chromomycin-a3 and Infertility--Male

To investigate testis-specific histone 2B (TSH2B) and its gene anomalies in infertile men.. Case-control study.. Basic science laboratory.. Fertile and infertile men.. Not applicable.. The histone and protamine status of sperm was studied by aniline blue and chromomycin A3 staining, respectively. Testis-specific histone 2B, total H2B, and phosphorylated TSH2B (pTSH2B) were estimated by Western blot analysis. The frequency of genetic polymorphisms and rare variants in H2BC1 was studied by Sanger sequencing. Phosphosites on TSH2B in sperm were identified by reverse-phase high-performance liquid chromatography purification of TSH2B followed by mass spectrometric analysis.. Aniline blue and chromomycin A3 staining revealed significantly higher histone retention and low protamine in sperm of infertile men. Sperm TSH2B and total H2B levels were significantly lower in oligozoospermic and oligoasthenozoospermic men (in both groups). The TSH2B levels were comparable in asthenozoospermic men; however, the pTSH2B level was significantly low. The H2BC1 gene sequencing identified 6 variants, of which 2 are rare variants (rs368672899 and rs544942090) and 4 (rs4711096, rs4712959, rs4712960 and rs4712961) are single nucleotide polymorphisms. Minor allele frequency of 5'-untranslated region variant rs4711096 was significantly lower in infertile men (OR = 0.65). The rare nonsynonymous variant, rs368672899, p.Ser5Pro was seen in 1 oligoasthenoteratozoospermic individual. Interestingly, mass spectrometric analysis identified a site on TSH2B to bear a phosphate group in the sperm of fertile men.. Our study reveals a defect in the replacement of somatic histones with testis-specific variants in infertile men. Chromatin compaction positively correlates with sperm motility, which is suggestive of its utility in diagnostic semen analysis of infertile individuals. Our observations with TSH2B and its cognate gene in sperm of infertile men indicate an essential role for TSH2B in meiosis and its phosphorylation in sperm motility, respectively.

Topics: Case-Control Studies; Chromomycin A3; Histones; Humans; Infertility, Male; Male; Meiosis; Protamines; Proteomics; Semen; Sperm Motility; Testis

What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes.. The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells.. Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation.. We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells.. We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa.. CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation.. The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique.. CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes.. The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare.. N/A.

Topics: Child; Chromomycin A3; Fertilization; Humans; Infertility, Male; Male; Semen; Spermatozoa

The absence of the acrosome causes the situation which is called globozoospermia. There are a few studies, mostly as case reports, about correlation between levels of sperm DNA damage in patients with total round-headed spermatozoa. We investigated this correlation as well as CMA3 positive spermatozoa in 20 globozoospermic men (with more than 90% round-headed spermatozoa) attending to Royan Institute. Semen samples divided into three parts to semen analysis, to measure DNA fragmentation index (DFI) using sperm chromatin structure assay (SCSA) and to detect CMA3(+) sperm cells by chromomycin A3 staining and fluorescent microscopy. Our results showed that there were significant differences in sperm concentration, total sperm motility, and normal morphology between patients and controls group (p < 0.001). Moreover, the average of DFI and CMA3 positive spermatozoa in patients group significantly increases compared with control group (p < 0.001). A significant correlation between DFI and CMA3(+) in total population was also detected in patients group (r = 0.45, p = 0.046). To our knowledge, this is the largest study about correlation between DNA damage levels and CMA3 positive spermatozoa with round head sperm cells in total globozoospermic men. It seems that the increase in DNA damage may be because of defective sperm DNA compaction, as we detected CMA3 positive sperm cells in these patients.

