Protamine Content Research Articles

Sperm Chromatin Status and DNA Fragmentation in Mouse Species with Divergent Mating Systems.

Sperm DNA integrity and chromatin status serve as pivotal indicators of sperm quality, given their intricate link to sperm function, embryo development, and overall fertility. Defects in chromatin compaction, which are often associated with compromised protamine content, can lead to damaged DNA strands. In this study, the chromatin status and possible correlation with DNA damage was assessed in males of three mouse species: Mus musculus, M. spretus, and M. spicilegus. We employed various staining methods, including aniline blue, methylene blue (Diff-Quik), toluidine blue, and chromomycin A3, to assess chromatin compaction in cauda epididymal sperm. Samples were also analyzed by the sperm chromatin structure assay (SCSA) to estimate DNA fragmentation (%tDFI, %HDS). Analyses were carried out on freshly collected sperm and cells incubated for 3 h in a HEPES-buffered modified Tyrode's medium simulating conditions of the female reproductive tract. Notably, the analysis of chromatin status yielded minimal abnormal values across all three species employing diverse methodologies. SCSA analyses revealed distinct variations in %tDFI between species. Following sperm incubation, the percentages of sperm stained with methylene blue exhibited differences among the species and were significantly correlated to the DNA fragmentation index. HDS demonstrated correlations with the percentages of sperm stained by aniline blue, methylene blue, and chromomycin A3. Overall, chromatin compaction was high across all species, with limited differences among them. The relationship between chromatin status and DNA integrity appeared to be related to levels of sperm competition among species.

SPAG17 mediates nuclear translocation of protamines during spermiogenesis.

Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.

L-Carnitine and Pentoxifylline Supplementation Improves Sperm Viability and Motility at Low Temperature.

In infertility clinics, preserving high-quality spermatozoa for a long time is a necessity. Pentoxifylline (PT) and L-carnitine (LC) are effective in improving sperm motility as well as protecting the sperm membrane. The present study aimed to investigate the protective impacts of PT and LC on the quality of the normal sperm motility, protamine content, and viability on prolonged storage for 12 days at 4-6°C. The present experimental work included 26 samples, which were first prepared based on the swim-up technique, of normozoospermic men. They were divided into three aliquots as untreated control, LC-treated, and PT-treated groups and incubated for up to 12 days at 4-6°C. Thereafter, chromatin maturity, sperm viability, and motility were assessed on 0, 1, 2, 5, 7, and 12 days. Data were analyzed using a one-way analysis of variance. The obtained data revealed that PT supplementation increased the percentage of motile spermatozoa in comparison with control and LC-treated specimens. On the other hand, LC supplementation increased the percentage of viable spermatozoa in comparison with the PT-treated and control samples. During the 12-day storage, the percentage of spermatozoa with a normal protamine content was nearly unchanged in the three groups (P>0.05). Although LC supplementation can be considered a better alternative than PT for preserving sperm viability, PT could better preserve sperm motility compared to LC during 12 days at 4-6°C.

Aerobic training mitigates the negative impact of diabetes on fertility.

Diabetes negatively affects the reproductive system. This present study investigated the effects of aerobic training on protamine 1 and 2mRNA expression, sex hormones, antioxidant defence and sperm quality in diabetic rats. Thirty-six male Wistar rats were randomly allocated into three groups including diabetic training (DT) group, diabetic (D) group and control (C) group. Rats in DT were exercised 5 times per week for 8weeks. Blood samples were collected for evaluation of sex hormones 48h after the last training session. Also, the testes were removed and subjected to histological evaluation and semen analysis. Testicular mRNA expressions of protamines were determined by RT-qPCR. Protamines 1 and 2, and the ratio of protamine 1 to protamine 2 were significantly lower in DT and D groups compared with C group (p<0.01). LH and testosterone levels were significantly lower in D group compared with DT and C group (p<0.01). Malondialdehyde was significantly lower in DT and C groups compared with D group (p<0.001). Sperm parameters were significantly lower in D group compared with C group (p<0.01). Our findings suggest that aerobic training may mitigate the negative impact of diabetes on sex hormones, oxidative stress, protamine content and sperm parameters in male rats.

Sperm chromatin protamination influences embryo development in unsexed and sexed bull semen.

Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.

The study investigating the determination of protamine in seminal plasma from azoospermic donors: Suggestion of new methods to diagnose obstructive azoospermia, and to capture childbearing sperm for testicular sperm extraction (TESE) and insemination sperm injection (ICSI).

