these procedures remain suboptimal. Programmed cell death (apoptosis) most likely contributes to failed ART and to the decrease in sperm quality after cryopreservation. There is a likelihood that some sperm selected for ART will display features of apoptosis despite their normal appearance, which may be partially responsible for the low fertilization and implantation rates seen with ART. One of the features of apoptosis is the externalization of phosphatidylserine (PS) residues, which are normally present on the inner leaflet of the sperm plasma membrane. Colloidal superparamagnetic microbeads ( approximately 50 nm in diameter) conjugated with annexin V bind to PS and are used to separate dead and apoptotic spermatozoa by magnetic-activated cell sorting (MACS). Cells with externalized PS will bind to these microbeads, whereas nonapoptotic cells with intact membranes do not bind and could be used during ARTs. We have conducted a series of experiments to investigate whether the MACS technology could be used to improve ART outcomes. Our results clearly indicate that integrating MACS as a part of sperm preparation techniques will improve semen quality and cryosurvival rates by eliminating apoptotic sperm. Nonapoptotic spermatozoa prepared by MACS display higher quality in terms of routine sperm parameters and apoptosis markers. The higher sperm quality is represented by an increased oocyte penetration potential and cryosurvival rates. Thus, the selection of nonapoptotic spermatozoa by MACS should be considered to enhance ART success rates.