Topics: Acrosome; Chromatin; Chromomycin A3; DNA; DNA Fragmentation; Humans; Infertility, Male; Male; Protamines; Semen Analysis; Sperm Count; Sperm Head; Sperm Motility; Spermatozoa

Seminal proteins can be considered as factors that control fertilization. Clusterin is one such protein that has been implicated in many activities, including apoptosis inhibition, cell cycle control, DNA repair, and sperm maturation. In this study, the relationship between human secretory clusterin (sCLU) in seminal plasma with sperm parameters, protamine deficiency, and DNA fragmentation was investigated. Semen samples were collected from 63 Iranian men, and semen analysis was performed according to World Health Organization criteria and computer aided semen analysis (CASA). The concentration of sCLU in seminal plasma was measured by enzyme-linked immunosorbant assay (ELISA), protamine deficiency was determined by chromomycin A3 staining (CMA3 ), and sperm DNA fragmentation was checked by sperm chromatin dispersion (SCD) assay. The level of sCLU in seminal fluid of fertile patients was 48.3 ± 38.6 ng/ml and in infertile patients was 15.5 ± 9.7 ng/ml; this difference was significant (P < 0.001). sCLU correlated negatively with protamine deficiency, sperm DNA fragmentation, and abnormal morphology. In conclusion, seminal clusterin can be considered as a marker for the quick assessment of semen quality in male infertility studies.

Topics: Biomarkers; Chromatin; Chromomycin A3; Clusterin; DNA Fragmentation; Enzyme-Linked Immunosorbent Assay; Humans; In Situ Hybridization, Fluorescence; Infertility, Male; Iran; Male; Protamines; Semen; Semen Analysis; Spermatozoa

Normal chromatin condensation is important for sperm fertilising ability. However, routine semen analysis does not identify defects in sperm chromatin structure. This study aimed to investigate the condensation of chromatin and DNA integrity in spermatozoa of infertile men and deduce the relationship with sperm quality, as measured by conventional semen parameters. Semen analysis was carried out to assess sperm quality according to World Health Organization criteria. The remaining aliquot of each sample was processed for transmission electron microscopy, chromomycin A3 (CMA3) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays. The ultrastructural analysis of spermatozoa from infertile men showed heterogeneity in sperm nuclear morphology. Some spermatozoa displayed a round nucleus with incomplete chromatin condensation. Immunoreactivity with antitransitional protein and antiprotamine antibodies indicated nuclear maturation defects in the spermatozoa of infertile men. Spearman's correlation analysis indicated a positive correlation between the percentages of CMA3- and TUNEL-positive spermatozoa. In addition, these two parameters were negatively correlated with sperm concentration, motility and normal morphology. This study demonstrated that men with morphologically normal spermatozoa of <30% have greater degree of protamine deficiency and DNA damage than men with morphologically normal spermatozoa of >30%. Evaluation of chromatin integrity appears to be a useful tool for assessing male fertility.

Topics: Adult; Chromatin; Chromatin Assembly and Disassembly; Chromomycin A3; DNA; DNA Damage; Humans; In Situ Nick-End Labeling; Infertility, Male; Male; Middle Aged; Protamines; Semen Analysis; Sperm Count; Spermatozoa

Sperm that bypass natural apoptosis and the ubiquitin-proteasome system may find their way into semen. In order to avoid the insemination of such sperm during an intracytoplasmic sperm injection (ICSI) treatment, novel sperm selection procedures such as the Zeta procedure have been implemented. Therefore, the aim of this study was to evaluate extent of ubiquitination and external phosphatidylserine (EPS) in sperm populations selected by combines density gradient centrifugation (DGC) and Zeta electric potential in comparison to DGC and neat semen samples.. Semen samples were collected from 51 infertile men and divided into control, DGC and DGC-Zeta groups. Semen analysis was carried out according to World Health Organization criteria. The percentages of protamine deficiency, DNA fragmentation, EPS and ubiquitinated sperm were assessed by chromomycin A3 (CMA3), TUNEL, Annexin V, and immunostaining, respectively.. Sperm selected by the DGC-Zeta procedure presented a lower percentage of sperm with protamine deficiency, abnormal morphology and DNA fragmentation while the percentage of annexin V and ubiquitin-positive sperm increased.. The results of this study reveal that, DGC-Zeta improves the quality of the selected spermatozoa for ICSI and increases ubiquitination and EPS rates. We propose these alterations are part of the normal physiological process of capacitation.

