Sperm Chromatin Maturity Research Articles

Age-related changes in human sperm DNA integrity.

Abnormal standard semen characteristics and reduced sperm chromatin maturity can appear with increasing male age. However, the influence of paternal age on semen parameters is still controversial. Therefore, this study was designed to estimate the influence of paternal age not only on conventional semen characteristics but also on sperm DNA integrity. This research was carried out on ejaculated sperm cells obtained from men (n = 1124) aged ≥40 y and <40 y. Our data revealed a decreased semen volume and an increased percentage of DFI (sperm DNA fragmentation index) in older men compared to younger men in the entire study cohort, in men with normozoospermia and in men with abnormal semen parameters. Moreover, there was a higher incidence of sperm DNA damage (>10% DFI, low fertility potential) in the groups of men aged ≥40 y than in the groups of men aged <40 y. Older men had over twice the odds ratio for high sperm DNA damage as younger men. Our findings suggest a detrimental effect of advanced paternal age on sperm chromatin integrity. The data show that the evaluation of sperm DNA has greater clinical utility than standard semen analysis in case of male fertility potential assessment.

The effect of paternal age on semen quality and fertilization outcome in men with normal sperm DNA compaction, reactive oxygen species, and total antioxidant capacity levels.

Studies showed a decrease of the semen analysis parameters and an increase in the average age of first-time fathers over the past several decades. The aim of the present study was to assess the influence of paternal age on semen quality and fertilization outcomes in men with normal sperm DNA fragmentation and chromatin maturation index (DFI and CMI), reactive oxygen species (ROS), and total antioxidant capacity (TAC) levels. The study was performed on 70 men with their wife's age ≤38 years and normal sperm DFI, CMI, ROS, and TAC levels. None of the couples had a history of genital inflammation, chronic diseases, endocrine abnormality, chromosomal aberrations, Y chromosome microdeletion, azoospermia, and leukocytospermia. These men were separated into 2 groups according to their age (group A: age <45 years and group B: age ≥45 years). Semen analysis and fertilization outcome after using the intracytoplasmic sperm injection were assessed in both groups. Sperm concentration showed a significant reduction in group B (p=0.04). Although semen volume, sperm normal morphology, and progressive motility were decreased in group B, the reduction was not significant when compared with group A (p=0.09, p=0.47, and p=0.77, respectively). In addition, the differences of embryo quality with grades A, B, and C and 8-cell embryo formation were not statistically significant between the 2 groups. These results demonstrated that in men with normal sperm DFI, CMI, ROS, and TAC levels, there were no significant changes in semen parameters and fertilization outcomes with an increasing age.

Study of Prognostic Factors affecting on the IUI Outcome of Infertile Patients

Objective: The main objective of the study is to determine of prognostic factors on the pregnancy rate after intrauterine insemination (IUI) of infertile patients. Methods: The study was carried out between January 2014 and October 2014 including one hundred and fifty five infertile men who attended the Fertility Center in Al – Sadr Medical City. Results: The of the study showed that there was statistically significant (p 0.01) was showed with type of infertility. Conclusion: Many factors may influence success rate after IUI treatment procedures such as potential sperm parameters, leukocyte concentration, MDA concentration and sperm chromatin maturity as well as age, infertility duration and smoking. Recommendation: Advance study to determine of the effect female factors on the IUI outcome.

Sperm DNA Fragmentation: Mechanisms of Origin.

Spermatozoa have the task to deliver an intact paternal genome to the oocyte and to support a successful embryo development. The high levels of sperm DNA fragmentation (sDF) found in sub-/infertile men threat human reproduction and health of the offspring. Strategies to prevent the onset of this type of sperm damage are extensively sought.sDF can be induced by factors like lifestyle-related habits, diseases, drugs, aging, infections and exposure to pollutants. At the cell level, all these factors induce sperm DNA breaks by three main mechanisms: apoptosis, impairment of sperm chromatin maturation and oxidative stress. Apoptosis and defects in maturation of sperm chromatin appear to act in the testis and account for DNA breaks found in dead ejaculated spermatozoa, whereas oxidative stress is likely inducing sDF during the transit through the male genital tracts and accounting for DNA breaks observed in viable spermatozoa of the ejaculate. Oxidative stress appears to be also the main mechanism responsible for induction of sDF after ejaculation, during in vitro manipulation of spermatozoa. Whether or not mature spermatozoa are able to trigger a cell death program is not yet clarified. In particular, it is not clear whether apoptotic nucleases or reactive oxygen species are responsible for producing DNA breaks in ejaculated mature spermatozoa. Knowledge of the mechanisms inducing sDF is a valuable starting point to define possible therapeutic options that however are still far to be established.