We analyzed seminal plasma of 88 normozoospermic, 40 oligozoospermic and 32 azoospermic donors. During this study, we focus to record the protamine concentration in the seminal plasma of azoospermic donors. The seminal protamine concentrations were found to be 19.6-62.8 IU/ml in normozoospermic donors; 25.4-100.8 IU/ml in oligozoospermic donors; and, most notably, 23.7-219.4 IU/ml in azoospermic donors. These results indicate that, based on seminal plasma protamine concentrations, even azoospermic donors were able to produce as much sperm as normo- and/or oligozoospermic donors. Using statistical analyses, significant differences were found between azoospermic and normozoospermic donors (p=0.0018). Protamine content was found to be a direct marker for the presence of sperm. The data from this study provided evidence for a new therapeutic approach for testicular varicose veins, which are found in obstructive or non-obstructive azoospermia. High seminal protamine concentrations indicated the future possibility of acquiring childbearing sperm for insemination sperm injection (ICSI) and testicular sperm extraction (TESE), even with azoospermic donors. Given these results, we also suggest a new cut-off value for acquisition of childbearing sperm in selection for ICSI.

Zinc deficient diet exacerbates the testicular and epididymal damage in type 2 diabetic rat: Studies on oxidative stress-related mechanisms

Zinc (Zn) is one of the most important trace elements in the body and is required for insulin secretion and release. Zn is also required for the growth and development of the reproductive system. Alteration in the Zn levels can cause moderate to severe damage to various organs, including the reproductive system. Most of type 2 diabetic patients have altered Zn levels/signaling. So, here we investigated the role of Zn-deficient diet (ZDD) in type 2 diabetes. Type 2 diabetes in the rat was induced by the combination of high-fat diet (HFD) and a single low dose of streptozotocin (STZ, 35 mg/kg, i.p.). Control animals were fed normal pellet diet throughout the study, while ZDD was given for four consecutive weeks to the diabetic rats, which were earlier kept on HFD for 16 weeks. The present findings showed that ZDD further decreased the serum Zn, plasma insulin and serum testosterone levels, whereas it increased cholesterol, triglycerides, BUN, %HbA1c in diabetic rats. Oxidative stress in testes was increased by ZDD as evidenced by decreased glutathione, catalase and SOD1 levels. ZDD-induced several abnormalities in sperm head morphology, altered sperm decondensation, sperm chromatin and protamine content, along with significant histopathological alterations in testes and epididymis. Further, ZDD altered protein levels of MT, MTF-1, Keap1, Nrf2, Nf-κB, GPX4 and GPX5 levels in the testes and epididymis of diabetic rat. The present results demonstrated that dietary Zn deficiency could exacerbate type 2 diabetes-induced germ cell damage.

Effects of cryopreservation on stallion sperm protamine messenger RNAs.

Protamines substitute DNA-binding histones during late spermatogenesis in sperm nucleus. Stallion sperm contains all three variants of these arginine-rich and positively charged nuclear proteins (P1, P2 and P3). Two variants of protamine-2, that is P2 and P3, constitute approximately 15% of the entire protamine content. Also, the ratio of protamine-1 to protamine-2 varies among different mammalian species, and abnormal protamine ratios and protamine content are correlated with male infertility. In this study, changes in protamine mRNA abundance for all three protamines were investigated in stallion sperm during cryopreservation. Twelve ejaculates were collected from six sexually mature stallions. Sperm samples were divided into two parts for total mRNA extraction: one as fresh and the other as cryopreserved sample. Levels of three protamine transcripts were determined by real-time reverse transcriptase polymerase chain reaction. Results of relative expression showed that cryopreservation can significantly alter protamine transcripts: protamine 2 was downregulated, while protamine 3 was upregulated in cryopreserved samples relative to the control. Changes in protamine 1 were not significant after cryopreservation. This study is the first to evaluate changes in mRNA abundance of protamine genes in stallion sperm following cryopreservation. Such evaluations are important in finding transcriptomic markers for success in fertilization and assisted reproductivetechniques.