Topics: Annexin A5; Centrifugation, Density Gradient; Chromomycin A3; DNA Fragmentation; Humans; Infertility, Male; Male; Phosphatidylserines; Protamines; Semen; Sperm Injections, Intracytoplasmic; Spermatozoa; Ubiquitin; Ubiquitination

The present study was undertaken to evaluate the effects of morphokinetic abnormalities of human spermatozoa on chromatin packing and DNA integrity and possible beneficial effects of sperm selection in ICSI.. Semen samples from 1002 patients were analysed for morphology and motility using CASA. Protamine status and DNA fragmentation were analysed by chromomycin A3 staining and sperm chromatin dispersion assay respectively.. Sperms with elongated, thin, round, pyri, amorphous, micro and macro forms were significantly higher in teratozoospermic and oligoasthenoteratozoospermic groups. Significant difference in chromatin packing and DNA fragmentation index was observed in these abnormal groups compared with normal. Similarly significant correlation was also seen between abnormal motility parameters and DNA fragmentation index in asthenozoospermic group compared with normal.. Specific abnormal morphological forms have higher incidence of chromatin packing abnormalities and DNA fragmentation. Using these sperms in ICSI might have an impact on fertilization, embryo development and abortion rates. These can be selectively avoided during ICSI procedure to improve ART outcome.

Topics: Adult; Antigens, Neoplasm; Chromatin; Chromomycin A3; DNA Fragmentation; Embryonic Development; Female; Humans; Infertility, Male; Male; Middle Aged; Pregnancy; Protamines; Sperm Injections, Intracytoplasmic; Spermatozoa

Failed fertilization post-ICSI has been mainly attributed to the sperm's inability to induce oocyte activation. Phospholipase C zeta (PLCζ) is considered to be one of the factors for the induction of oocyte activation. The aim of this study was to quantitatively assess the expression of PLCζ in globozoospermic men or those with previously low or failed fertilization in comparison with fertile men or those with high fertilization potential. In addition, the relationship between expression of PLCζ and that of other sperm markers was evaluated.. Real-time PCR was carried out to evaluate relative expression of PLCζ mRNA. Chromatin maturity and acrosin activity were assessed by CMA3 staining and a colorimetric method.. The expression of PLCζ was significantly lower in globozoospermic men (P< 0.01, n= 8) or individuals with previously low or failed fertilization (P< 0.01, n= 36) in comparison to fertile men (n= 24). In addition, a significant difference was observed between globozoospermic (P< 0.01) and individuals with previously low or failed fertilization (P= 0.003) in comparison to high fertilization individuals (n= 17). Expression of PLCζ was not correlated with either chromatin maturity or acrosin activity. However, a significant correlation was observed between the percentage of fertilization and relative expression of PLCζ (r= 0.4, P< 0.01).. In this study, for the first time, we have shown that assessment of relative expression of PLCζ may provide a useful marker for the ability of sperm to induce oocyte activation after ICSI.

Topics: Acrosin; Adult; Chromatin; Chromomycin A3; Fertility; Fertilization; Humans; Infertility, Male; Male; Oocytes; Phosphoinositide Phospholipase C; Protamines; Real-Time Polymerase Chain Reaction; Semen; Spermatozoa

To compare the extent of sperm DNA damage with the degree of protamine deficiency in spermatozoa of normal and subfertile individuals, 30 semen samples from three groups of subfertile individuals (oligozoospermic, asthenozoospermic and oligoasthenozoospermic) and 14 samples from normal individuals were collected from men referred to the Fertility and Infertility Centre of Shariati Hospital, Tehran. DNA damage was measured using the alkaline Comet assay, and protamine deficiency was measured using chromomycin A3 (CMA3) staining. Results indicated a significant difference in the extent of DNA damage in spermatozoa of subfertile patients compared with normal patients (P < 0.01). Spermatozoa from oligoasthenozoospermic patients showed a higher level of DNA damage compared with the other two study groups of subfertile men. The percentage of CMA3-positive spermatozoa was also higher in subfertile individuals compared with normal patients (P < 0.01), with the highest level occurring in oligoasthenozoospermic patients. A direct correlation between protamine deficiency and sperm DNA damage was found for all subfertile patients studied.