The use of aniline blue chromatin condensation test on prediction of pregnancy in mild male factor and unexplained male infertility

The aim of this study was to investigate the possibility of using sperm function tests (hypoosmotic swelling test [HOS], aniline blue [AB] staining test, and sperm chromatin dispersion [SCD]) to predict intrauterine insemination [IUI] success rate. A total of 243 couples with mild male factor or unexplained male infertility who underwent IUI were evaluated prospectively. The results of basic sperm analysis and sperm function tests were compared between pregnant or nonpregnant groups. The HOS (11.9±9.6% vs. 10.1±8.5%, p=0.35) and SCD tests (32.9±21.0% vs. 29.9±19.0%, p=0.48) were not significantly different between pregnant (n=22) and nonpregnant (n=221) groups. However, the AB staining negativity rate was significantly higher in the pregnant group compared to the nonpregnant group (35.2±20.8% and 24.4±18.0%, p=0.008). On ROC analysis, a cut-off value of 24% for AB negativity showed a sensitivity and a specificity value of 82.35% and 51.38% (AUC)=0.653; 95% confidence interval: 0.571-0.72 P (Area=0.5)=0.0267, respectively, for prediction of pregnancy. Our study showed that the sperm chromatin maturity, assessed by AB stain, may predict the pregnancy in couples with unexplained female infertility plus mild male factor or unexplained male infertility. The HOS and SCD failed to predict the pregnancy in this group of couples.

Upstream or swim up processing technique: which one is more effective to select human sperm with high chromatin integrity

Sperm processing methods separate motile sperms with good morphology from dead and abnormal forms of sperms, immature germ cells, and non-sperm cells. The propose of this study was to compare the efficacy of upstream and swim-up processing techniques to separate sperms with the high quality especially in relation to sperm chromatin integrity. This experimental study used semen samples from 60 normozoospermic men. Specimens were divided into equal aliquots for processing by swim up (group A), and upstream (group B) methods and compare with control by raw semen (group C). Sperm concentration, morphology, motility, DNA fragmentation and chromatin maturation were measured in these three groups. The results revealed that sperm concentration in the swim up samples was significantly greater than upstream samples (p≤0.04). as addition, motile sperm recovery including the percentage of progressive motility and a total number of motile sperm was better in the swim-up compared to an upstream method and raw semen (p≤0.001). The cell debris and seminal fluid were equally removed by both methods and the percentage of normal forms was also similar in both procedures (p≥0.4). In addition, sperm DNA fragmentation and chromatin maturation were not significantly different between the three groups (p≥0.1). According to results, apparently the upstream method had no significant efficiency to separate good quality sperms compare to swim up. Therefore, swim up seems to be a simple, inexpensive, reliable and widely available method with an efficient yield to separate motile sperm with good morphology and better chromatin integrity for insemination in the infertility clinics.

The effect of human sperm chromatin maturity on ICSI outcomes.

Because sperm chromatin may play a key role in reproductive success, we verify the associations between sperm chromatin abnormalities, embryo development and the ability to achieve pregnancy. The evaluation of sperm chromatin maturity using aniline blue (AB), chromomycin A3 (CMA3) and toluidine blue (TB) staining were carried out in group of males from infertile couples that underwent ICSI. Low levels of sperm chromatin abnormalities (<16%) were found in most subjects (>50%). A higher percentage of TB-positive sperm cells were discovered in the men from couples who achieved≤50% fertilized oocytes compared to men who achieved>50%. No significant differences were discovered by the applied tests between the men from couples who achieved≤50% and those who achieved>50% high-quality embryos on the 3rd or 5th day after fertilization, nor between the men from couples who achieved pregnancy and those who failed. The sperm chromatin maturity did not correlate with the ICSI results. However, the ROC analysis revealed a significant predictive value of TB-positive spermatozoa only for fertilization. Therefore, the TB assay can be considered as a useful test for the prediction of fertilization. Our findings suggest that the level of sperm chromatin abnormalities of the examined men was not clinically significant. No found associations between sperm chromatin maturity and embryo development and the ability to achieve pregnancy. We could not exclude the effects of the repairing processes in the fertilized oocyte. The use of complementary tests that verify the status of the sperm chromatin seems justified.

Impact of diet on sperm characteristics, chromatin maturity and DNA fragmentation

Impact of diet on sperm characteristics, chromatin maturity and DNA fragmentation

Effect of erythrocyte-sperm separation medium on nuclear, acrosomal, and membrane maturity parameters in human sperm.

