Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions

The aim of this study was to examine the possible relationship between sperm DNA integrity and chromatin packaging evaluated by cytochemical assays, traditional sperm parameters and recurrent spontaneous abortion (RSA) of unknown origin. In this cohort study, 40 couples with a history of RSA and 40 couples with proven fertility were considered as case and control groups respectively. The semen samples of all husbands were analysed for sperm parameters and also sperm chromatin and DNA integrity assessed using cytochemical tests including aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), acridine orange (AOT) and nuclear chromatin stability assay. Among different sperm parameters, only slow motility was significantly different between the two groups. In sperm chromatin evaluations, there were significant differences between the two groups in all of the tests. In addition, the majority of semen samples in RSA patients exhibited upper percentages of abnormal spermatozoa than the cut-off values regarding different cytochemical assays. Our study showed that in the cases of RSA, slow motility had a significant reduction in comparison with controls and also spermatozoa of men from RSA group had less chromatin condensation and poorer DNA integrity than spermatozoa that obtained from fertile men with no history of RSA.     

Extra-epididymal spermatozoa express nuclear abnormalities

Extra-epididymal spermatozoa account for approximately a third of all spermatozoa found in the normal human ejaculate. Whilst remaining outside of the testes at core body temperature, the functional competence of spermatozoa, including cell motility and fertilizing capacity, diminishes. By examining spermatozoa found in the seminal fluid of recently vasectomized men, this study has investigated the nuclear changes that occur in spermatozoa whilst persisting in sites distal to the epididymis. Spectral recordings of spermatozoa stained with the nucleic acid dye, toluidine blue and the sperm chromatin structure assay (SCSA) were performed. Toluidine blue staining of human sperm DNA is an effective predictor of abnormal protamine disulphide crosslinking and chromatin condensation. Using flow cytometry, the SCSA determines the sensitivity of sperm DNA to acid-induced denaturation, providing a measure of chromatin and DNA damage. Abnormal protamine disulphide crosslinking and chromatin condensation was significantly higher in spermatozoa from patients after vasectomy when compared to normozoospermic controls (p < 0.01). Additionally, spermatozoa from vasectomized donors were significantly more sensitive to acid-induced denaturation than were normozoospermic donors (p < 0.05). The results indicate that spermatozoa surviving in extra-epididymal sites are more likely to possess DNA and chromatin abnormalities than those present in the testes and epididymis. These changes may partly explain the depletion of cell viability and fertilizing capacity of extra-epididymal spermatozoa which has been reported previously.     

Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests

Varicocele occurs in approximately 15% to 20% of the general male population and it is the most common cause of poor semen production and decreased semen quality. It has been demonstrated that patients with varicocele have a significantly higher DNA fragmentation index (DFI) and spermatozoa with nuclear anomalies than healthy fertile men. Therefore, the aim of this study was to evaluate sperm chromatin integrity in these patients. Sixty men referring to the andrology laboratory were categorised into three different groups: 20 infertile men with varicocele, 20 infertile men with abnormal semen parameters and 20 fertile men who had normal spermatogram were considered as control group. Semen analysis was performed according to WHO criteria. To evaluate sperm chromatin quality and DNA integrity, after fixation of sperm smears, aniline blue, toluidine blue, chromomycin A₃ and acridine orange staining were applied in three groups. The slides were analysed by light and fluorescent microscopy and to determine the percentage of mature or immature spermatozoa, 200 spermatozoa were counted in each slide. The results showed that the rates of aniline blue-reacted spermatozoa were significantly higher in infertile and varicocele patients than in the normal group ( P  < 0.001). In addition, with regard to chromomycin A₃, acridine orange and toluidine blue staining, there was a significant difference between the three groups ( P  < 0.001). The results showed that the varicocele samples contain a higher proportion of spermatozoa with abnormal DNA and immature chromatin than those from fertile men as well as infertile men without varicocele. Therefore, varicocele results in the production of spermatozoa with less condensed chromatin and this is one of the possible causes of infertility due to varicocele.     