Effects of N-acetyl-cysteine supplementation on sperm quality, chromatin integrity and level of oxidative stress in infertile men

BackgroundInfertile men have higher levels of semen reactive oxygen species (ROS) than fertile men. High levels of semen ROS can cause sperm dysfunction, sperm DNA damage and reduced male reproductive potential. This study investigated the effects of supplementation with N-acetyl-cysteine (NAC) on the sperm quality, chromatin integrity and levels of oxidative stress in infertile men.MethodsThe study was carried out in the unit of ACECR Infertility Research Center, Qom, Iran. The patients consisted of 50 infertile men with asthenoteratozoospermia who received NAC (600 mg/d) orally for 3 months, after which they were compared with pre-treatment status. Semen was analyzed according to WHO (2010), followed by the assessment of protamine content [chromomycin A3 (CMA3)] and DNA integrity [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)]. Oxidative stress markers, i.e. total antioxidant capacity (TAC) and malondialdehyde (MDA), as well as hormonal profile (LH, FSH, Testosterone and Prolactin) were determined by ELISA kit.ResultsAfter NAC treatment, patients’ sperm count and motility increased significantly whereas abnormal morphology, DNA fragmentation and protamine deficiency showed significant decreases compared to pre-treatment levels (P < 0.05). Hormonal profile improvement was associated with lowered FSH and LH levels and increased amount of testosterone (P < 0.05). TAC significantly increased and MDA decreased with an inverse significant correlation between TAC and MDA (P < 0.05).ConclusionNAC oral supplementation may improve sperm parameters and oxidative/antioxidant status in infertile males.

Sperm morphometry and chromatin condensation in Nelore bulls of different ages and their effects on IVF

The aim of this study was to evaluate the chromatin packing and sperm head morphometry of cryopreserved semen of Nelore bulls (Bos taurus indicus) of different ages. Furthermore, the influence of the degree of chromatin compaction on in vitro embryo production (IVP) was investigated. Forty bulls were divided into three groups: young (1.8–2 years), adult (3.5–7 years), and senile (8–14.3 years). The ejaculates were frozen according to standards established by the Artificial Insemination Center located in the Southeast of Brazil. Toluidine blue staining was used for simultaneous evaluation of the sperm chromatin and sperm head morphometry. Chromomycin A3 (CMA3) was applied to analyze sperm protamination and IVP for embryonic development. Spermatozoa of young bulls presented higher values for area (A, pixels), perimeter (P, pixels), and width (W, pixels) compared to adults and senile (young: A = 1848.5 ± 119.79, P = 10.23 ± 0.29, and W = 1.95 ± 0.1; adults: A = 1672.9 ± 104.46, P = 9.86 ± 0.33, and W = 1.81 ± 0.06; senile: A = 1723.1 ± 124.41, P = 9.97 ± 0.33, and W = 1.83 ± 0.09; P < 0.0001) and showed higher protamination deficiency when analyzed by CMA3 (young: 1.57 ± 0.76; adults: 1.09 ± 0.63, and senile: 0.90 ± 0.59; P < 0.05). Likewise, variables of sperm head size (A, P, and W) and protamination assessed by CMA3 showed negative correlation with age and positive correlation with ellipticity, evaluated by toluidine blue method (P < 0.05). Sperm head area was larger in spermatozoa presenting chromatin instabilities than spermatozoa without chromatin alteration (P < 0.0001). There was no difference in IVP when using semen with larger or smaller portions of spermatozoa with chromatin instabilities, indicating that the proportion of sperm with abnormal chromatin compaction (4%–16.15%) did not interfere with early embryonic development. From our results, it can be concluded that sperm of young Nelore bulls have larger heads compared to adults and senile due to reduced protamine content when evaluated by CMA3 and higher proportion of major sperm defects assessed by differential interference contrast microscopy.

A Preliminary Study: N-acetyl-L-cysteine Improves Semen Quality following Varicocelectomy.

Surgery is considered the primary treatment for male infertility from clinical varicocele. One of the main events associated with varicocele is excessive production of reactive oxygen species (ROS). N-acetyl-L-cysteine (NAC), an antioxidant that scavenges free radicals, is considered a supplement to alleviate glutathione (GSH) depletion during oxidative stress. Despite beneficial effects of NAC in other pathological events, there is no report on the effect of NAC in individuals with varicocele. Therefore, the aim of this study is to evaluate the outcome of NAC on semen quality, protamine content, DNA damage, oxidative stress and fertility following varicocelectomy. This prospective clinical trial included 35 infertile men with varicocele randomly divided into control (n=20) and NAC (n=15) groups. We assessed semen parameters, protamine content [chromomycin A3 (CMA3)], DNA integrity [terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)] and oxidative stress [2', 7'-dichlorodihydrofluorescein-diacetate (DCFH-DA)] before and three months after varicocelectomy. Percentage of abnormal semen parameters, protamine deficiency, DNA fragmentation and oxidative stress were significantly decreased in both groups compared to before surgery. We calculated the percentage of improvement in these parameters compared to before surgery for each group, then compared the results between the groups. Only percentage of protamine deficiency and DNA fragmentation significantly differed between the NAC and control groups. The results of this study, for the first time, revealed that NAC improved chromatin integrity and pregnancy rate when administered as adjunct therapy post-varico- celectomy (Registeration Number: IRCT201508177223N5).