The purpose of this study is to investigatewhether erythrocyte-sperm separation medium (ESSM) has effects on human sperm motility, morphology, viability, membrane maturity, acrosome integrity, and nuclear attributes before and after cryopreservation. Semen samples from normozoospermic (n = 36) and oligozoospermic (n = 9) patients were analyzed. Samples from the same patient were divided into three aliquots: group 1 and group 2 were resuspended in sperm washing media and ESSM, respectively. Group 3 was resuspended in ESSM with blood sample to mimic the extensive number of erythrocytes in the testicular sperm extraction (TESE) material. All groups were evaluated for sperm concentration, motility, Kruger/Tygerberg strict morphology, viability by eosin-nigrosin staining, membrane maturity by hyaluronic acid-binding assay (HBA), acrosomal integrity by Pisum sativum lectin staining, chromatin maturity by aniline blue staining, and DNA integrity by TUNEL assay before and after cryopreservation. No significant difference was determined between ESSM-treated and ESSM-untreated sperm samples for the sperm parameters tested (p > 0.05). After cryopreservation, total sperm motility and viability decreased regardless of ESSM used. The percentages of sperm with Tygerberg normal morphology, intact acrosome, and HA-bound sperm were found to be lower in oligozoospermic samples before cryopreservation in each group. However, no statistically significant differences were found between oligozoospermic and normozoospermic samples when all groups were compared. Thus, ESSM treatment did not cause a significant change on sperm motility, normal morphology, viability, HA-binding capacity, chromatin maturity, and DNA fragmentation. ESSM can enhance the efficiency of sperm retrieval protocol and can also decrease the time required to collect spermatozoa while not affecting sperm morphogenetic properties.

Chromatin Protamination and Catsper Expression in Spermatozoa Predict Clinical Outcomes after Assisted Reproduction Programs

Identification of parameters predicting assisted reproductive technologies (ARTs) success is a major goal of research in reproduction. Quality of gametes is essential to achieve good quality embryos and increase the success of ARTs. We evaluated two sperm parameters, chromatin maturity and expression of the sperm specific calcium channel CATSPER, in relation to ART outcomes in 206 couples undergoing ARTs. Chromatin maturity was evaluated by Chromomycin A3 (CMA3) for protamination and Aniline Blue (AB) for histone persistence and CATSPER expression by a flow cytometric method. CMA3 positivity and CATSPER expression significantly predicted the attainment of good quality embryos with an OR of 6.6 and 14.3 respectively, whereas AB staining was correlated with fertilization rate. In the subgroup of couples with women ≤35 years, CATSPER also predicted achievement of clinical pregnancy (OR = 4.4). Including CMA3, CATSPER and other parameters affecting ART outcomes (female age, female factor and number of MII oocytes), a model that resulted able to predict good embryo quality with high accuracy was developed. CMA3 staining and CATSPER expression may be considered two applicable tools to predict ART success and useful for couple counseling. This is the first study demonstrating a role of CATSPER expression in embryo development after ARTs programs.

Investigation of the relationship between HA binding and sperm function tests including sperm DNA damage and chromatin maturity

Investigation of the relationship between HA binding and sperm function tests including sperm DNA damage and chromatin maturity

Sperm chromatin maturity and integrity correlated to zygote development in ICSI program

ABSTRACTThis study aimed to evaluate sperm chromatin maturity and integrity of that injected into good-quality oocytes in an in vitro fertilization-intra cytoplasmic sperm injection (IVF-ICSI) program. A cut-off value of sperm chromatin maturity and integrity was developed as a function of their correlation to the zygote development, i.e., embryo formation and cleavage rate. The study assessed sperm chromatin maturity using aniline blue (AB) staining, whereas toluidine blue (TB) staining was used to assess sperm chromatin integrity. Ejaculates from 59 patients undergoing ICSI and 46 fertile normozoospermic donors for determination of normal values of sperm chromatin status were used in this study. Embryo formation and cleavage rates were observed for the period of 3 days after ICSI. There was a significant difference in the percentage of sperm with mature chromatin between ejaculate from ICSI patients and fertile donor (p=0.020); while there was no significant difference in sperm chromatin integrity of both samples (p=0.120). There was no significant correlation between sperm chromatin maturity and either embryo formation or cleavage rate; as well as sperm chromatin integrity to both parameters of zygote development (p>0.05). Furthermore, we found that the cut-off value of sperm chromatin maturity and integrity of the fertile normozoospermic ejaculates were 87.2% and 80.2%, respectively. Using the cut-offs, we found that low sperm chromatin maturity at the level of <87% correlated significantly with the cleavage rate of the zygote (p=0.022; r=0.371); whereas poor sperm chromatin integrity at the level of <80% correlated with embryo formation (p=0.048; r=0,485). In conclusion, this study showed that poor maturity and integrity of sperm chromatin (AB<87% and TB<80%, respectively), could affect zygote development following ICSI.Abbreviations: AB: aniline blue; CMA3: chromomycin A3; ICSI: intra cytoplasmic sperm injection; IVF: in vitro fertilization; PBS: phosphate buffer saline; SPSS: Statistical Package for Social Science; TB: toluidine blue; WHO: World Health Organization