A human morphologically normal spermatozoon may have noncondensed chromatin

According to numerous assisted reproductive medicine practitioners, semen with normal characteristics might not require further investigation. However, on the scale of the individual spermatozoon, it is well known that normal morphology does not guarantee optimal nuclear quality. Here, for 20 patients with normal sperm characteristics and a high proportion of spermatozoa with noncondensed chromatin, we subsequently assessed chromatin condensation status (aniline blue staining) and morphology (Papanicolaou staining) of the same 3749 spermatozoa. Although the overall proportion of morphologically normal spermatozoa was not correlated with the overall proportion of spermatozoa with noncondensed chromatin, an individual spermatozoon's morphology appeared to be closely related to its chromatin condensation status. Morphologically normal spermatozoa with noncondensed chromatin were seen in all patients; the proportion averaged 23.3% [min 10.9%–max 44.4%]. Morphologically abnormal spermatozoa were more likely to have noncondensed chromatin than morphologically normal ones (P < 0.0001). Small‐, large‐ or multiple‐headed spermatozoa presented the highest degree of noncondensation (>80% for each type), and more than half the vacuolated spermatozoa also presented noncondensed chromatin. However, a morphologically normal spermatozoon may also have a noncondensed chromatin.     

Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests,and assisted reproduction techniques

The main purpose of this prospective study is to examine possible influences of abnormalities of sperm nuclear condensation and chromatin decondensation with sodium dodecyl sulphate (SDS)‐EDTA on outcomes of intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI) cycles. Semen samples from 122 IUI and 236 ICSI cycles were evaluated. Before semen preparation for IUI or ICSI, basic semen analysis was performed and a small portion from each sample was spared for fixation. The condensation of sperm nuclear chromatin was evaluated with acidic aniline blue, followed by sperm chromatin decondensation by SDS‐EDTA and evaluation under light microscope. Ongoing pregnancy rate was 24% and 26.2% in the IUI and ICSI groups respectively. The chromatin condensation rate was significantly higher in the ongoing pregnancy‐positive group compared to the negative group, both in IUI (P = 0.042) and ICSI groups (P = 0.027), and it was positively correlated with ongoing pregnancy rate in both IUI and ICSI groups (P = 0.015, r = 0.214 and P = 0.014, r = 0.312 respectively). Chromatin decondensation rates were not significantly different in neither of the groups. These results indicate that IUI and ICSI outcome is influenced by the rate of spermatozoa with abnormal chromatin condensation. Sperm chromatin condensation with aniline blue is useful for selecting assisted reproduction techniques (ART) patients.     

Effect of freeze–thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men

Cryopreservation is known to impair sperm motility and decrease the fertilization rate by detrimental effects on acrosomal structure and acrosin activity. However, the consequences of cryopreservation on the integrity of the sperm nucleus, chromatin stability and centrosome are less clear. The present study was designed to determine the effect of the freeze-thawing procedure on chromatin condensation (aniline blue staining) and the morphology (strict criteria) and membrane integrity of human spermatozoa. The structural and functional characteristics of the sperm plasma membrane were measured by the eosin-test and hypo-osmotic swelling test which were done separately. Sperm cryopreservation was performed on semen samples from two groups of men classified as fertile (n = 20) and subfertile (n = 72), based on their reproductive history and semen analysis according to WHO guidelines. The mean percentage of condensed chromatin, morphologically normal spermatozoa and membrane integrity in all semen samples investigated (n = 92) decreased significantly (p = 0.0001) after freeze-thawing, in comparison to the value observed prior to freezing. By comparing the semen samples between fertile and subfertile patients, significantly (p = 0.0009) greater damage was demonstrated in the subfertile than in the fertile group. Furthermore, no significant difference was observed between the two groups with regard to the morphological alteration and structural as well as functional damage of the sperm membrane. In conclusion, the freeze-thawing procedure significantly affects chromatin structure and sperm morphology, especially in the head and the tail regions, and this may explain the lower fertilization rate and IVF/ICSI outcome when frozen-thawed spermatozoa are used. In addition, this study demonstrates that chromatin condensation is a sensitive parameter for the evaluation of cryodamage of semen samples from fertile and subfertile patients, though subfertile patients with very poor semen characteristics have yet to be studied. It is therefore recommended that chromatin condensation be used as an additional parameter for the assessment of sperm quality after freeze-thawing.     