Shedding Light on the Nature of Seminal Round Cells.

IntroductionIn this investigation we assess the incidence of round cells (RCs) in semen samples in our infertile patient population and their significance on intracytoplasmic sperm injection (ICSI) cycle outcomes. We also evaluate the usefulness of RCs as indicators of bacterial infection and highlight the origin of this cell-type, as well as its role in the human ejaculate.Patients and MethodsIn a prospective fashion, a total of 4,810 ejaculated samples were included in the study during a period of 24 months. RCs were characterized for white blood cell (WBC) components versus exfoliated germ cells by testing for multiple markers of ploidy as well as protamine assays. Cases displaying ≥ 2 x 106/ml RCs were screened for bacteria. Raw specimens containing RC were processed by peroxidase and other leukocyte assays, specific stains for protamines were used to identify spermiogenic stage, aneuploidy (FISH) assessment was carried out, and the presence of various Sertoli-cell cytoplasmic remnants was analyzed to identify and characterize immature germ cells. The effect of RC on clinical outcome was assessed in specimens used for ICSI.ResultsThe average age of the men involved was 39.2 ± 7 years. Semen samples had a mean concentration of 40.7 ± 31 x 106/ml, motility of 42.6 ± 35%, and morphology of 2.3 ± 2%. RCs were identified in 261 specimens, representing a proportion of 5.4%. Men with RCs had comparable age but lower sperm concentration and morphology than the control group (P<0.001). The aneuploidy rate of 4.3% in RCs group was remarkably higher than the control group (2.3%; P<0.001). Sperm aneuploidy rate positively correlated with the number of RCs (P<0.001). Of 44 men, 17 of them in 18 cycles had up to 1.9 x 106/ml RCs without affecting fertilization and clinical pregnancy rates when compared to controls (n = 365 cycles). In 27 men undergoing 33 ICSI cycles with ≥ 2 x 106/ml RCs, the fertilization rate trended lower and the miscarriage rate was significantly increased (P = 0.05). There was lack of correlation between RC and bacteriological growth. Specific markers indicated that seminal RCs are mostly immature germ cells encased in the remnants of Sertoli cell cytoplasm. Moreover, their modest protamine content and their haploid status confirm that they are post-meiotic. Sequential observation in the same man showed that RC episodes were followed by an amelioration of semen parameters, and interestingly, the episodic occurrence of RCs often coincides with flu season peaks.ConclusionsSeminal RCs are not a marker of infectiousness but rather a transient indicator of spermatogenic insult that possibly occurs in most men following a mild and transient ailment such as the flu.

The effect of tetrabromobisphenol A on protamine content and DNA integrity in mouse spermatozoa.

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant of increasing concern to human health because of its action as an endocrine disruptor. We have previously demonstrated that TBBPA is able to increase apoptosis of testicular cells and other changes in the first and second generations of mice exposed to TBBPA. However, the potential effects of TBBPA on mouse epididymal spermatozoa have not yet been investigated. Therefore, we initiated this study to determine whether TBBPA exposure could also result in increased DNA fragmentation in epididymal spermatozoa and whether it had an effect on the protamines as the major nuclear proteins. C57Bl/6J mouse pups (n=10) were exposed to TBBPA (experimental group) during the gestation, lactation, pre-pubertal and pubertal periods up to the age of 70days as previously described and compared to control mouse pups (n=10) that were not exposed. The results demonstrate that TBBPA treatment results in a significantly decreased protamine 1/protamine 2 ratio (0.362 vs. 0.494; p<0.001), increased total protamine/DNA ratio (0.517 vs. 0.324; p<0.001) and increased number of terminal deoxynucleotidyl transferase dUTP nick end labelling positive spermatozoa (39.5% vs. 21.2%; p<0.05) observed between TBBPA and control mice respectively. These findings indicate that TBBPA exposure, in addition to the resulting increased sperm DNA damage, also has the potential to alter the epigenetic marking of sperm chromatin through generation of an anomalous content and distribution of protamines. The possibility is now open to study whether the detected altered protamine content and DNA integrity are related to the previously observed second-generation effects upon TBBPA exposure.