Effect of varicocelectomy on sperm parameters, oxidative stress and Chromatin maturity in seminal fluid of infertile patients with varicocele

This study aimed to determine the effect of varicocelectomy on sperm parameters, oxidant- antioxidant status and chromatin maturity percent. The current study has been conducted on 154 infertile patients complaining from varicocele and varicocelomized men in addition to 25 fertile men as control. The results revealed significant decrease (P

Sperm Chromatin Immaturity Observed in Short Abstinence Ejaculates Affects DNA Integrity and Longevity In Vitro.

BackgroundThe influence of ejaculatory abstinence (EA) on semen parameters and subsequent reproductive outcome is still debatable; hence understanding the impact of EA on sperm structural and functional integrity may provide a valuable information on predicting successful clinical outcome.ObjectiveTo understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status.MethodsThis experimental prospective study included 76 ejaculates from 19 healthy volunteers who provided ejaculates after observing 1, 3, 5 and 7 days of abstinence. Sperm chromatin maturity, DNA integrity and global methylation status were assessed in the neat ejaculate. Sperm motility, DNA integrity and longevity were assessed in the processed fraction of the fresh and frozen-thawed ejaculates to determine their association with the length of EA.ResultsSpermatozoa from 1 day ejaculatory abstinence (EA-1) displayed significantly higher level of sperm chromatin immaturity in comparison to EA-3 (P < 0.05) and EA-5 (P < 0.01) whereas; the number of 5-methyl cytosine immunostained spermatozoa did not vary significantly across groups. On the other hand, in vitro incubation of processed ejaculate from EA-1 resulted in approximately 20 and 40 fold increase in the DNA fragmented spermatozoa at the end of 6 and 24h respectively (P < 0.01–0.001).ConclusionUse of short-term EA for therapeutic fertilization would be a clinically valuable strategy to improve the DNA quality. However, use of such spermatozoa after prolonged incubation in vitro should be avoided as it can carry a substantial risk of transmitting DNA fragmentation to the oocytes.

Effect Invitro Activation on the Spermiograms of ligozoospermic Patients

Objective: The objective of this study was to improve certain sperm function parameters in vitro of oligozoospermic patients and other mild male factors using sperm activation techniques such as swim-up and density gradient. Methodology: The study was carried out between January 2014 and October 2014 including (30) semen samples of oligozoospermic patients who attended the Fertility Center in AL- Sadr Medical City. The liquefied semen was then carefully mixed for few second and the semen was analyzed by macroscopic and microscopic examination. Two method of IUI sperm activation have been used in this study to separate the motile spermatozoa in seminal plasma. Data analyzed using the SPSS version(20) Results: The results of the study showed that there was statistical significant ( p&lt;0.05) decrease in the sperm concentration, leukocyte concentration, malondialdehyde(MDA) concentration and sperm chromatin maturity after activation by both swim-up and density gradient techniques compared with before activation. Also the results was appeared a significant (p&lt;0.05) improvement in progressive motility (A+B) and normal morphology after activation by both techniques while, there was a significant (p&lt;0.05) improvement in progressive motility (A+B) was noticed by using density gradient technique compared to swim-up technique. Conclusions: It was concluded that the density gradient and swim-up techniques were appeared clear improvement in the certain sperm parameters of oligozoospermic patients after activation. Recommendation: The density gradient techniques with intrauterine insemination to treatment Oligozoospermic patients.

Effect of varicocelectomy on sperm parameters, oxidative stress and Chromatin maturity in seminal fluid of infertile patients with varicocele

This study aimed to determine the effect of varicocelectomy on sperm parameters, oxidant- antioxidant status and chromatin maturity percent. The current study has been conducted on 154 infertile patients complaining from varicocele and varicocelomized men in addition to 25 fertile men as control. The results revealed significant decrease (P

Sperm protamine-status correlates to the fertility of breeding bulls.

During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin structure, coinciding with reduced fertility in bulls. These findings are highly significant because they reveal molecular and morphological phenotypes of mammalian spermatozoa that influence fertility.