Use of acridine orange to evaluate chromatin integrity of human spermatozoa in different groups of infertile men

To study the sperm chromatin compactness various methods, such as acidic aniline blue or acridine orange staining, have been applied. Due to its metachromatic properties, acridine orange dye fluoresces green with double- and red with single-stranded DNA. Samples (n = 181) were evaluated and grouped as follows: group I, normal recently fertile; group II, male having female partner with repeated early pregnancy loss; group III, male with varicocele; and group IV in-vitro fertilization and intrauterine insemination failures. Routine semen analyses were carried out in all the cases. Amorphous particulate matter as observed under phase contrast microscope was graded on the scale of nil to +4. Fixed smears were stained with an aqueous solution of acridine orange and viewed under a fluorescence microscope. Two hundred cells were counted and the percentage of fluorescence calculated. Groups II, III and IV exhibited significantly low green fluorescence compared with the control group. The study also indicates that increased amorphous particulate matter (indicating infection) might be one of the contributing factors to lower acridine orange stainability. Thus acridine orange staining can be used to evaluate the integrity of the nucleus, disorders of which can cause unexplained infertility or lower fertilization potential that may go undetected by routine analysis.     

Human sperm chromatin undergoes physiological remodeling during in vitro capacitation and acrosome reaction

Capacitation (CAP) and acrosome reaction (AR) are sequential processes of sperm activation. Beside the known ionic, membrane, and transduction events and final release of proteolytic enzymes that help sperm movement toward the egg, chromatin changes, such as a physiological remodeling, are also possible. Our aims were to ascertain that CAP and AR do not induce DNA damage and to evaluate changes occurring in the human sperm head during these physiological processes using cytochemical stains. Percollpurified spermatozoa from normal donors were incubated in Biggers, Whitten, and Whittingham medium ± fetal cord serum ultrafiltrate (CAP inducer) and then with lysophosphatidylcholine (AR inducer). CAP and AR were associated with increases in aniline blue (AB, for histones; ～70%) and toluidine blue (TB, for chromatin compaction; ～40%) staining but had no influence on that of chromomycin A3 (for protamines). The increase (～40%) in iodoacetamide-fluorescein (IAF, for sulfhydryl groups) staining observed during CAP was absent after AR. CAP and AR did not damage DNA (percentage of DNA fragmentation index remained low) nor affect histone content. CAP, and even more AR, primed sperm heads to decondense (～80% and ～140% increases, respectively) when challenged with sodium dodecyl sulfate + dithiothreitol. Interestingly, induced decondensation correlated with all other tests (CAP, AB, TB, and IAF). Therefore, the data strongly support a physiological remodeling of nondamaged human sperm chromatin during CAP and AR, and modifications are probably interlinked and help prepare chromatin for postfertilization events.     

Evaluation of gram stain as an alternative in the assessment of human spermatozoa quality

INTRODUCTION: During spermiogenesis, protaminosis and sperm chromatin condensation are important prerequisites for the preservation of DNA integrity in spermatozoa. The aim of this study is to assess Gram stain as an alternative technique for the evaluation of human sperm chromatin condensation status. Patients and METHODS: Aniline blue and Gram staining were applied to semen samples from 34 donors in order to determine the relationship between sperm chromatin condensation and infertility. In addition, the possible correlation between morphology and vitality (eosin-Y staining) of spermatozoa compared with their nuclear status (aniline blue and Gram staining) was studied. RESULTS: Chromatin condensation and sperm vitality were significantly higher in fertile men compared to the subfertile. A significant correlation was found between chromatin condensation and (a) sperm vitality (p < 0.01), and (b) nuclear protein status (p < 0.01). CONCLUSIONS: Gram staining may be used as a routine method in assisted reproduction laboratories and could assist in the evaluation of sperm quality as well as in the selection of the appropriate fertilization technique.     