Evaluation of heat-shock protein A2 (HSPA2) in male rats before and after varicocele induction.

Varicocele is a major cause of infertility and may impair spermatogenesis. This study evaluated the molecular consequences of varicocele on the induction of heat-shock proteins, intracellular chaperones involved in stress responses, and of the ubiquitin-proteasome system, which is participates in the removal of defective sperm in the testis and epididymis. Wistar rats were randomly divided into three groups: surgically induced left varicocele, sham-operated, and untreated controls. Two months after surgery, we observed significantly reduced sperm parameters, DNA integrity, and protamine content in the sperm retrieved from the left epididymis compared to the right epididymis in the varicocele group, as well as compared to sperm retrieved from the left epididymis of the sham and control groups. According to Western blot analysis, we observed significantly higher HSPA2 expression in testicular tissue from the left testis compared to the right testis in the varicocele group or the left testis of the control group. Immunohistochemical analysis showed that expression of HSPA2 was higher in the round spermatid and sperm from the left varicocele compared to the control group. There was normally less HSPA2 expressed in the caput and corpus compared to the cauda of the epididymis in the control group, but this pattern was altered in the caput epididymis of the varicocele group. Levels of ubiquitination were also remarkably lower in the left testis of the varicocele group. Therefore, varicocele impacts expression of HSPA2 and ubiquitination.

Sperm protamine deficiency correlates with spermDNAdamage inBos indicusbulls

The primary purpose of spermatozoa is to deliver the paternal DNA to the oocyte at fertilization. During the complex events of fertilization, if the spermatozoon penetrating the oocyte contains compromised or damaged sperm chromatin, the subsequent progression of embryogenesis and foetal development may be affected. Variation in sperm DNA damage and protamine content in ejaculated spermatozoa was reported in the cattle, with potential consequences to bull fertility. Protamines are sperm-specific nuclear proteins that are essential to packaging of the condensed paternal genome in spermatozoa. Sperm DNA damage is thought to be repaired during the process of protamination. This study investigates the potential correlation between sperm protamine content, sperm DNA damage and the subsequent relationships between sperm chromatin and commonly measured reproductive phenotypes. Bos indicus sperm samples (n = 133) were assessed by two flow cytometric methods: the sperm chromatin structure assay (SCSA) and an optimized sperm protamine deficiency assay (SPDA). To verify the SPDA assay for bovine sperm protamine content, samples collected from testis, caput and cauda epididymidis were analyzed. As expected, mature spermatozoa in the cauda epididymidis had higher protamine content when compared with sperm samples from testis and caput epididymidis (p < 0.01). The DNA fragmentation index (DFI), determined by SCSA, was positively correlated (r = 0.33 ± 0.08, p < 0.05) with the percentage of spermatozoa that showed low protamine content using SPDA. Also, DFI was negatively correlated (r = -0.21 ± 0.09, p < 0.05) with the percentage of spermatozoa with high protamine content. Larger scrotal circumference contributes to higher sperm protamine content and lower content of sperm DNA damage (p < 0.05). In conclusion, sperm protamine content and sperm DNA damage are closely associated. Protamine deficiency is likely to be one of the contributing factors to DNA instability and damage, which can affect bull fertility.

Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment

Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)? Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study. Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown. Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013. Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated. Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P = 0.013), men's age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P < 0.05), embryo quality (P < 0.05) and implantation rate (P < 0.05). A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage. Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE. No personal or direct financial support has been received for any of this work. The authors declare no competing interests. N/A.

Zeta potential vs apoptotic marker: which is more suitable for ICSI sperm selection?

Selection of appropriate sperm is considered as a decision making point in the ICSI procedure. Canonically, sperm selection is based on morphology and motility. Recent advances in this field, have shown that, this procedure can be assisted by further selection based on membrane surface charge (Zeta potential) and surface apoptotic marker (phosphatidylserine externalization) using magnetic activated cell sorter (MACS). Based on the literature, both these procedures improve quality of selected sperm population. Therefore, this study aims to compare the efficiency of these two procedures. Semen samples were collected from 36 fertile and infertile (teratozoospermic and /or asthenozoospermic) individuals. Sperm DNA fragmentation, protamine deficiency and morphology were assessed by TUNEL, CMA3 and papanicolaou staining in unprocessed, MACS and Zeta processed samples. Although both MACS and Zeta were able to separate a higher percentage of sperm with normal morphology, and lower DNA fragmentation and protamine deficiency compared to unprocessed, MACS procedure could significantly isolate, a greater percentage of sperm with normal acrosome and protamine content compared to Zeta procedure. Both MACS and Zeta procedures improve the quality of the selected spermatozoa for ICSI. However, MACS procedure is more efficient in individuals with severe male factor infertility to select sperm with normal acrosome and protamine content but concern regarding transfer of MACS beads into the oocyte remains to be resolved.