Topics: Adult; Chromomycin A3; Comet Assay; DNA Damage; Humans; Infertility, Male; Iran; Male; Protamines; Spermatozoa; Statistics, Nonparametric

Although the incidences of testicular cancer and Hodgkin's lymphoma have increased in young men over the past decade, combination chemotherapy has improved survival. As fertility is of importance to these patients, characterization of sperm chromatin structure is needed. We assessed sperm chromatin in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy, in comparison with control community and idiopathic infertile volunteers.. DNA damage was assessed with the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and comet assays; reactive thiols (SH) and DNA compaction were determined with the monobromobimane (mBBr) and chromomycin A3 (CMA3) assays, respectively.. Both testicular cancer (37%) and Hodgkin's lymphoma (81%) patients had normospermic samples with increased DNA damage, compared with controls. Cancer patients also had higher reactive thiols and CMA3 staining, indicating low DNA compaction.. Sperm DNA integrity and compaction were affected in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy. Although SCSA, TUNEL and comet assays all detected DNA damage, the latter was optimal for use in cancer patients. A combination of the comet assay with tests that evaluate sperm DNA compaction, such as flow cytometry-based CMA3 and mBBr assays, is a reliable strategy to characterize sperm chromatin quality in cancer patients at the time of sperm banking.

Topics: Adult; Bridged Bicyclo Compounds; Chromatin; Chromomycin A3; Cohort Studies; Comet Assay; DNA Damage; Flow Cytometry; Hodgkin Disease; Humans; In Situ Nick-End Labeling; Infertility, Male; Male; Middle Aged; Sensitivity and Specificity; Spermatozoa; Testicular Neoplasms

The compaction of human sperm chromatin is the result of replacement of approximately 85% of histones with protamines. Germ-line testis/sperm-specific histone 2B (TSH2B) has been detected in only approximately 30% of mature spermatozoa. Its level in the semen of subfertile patients varies; its function is unknown. We evaluated TSH2B in the sperm samples of 23 donors and 49 subfertile patients and assessed its association with chromatin compaction status.. TSH2B level was measured using immunoblotting. Chromatin packaging quality was evaluated by staining with chromomycin A3 (CMA3) which marked spermatozoa with defective packaging. To assess both TSH2B and chromatin status in the same spermatozoon, CMA3 staining and TSH2B immunolocalization were performed sequentially.. A significant correlation (r = 0.55, P = 0.0027) was found between TSH2B level and percentage of CMA3-positive sperm in patient and donor semen samples. When individual spermatozoa were assessed for these parameters, 92% of TSH2B-containing cells were also CMA3 positive. Variation in the total sperm TSH2B level was less in donors than in patients.. CMA3 positive staining of TSH2B-containing individual spermatozoa and a significant correlation between the total TSH2B level and CMA3 percentage in semen samples suggest a structural role for TSH2B in sperm chromatin organization. Low variability of TSH2B level in donors implies a mechanism (however unknown) regulating this parameter.

Topics: Chromatin; Chromatin Assembly and Disassembly; Chromomycin A3; Fluorescent Antibody Technique; Histones; Humans; Infertility, Male; Male; Protamines; Semen; Spermatozoa; Testis; Tissue Donors

Our purpose was to investigate the association between percentage chromomycin A3 (CMA3) positivity of spermatozoa with some sperm parameters and in vitro fertilization rate.. Spermatozoa were collected from 139 men, washed in PBS, fixed in methanol/glacial acetic acid (3:1), and then spread on slides. CMA3 positivity is expressed as the percentage in 200 spermatozoa.. Percentage of CMA3 positivity showed not only a negative correlation with fertilization rate but also a significant difference between fertilizing and nonfertilizing patients. Moreover, percentage of CMA3-positive spermatozoa showed a negative correlation with count and percentage motility and a positive correlation with percentage of abnormal morphology. Percentage of CMA3 positivity also had a positive correlation with some abnormalities of head such as amorphous and macrocephaly. Ultrastructural study showed chromatin unpackaging in high CMA3-positive semen samples in comparison with low CMA3-positive semen samples.. There is a close relationship among fertilization rate, sperm parameters, and CMA3 positivity and CMA3 could be considered as a useful tool for evaluation of male fertility prior to infertility treatment.