Effect of human sperm chromatin anomalies on fertilization outcome post-ICSI

The aim of this study was to evaluate the effect of sperm chromatin anomalies on fertilization outcome post-intracytoplasmic sperm injection (ICSI). Therefore, along with semen parameters, Chromomycin A3 (CMA3) staining for protamine deficiency, aniline blue staining for excessive histones, SDS for sperm chromatin stability and SDS + EDTA for the ability of sperm to undergo decondensation were carried out on 55 semen samples from patients referred to the Isfahan Fertility and Infertility Center for ICSI. The results showed that among the aforementioned tests and semen parameters only CMA3 showed a significant correlation with fertilization outcome post-ICSI. Patients were also grouped according to CMA3 level of <30% or >30% or fertilization rate of <50% or >50%. The results show that the mean percentage fertilization and mean percentage of CMA3 positivity is different in both groups, respectively. The area under receiver operator characteristics curve shows that CMA3 is a highly sensitive and specific test for prediction of fertilization outcome post-ICSI. In conclusion, that sperm protamine deficiency has profound effect on fertilization failure in ICSI.     

Assessment of sperm functions in infertile patients with varicoceles

Acrosin activity, acrosome reaction and nuclear chromatin condensation were studied in 24 infertile patients with varicoceles and 26 fertile men with or without varicocele. Chromatin condensation, assessed by aniline blue staining, and acrosin activity, evaluated by gelatinolysis technique, were significantly affected in the group of infertile patients. Defective chromatin condensation and defective acrosin activity were detected in 67% and 50% of the infertile patients, respectively. No significant difference was found between the two groups regarding the acrosome reaction, which was assessed by the triple staining technique after exposure of spermatozoa to low temperature (4 degrees C). This study identified a subgroup of infertile patients with normal standard semen parameters but impaired sperm functions. Results of the sperm function tests and standard semen parameters were not correlated. Therefore, it is concluded from this study that important sperm functions are impaired in patients with varicocele and that the gelatinolysis technique and aniline blue staining are effective tools for assessment of the fertilization potential of varicocele patients.     

Male obesity and age in relationship to semen parameters and sperm chromatin integrity

Obesity can adversely affect human health, including fertility. While obesity can disturb the hormonal profile of the female organism and is associated with fertility loss, little is known about what effect male obesity has on fertility. The present study analysed sperm samples of 153 donors. The men were selected from couples attending an infertility clinic, who had tried for 12 months or more to achieve pregnancy without success. The age of the men under investigation was recorded, and their body mass index (BMI) was calculated. All semen samples were assessed for volume, concentration, motility and morphology. Sperm chromatin integrity was measured by sperm chromatin structure assay. Quality of sperm chromatin condensation was assessed by toluidine blue, aniline blue and chromomycin A3 staining. We can conclude that the impact of elevated BMI on the parameters investigated (basic semen parameters, chromatin integrity and chromatin condensation) was not proven in this study. On the other hand, ejaculate quality appeared to be affected by ageing. The impact was reflected by chromatin integrity, which is a factor that can substantially affect fertility in men, rather than by basic sperm parameters.     

Probable spermatozoal diploidy in the semen of a golden retriever

Summary. The semen of a 3-year-old golden retriever was examined for breeding purposes. When the morphology of the spermatozoa was analysed for the first time, 37% were observed to have giant heads. In most of the giant heads, a diadem defect was also found. The dog was successfully used for breeding. On re-examination, the percentage of giant heads was found to be greater than before. The right testicle exhibited tissue softening. To determine the reason for the defect, an aspiration needle biopsy was performed and ultrasound examination undertaken. In the biopsy smears, both normal spermatozoa and spermatozoa with giant heads were found. On ultra-sonography, the echogenicities of both testicles were the same, and normal. DNA flow cytometry was performed to determine the DNA content of the spermatozoa. Two populations of sperm cells were detected, one having a median fluorescent intensity twice as high as that of normal spermatozoa, suggesting a diploid DNA content. Transmission electron microscopy (TEM) was performed to find out whether the altered intensity correlated with the ultrastructure of the spermatozoa. The nuclei of the sperm heads showed a normal chromatin condensation. Semen quality became worse over a period of 2 years, with 60% giant heads in the last sample. The process was considered to be progressive spermatogenic degeneration with diploidy. Relatives examined did not suggest any hereditary predisposition to the problem. The male was still fertile at the time of the last sample collected and sired a litter of 10 healthy puppies.     