Effects of supplement therapy on sperm parameters, protamine content and acrosomal integrity of varicocelectomized subjects.

Surgical repair of varicocele has long been a procedure to correct spermatogenesis. However, the outcome has been reported to be inadequate. We combined varicocelectomy with supplement therapy to evaluate the concurrent effect of these procedures. A prospective randomized controlled study was undertaken to investigate the effects of zinc sulfate, folic acid and zinc sulfate/folic acid on sperm quality, protamine content and acrosomal integrity following surgical repair of varicocele. Male subjects with palpable varicocele were included in the study and randomized into four groups. Subjects received Zinc sulfate, Folic acid, Zinc sulfate/Folic acid or placebo for 6 months. A semen sample was obtained before surgery and 3 and 6 months after surgical repair. Semen samples were evaluated for sperm parameters as well as chromatin content and acrosomal integrity. Most of the evaluated parameters showed a mild improvement after varicocelectomy in the placebo group. Interestingly, co-administration of Zinc sulfate and folic acid improved most factors significantly. Folic acid administration but not zinc sulfate could increase sperm number. Hence, Zinc sulfate was better than folic acid when change in morphology was assessed, and none of them was significantly effective in sperm motility. In Zinc sulfate and Folic acid groups, protamine content and halo formation rate significantly improved. We may conclude that co-administration of zinc and folic acid significantly improved sperm parameters and increased varicocelectomy outcomes. So, medical treatment with compatible drugs after surgery might be advantageous for obtaining acceptable results.

Combination of running exercise and high dose of anabolic androgenic steroid, nandrolone decanoate, increases protamine deficiency and DNA damage in rat spermatozoa

High doses of anabolic-androgenic steroids (AAS) are used by some athletes to increase muscle mass, that is often associated with male infertility. The aim of this study was to investigate the possible cause/s of male infertility using a rat model by analysing sperm quality, including its protamine content and DNA integrity, as well as pregnancy rate. Five groups of male Wistar rats were treated for 10 weeks as follows: nandrolone decanoate (10 mg kg(-1) per week) (ND); running exercise (50 min per day, 5 days a week) (EX); Combination of ND and exercise (ND-EX); nandrolone decanoate solvent (Sham); and control without any injection or exercise (CO). Deterioration in sperm quantity was observed in all test groups (P ≤ 0.01). The frequency of fertile rats was decreased in the ND-EX and ND groups (P ≤ 0.05). Chromomycin-A3 staining showed a protamine deficiency in the epididymal spermatozoa in the ND-EX rats (P ≤ 0.05). Chromatin analysis indicated an abnormal maturation of the sperm nuclei in all test groups compared with the controls (P ≤ 0.05). TUNEL analyses showed a highly significant increase in apoptosis in the EX, ND, and ND-EX groups (P ≤ 0.01). Our data show that a combination of exercise and high doses of nandrolone decanoate negatively influences the DNA integrity and protamine content resulting in lower sperm quality and reduced pregnancy rate.

Correlation of CMA3 Staining with Sperm Quality and Protamine Deficiency

Objective: Protamines cause sperm chromatin to compact and its deficiency is associated with poor fertilizing ability and low quality of spermatozoa. However, the threshold for deficiency has not been well established. The aim of this study was to determine the threshold of sperm protamine content that correlates with diminished sperm quality. Methods: Semen samples were obtained from 238 idiopathic oligoasthenoteratozoospermic (OAT) and 208 normospermic men. Semen was analyzed according to WHO guidelines. Sperm protamine content was assessed by chromomycin A3 (CMA3) staining. Results: The mean level of CMA3 staining was significantly higher in OAT than in men with normal sperm (P<0.001). CMA3 staining correlated negatively with sperm concentration (r=-0.338, P<0.001), motility (r=-0.273, P<0.001), and normal morphology (r=-0.303, P<0.001). Using ROC analysis we showed that 31% of stained sperm represents an appropriate cut-off point for protein deficiency (men with higher than 31% stained sperm are protamine deficient). Conclusions: High odds ratio of OAT men followed by ROC analysis with a cut-off point of 31% for CMA3 staining revealed that this test is a sensitive and specific for evaluating of protamination, maturity, and quality of spermatozoa.