Topics: Chromatin; Chromomycin A3; Female; Fertilization in Vitro; Humans; Infertility, Male; Male; Sperm Count; Sperm Motility; Spermatozoa; Staining and Labeling

The nick translation and terminal transferase assays have been compared to test their relative efficiency in detecting DNA breakage in ejaculated human spermatozoa. The results have been correlated with the percentage of chromomycin A3 positive sperm, a fluorochrome that is indicative of the protamination state of sperm. Examination of the ejaculated sperm of 30 subjects revealed that the percentage of positivity to the nick translation and terminal transferase assays did not differ, even when using different fixatives. It is concluded that the inability of the two assays to distinguish the type of DNA damage, as is possible in somatic nuclei, is most probably linked to the unique nature of sperm chromatin. It is proposed that the presence of the damaged DNA may be the remnants of an imperfect spermiogenesis, probably related to an inadequate protamine deposition. This is supported by the strong correlation between the presence of DNA damage and underprotamination as evidenced by chromomycin A3.

Topics: Chromomycin A3; DNA Damage; Ejaculation; Fluorescent Dyes; Genetic Techniques; Humans; Infertility, Male; Male; Spermatozoa

This study aimed to investigate the association between anomalies in sperm chromatin packaging, morphology and fertilization in patients undergoing routine in-vitro fertilization (IVF) or subzonal insemination (SUZI). Sperm chromatin packaging was assessed using chromomycin A3 (CMA3), a fluorochrome specific for guanine-cytosine rich sequences of DNA. One hundred to 150 sperm cells were assessed in 55 patients to compare sperm chromatin packaging and morphology to fertilization after IVF or SUZI. When the morphology and CMA3 fluorescence of individual spermatozoa was assessed, > 75% of the macrocephalic sperm fluoresced in all patients. In contrast, a mean of 37% of the spermatozoa with normal morphology fluoresced in IVF patients compared with 58% of the normal spermatozoa in male factor patients treated by SUZI. SUZI patients displaying a high fluorescence (> 70%) in their spermatozoa also had a significantly lower fertilization rate. Lower packaging quality in morphologically normal spermatozoa may represent a major limiting factor in the fertilizing ability of male factor patients. This study confirms that a high percentage of CMA3 positivity is present in certain forms of male factor infertility and that such a test may be used to distinguish separate populations in morphologically normal spermatozoa.

Topics: Chromatin; Chromomycin A3; Embryo, Mammalian; Female; Fertilization in Vitro; Humans; Infertility, Male; Insemination, Artificial; Male; Microscopy, Fluorescence; Pregnancy; Sperm Motility; Spermatozoa; Treatment Outcome

A major event in enhancing sperm chromatin stability is the replacement of the histones by protamines during spermiogenesis. In this study, we present results indicating that chromomycin A3 (CMA3) can be used to show protamine deficiency in sperm chromatin. Fixed chromatin of mature mouse spermatozoa showed high fluorescence after treatment with ethidium bromide (EB), but was completely unstained after treatment with CMA3. The same chromatin was found to be highly resistant to in situ nick-translation. In contrast, a substantial fraction of human spermatozoa were positive for CMA3. The accessibility of CMA3 to the DNA of human sperm was eliminated if the slides were previously treated with protamine in situ. This treatment did not affect the accessibility of EB to the chromatin. Individual human sperm samples revealed a substantial frequency of spermatozoa with endogenous nicks, which was found to be the same as the frequency of spermatozoa responding positively to CMA3 staining. Treatment of preparations with protamines prevented the identification of the endogenous nicks. These data as a whole suggest that CMA3 could represent a useful tool for the detection of protamine deficiency in sperm chromatin. Furthermore, confirmation of experiments relating sensitivity to nick translation and positivity to CMA3 may allow an indirect in situ visualization of nicked and partially denatured DNA, which could correlate with certain forms of male factor infertility.

Topics: Animals; Chromatin; Chromomycin A3; DNA; DNA Damage; Ethidium; Fluorescent Dyes; Genetic Techniques; Humans; In Vitro Techniques; Infertility, Male; Male; Mice; Protamines; Spermatogenesis; Spermatozoa