Total globozoospermia associated with increased frequency of immature spermatozoa with chromatin defects and aneuploidy: a case report

Globozoospermia, characterised by the presence of round spermatozoa lacking acrosomes in an ejaculate, is a known cause of male infertility. Semen analysis, including sperm chromatin structure assay, toluidine blue, chromomycin A3 and aniline blue staining and fluorescence in situ hybridisation, was performed in an infertile globozoospermic patient to establish to which extent these genetic factors contributed to his infertility. No spermatozoa capable of hyaluronan (HA) binding were detected in the HA binding assay. Increased rates of immature spermatozoa with defective replacement of histones by protamines, DNA breaks and disturbed chromatin integrity and sperm aneuploid for the sex chromosomes were observed. Intracytoplasmic sperm injection (ICSI) was used in three in vitro fertilisation (IVF) cycles, and enough morphologically well‐developing embryos were obtained in each cycle. However, no pregnancy was achieved. The infertility of our couple, resistant to IVF/ICSI treatment, was most probably caused by a combination of male and female factors.     

Use of the acridine orange staining on smears of human spermatozoa after heat-treatment: evaluation of the chromatin condensation

A simple, rapid procedure was described to evaluate the degree of chromatin compactness in human spermatozoa. Smears of fixed spermatozoa were heat-treated and stained with acridine orange. Smears from the same sperm samples were also stained with acidic aniline blue. Three groups of semen could be distinguished according to the percentage of red sperm heads observed under fluorescence microscope. 1) Semens with spermatozoa whose chromatin appeared normal before and after heat-treatment. 2) Semens with spermatozoa whose chromatin appeared normal before and abnormal after heat-treatment. 3) Semens with spermatozoa whose chromatin appeared abnormal before and after heat-treatment. The positive correlation between the percentages of red heads and the percentages of blue stained heads suggests that modifications in the biochemical composition of the basic protein component associated with DNA are responsible for the denaturation process.     

Standardization of sample preparation, staining and sampling methods for automated sperm head morphometry analysis of boar spermatozoa

The accuracy of computer-assisted sperm morphometry analysis (CASMA) depends on the careful preparation, fixation and staining of spermatozoa. The efficiency of CASMA may be enhanced by developing optimized protocols. The aim of the present study was to evaluate the influence of sperm washing and the use of three staining techniques [rapid Panoptic, Hemacolor and Harris's Haematoxylin (HH)] on image-processing accuracy and boar sperm head morphometry. Sperm washing had a significant effect on samples stained with rapid Panoptic, increasing the percentage of correctly binarized sperm heads and the contrast between cells and background. However, rapid Panoptic yielded the lowest percentage of properly digitized sperm heads. HH provided the highest cell/background contrast, and also greater sperm head staining intensity, but discrimination of sperm midpieces was considered insufficient. Hemacolor occupied an intermediate position, providing acceptable colour intensity and satisfactory cell/background contrast. Use of different staining procedures prompted dimensional differences in sperm head morphometry. Significant differences between animals were observed for all morphometric parameters. Low within-animal variation coefficients reflected a homogeneous sperm head population. Between-animal variation coefficients were relatively high for Hemacolor and HH, and significantly high for the rapid Panoptic stain. Using Panoptic and HH, stable morphometric measurements required at least 100 properly digitized sperm heads rather than 200, while Hemacolor required only 50 spermatozoa. These results indicate that both washing of semen and staining procedures significantly affect the accuracy of image processing and sperm head dimensions. Hemacolor and HH proved to be the best staining techniques for evaluating sperm head dimensions in boar.     

Sperm chromatin quality and DNA integrity in partial versus total globozoospermia

Globozoospermia is a severe form of teratozoospermia with low incidence in infertile patients, considered as one of the important causes of male infertility. The objective was to investigate the chromatin/DNA integrity as well as apoptosis in ejaculated spermatozoa of cases with partial or total globozoospermia. Fifty‐seven semen samples were divided into three groups of partial globozoospermia (n = 17), total globozoospermia (n = 10) and normozoospermia (control; n = 30). Sperm chromatin condensation, DNA integrity and apoptosis were assessed using cytochemical assays. The results showed significant differences in sperm parameters of count and motility between two case groups versus controls. The percentages of spermatozoa with abnormal chromatin packaging and protamine deficiency were significantly higher in total and partial globozoospermic men compared to normozoospermic samples. Also, the rates of TUNEL‐positive spermatozoa were significantly increased in both globozoospermic cases with respect to the control (18.3 ± 10.1 and 12.3 ± 9.2 versus 5.9 ± 3 respectively). However, no significant differences were noticed between two subgroups of patients with regard to sperm DNA denaturation, DNA fragmentation and apoptosis. Abnormal chromatin packaging, DNA damage and apoptosis were significantly higher in cases than controls. The sperm chromatin/DNA anomalies may be considered as one of the main aetiology of ART failure in globozoospermic patients.     