Detection of single nucleotide polymorphism of holandric genes and association with sperm nuclear maturity in bovines

Implementation of a successful cattle breeding program relies on selection of genetically superior and tested males against various andrological anomalies. In the present project, single stranded conformation polymorphism (SSCP) of holandric genes viz. ubiquitin specific protease, Y-linked (USPY) and RNA binding motif Y-linked (RBMY) was studied for associating with spermatological attributes (sperm chromatin maturity, semen volume, initial motility) in cattle (10 HF crossbred) and buffalo (10 Murrah). The SSCP band patterns revealed species specific variation only in two primer-pairs (US2 and US3) of USPY gene. The maximum and minimum values of percent immature sperm were ranging between 0.392 to 2.586 in cattle and from 0.901 to 4.000 in buffalo. The per cent immature spermatozoa in the ejaculates did not differ significantly either for species or for the categories (acceptable and unacceptable semen donors). The average immature sperm count was the least in buffalo ejaculates (1.383 ± 0.176). The correlation coefficient was found negative between the spermatological parameters (volume and initial motility) and the immature sperm yield in both cattle and buffalo ejaculates. It indicates that the immature sperm production might be negatively associated with semen volume and initial motility of the spermatozoa.

Impact of cigarette smoking on histone (H2B) to protamine ratio in human spermatozoa and its relation to sperm parameters.

Smoking is strongly associated with abnormalities in histone-to-protamine transition and with alteration of protamine expression in human spermatozoa. A proper protamine to histone ratio is, however, essential for sperm chromatin maturity and DNA integrity. Alterations in these sperm nuclear proteins were observed in infertile men. The present prospective study is aimed at evaluating the possible relationship among smoking, semen quality and the histone-to-protamine transition ratio in mature spermatozoa. Histone H2B and protamine 1 (P1) and 2 (P2) were quantified using acid-urea polyacrylamide gel electrophoresis in the spermatozoa of 35 smokers and 19 non-smokers. Levels of lipid peroxidation marker malondialdehyde (MDA) were measured in seminal plasma by thiobarbituric acid assay. Cotinine concentrations were determined in seminal plasma using an enzyme-linked immunosorbent assay. Histone H2B levels in smokers (292.27 ± 58.24 ng/10(6)) were significantly higher (p = 0.001) than that of non-smokers (109.1 ± 43.70 ng/10(6)), besides, a significant difference (p > 0.0001) was found for the P1 and P2 ratio between smokers (1.71 ± 0.071) and non-smokers (1.05 ± 0.033). The H2B/(H2B+P1 + P2) ratio (0.29 ± 0.71) of smokers were significantly higher (p = <0.0001) than that of non-smokers (0.12 ± 0.01). The concentrations of MDA (μm) (7.13 ± 1.15) and cotinine (ng/mL) (60.44 ± 31.32) in seminal plasma of smokers were significantly higher (p = 0.001) than those in the samples of the non-smoker group (4.42 ± 1.16 and 2.01 ± 2.84 respectively). In addition, smokers showed significantly (p ≤ 0.002) lower sperm count, motility (p = 0.018), vitality (p = 0.009) and membrane integrity (p = 0.0001) than non-smokers. These results reveal that patients who smoke possess a higher proportion of spermatozoa with an alteration of the histone to protamine ratio than patients who do not smoke, and suggest that cigarette smoking may inversely affect male fertility.

The association of seminal plasma antioxidant levels and sperm chromatin status with genetic variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) in infertile men with oligoasthenoteratozoospermia.

In this study we aimed to examine the effects of genetic variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) on GST activity, seminal oxidative stress and sperm chromatin status in infertile men with oligoasthenoteratozoospermia (OAT). The study population (n = 121) consisted of 95 infertile men with OAT and 26 controls with normozoospermia. Multiplex polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were utilized to detect the aforesaid genetic variants. We measured GST activity and total antioxidant capacity (TAC) of seminal plasma by spectrophotometry. Sperm chromatin integrity and maturity were assessed using toluidine blue and chromomycin A3 (CMA3-positive sperm) staining, respectively. The analysis showed that subgroups of GSTM1 null and GSTP1 C/T+T/T genotypes in comparison with GSTM1 present and GSTP1 wild type (C/C) genotypes did not have statistically significant differences in both OAT or normozoospermic men considering sperm concentration and motility, percentage of CMA3-positive sperm, seminal plasma TAC, sperm chromatin integrity and GST activity. Thus, the findings of our study suggest that there are no significant associations between GSTM1 and GSTP1 polymorphisms and sperm parameters at conventional or at molecular levels including OS status, sperm chromatin integrity or maturity in Iranian infertile men with OAT and normozoospermia. However, these polymorphisms could be related to the fertility status of the studied population but not evaluated in this study.