Chromatin condensation in cat spermatozoa during epididymal transit as studied by aniline blue and acridine orange staining

Summary.  Chromatin stability and DNA-resistance to acidic denaturation was evaluated by acidic aniline blue and acridine orange staining of cat sperm from different regions of the epididymis. The results were related to conventional sperm parameters. The percentage of aniline blue-stained spermatozoa (persisting histones) decreased significantly from the caput to the cauda region (31.8% and 7.8%, respectively; P <0.0001). The percentage of stained heads of cauda sperm was much lower in populations of morphologically normal forms than in those with abnormal forms (4.1% and 13.8%, respectively, P <0.0001). Among spermatozoa with abnormalities, the percentage of stained heads was significantly higher in cells with head abnormalities than in sperm with only tail abnormalities (87.1% and 10.3%, respectively; P >0.0001). With acridine orange fluorescence staining, 86.5% of cauda epididymal sperm were found with well-condensed chromatin, stabile against acid-induced denaturation. Chromatin stability increased significantly from the caput epididymal region (51.1%) to the cauda epididymal region (86.5%). The percentage of cauda epididymal sperm with normal condensed chromatin was neither linked to testicular sperm count, motility nor to age of the cats. The parameter of chromatin condensation and stability can be a valuable index of sperm quality, reflecting the possible disorders of spermatogenesis and epididymal sperm maturation, frequently observed in feline species.     

Chromatin alterations induced by freeze-thawing influence the fertilizing ability of human sperm

A previous cytochemical study revealed that chromatin alterations could be induced in frozen-thawed human sperm. In order to evaluate the biological consequences of such chromatin alterations, we evaluated the relationship between these chromatin alterations and the drastic decrease or loss of sperm fertilizing ability after freeze-thawing. Using acridine orange staining and Feulgen-DNA quantitative microspectrophotometry, the nuclear variables of sperm were compared using samples which had either recovered, or lost their fertilizing ability after freezing-thawing during artificial inseminations, despite good post-thaw motility. There was a clear decrease in Feulgen-DNA content and nuclear surface values in sperm which exhibited a loss of fertilizing ability. Thus freeze-thawing may alter the DNA/nuclear protein relationships and impair the fertilizing ability of human sperm.     

Evaluation of nuclear maturity in human spermatozoa obtained by sperm-preparation methods

Summary. Change in the nuclear maturity has been considered as an index of sperm quality. This is done by using aniline blue staining that stains spermatozoa with disturbances in the chromatin condensation. Therefore, this technique was used to evaluate the quality of sperm obtained by sperm preparation methods. In 14 semen samples from healthy donors with normal semen analysis and normal forms, the swim-up (SU), Percoll gradient centrifugation (PG), glass wool column filtration (GW) and sedimentationmigration (SM) were performed. GW and SM methods presented the lowest number of spermatozoa with alteration in the nuclear maturity (ANM), 11, 14% and 12, 42% compared to native semen (19%) ( P < 0.005 and P < 0.05 respectively). SU and PG did presented no significant difference compared to native semen. In tests using the four methods, approximately 60% of the ANM occurred in normal spermatozoa. In the cells with pathologic abnormalities and ANM, 74.5% corresponded to macrocephalic forms, as this abnormality correlated mainly with ANM. It is concluded that in a semen sample with a higher percentage of normal forms, approximately 19% will have an ANM. GW and SM methods obtain the lowest percentage of ANM. ANM presents itself in 98% of the macrocephalic forms and they are effectively isolated with the PG method. The practice of this simple technique may orientate towards the sperm preparation methods to be used in assisted reproduction.