Molecular andrology as related to sperm DNA fragmentation/sperm chromatin biotechnology

Genetic male infertility occurs throughout the life cycle from genetic traits carried by the sperm, to fertilization and post-fertilization genome alterations, and subsequent developmental changes in the blastocyst and fetus as well as errors in meiosis and abnormalities in spermatogenesis/spermatogenesis. Genes encoding proteins for normal development include SRY, SOX9, INSL3 and LGR8. Genetic abnormalities affect spermatogenesis whereas polymorphisms affect receptor affinity and hormone bioactivity. Transgenic animal models, the human genome project, and other techniques have identified numerous genes related to male fertility. Several techniques have been developed to measure the amount of sperm DNA damage in an effort to identify more objective parameters for evaluation of infertile men. The integrity of sperm DNA influences a couple's fertility and helps predict the chances of pregnancy and its successful outcome. The available tests of sperm DNA damage require additional large-scale clinical trials before their integration into routine clinical practice. The physiological/molecular integrity of sperm DNA is a novel parameter of semen quality and a potential fertility predictor. Although DNA integrity assessment appears to be a logical biomarker of sperm quality, it is not being assessed as a routine part of semen analysis by clinical andrologists. Extensive investigation has been conducted for the comparative evaluation of these techniques. However, some of these techniques require expensive instrumentation for optimal and unbiased analysis, are labor intensive, or require the use of enzymes whose activity and accessibility to DNA breaks may be irregular. Thus, these techniques are recommended for basic research rather than for routine andrology laboratories.     

SCSA和SCD检测精子DNA完整性结果比较及与精子质量参数的相关性分析

目的:近年来精子DNA碎片预测男性生育受到广泛关注,其检测方法较多,但方法学的比较研究很少,本文旨在比较精子DNA完整性常用检测方法之间的差异,并分析DNA碎片与精子质量的相关性。方法:选择108例精液样本,采用精子染色质结构分析试验(SCSA)和精子染色体扩散实验(SCD)对样本分别进行精子DNA碎片检测分析,对两种方法的结果进行比较,并与精液常规、精子形态以及患者年龄进行相关性分析。结果:两种方法检测的精子DNA碎片化指数(DFI)存在显著的一致性(P0.01),DFI与精子活动率、前向运动精子百分率以及正常形态精子百分率呈显著负相关(P0.01),与畸形精子指数呈显著正相关(P_(SCSA)0.01,P_(SCD)0.05),SCSA得出的DFI与年龄呈显著正相关(P0.01),而SCD法的DFI并未发现类似的现象。结论:两种方法检测的结果对精子质量均有较好的预测价值,精子DFI作为一个检测指标,对男性生育力预测有一定的临床价值。但不同方法之间的检测结果差异较大,检测方法的标准化有待进一步研究。     

The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation

Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test for the screening of sperm DNA fragmentation in the andrology laboratory.     

Evaluation of sperm DNA fragmentation and chromatin structure in infertile men with immotile short-tail sperm defect

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients’ samples (22.6 ± 6.9) compared with controls (16.3 ± 4.2) (p < .05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45 ± 4.60 vs. 7.03 ± 2.86, p < .05) and SCSA (24.80 ± 13.1 vs. 15.2 ± 7.2, p < .05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis.     

Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation

Male infertility has a complex etiology, and many times, the cause is unknown. While routine semen analysis provides an overview of basic semen parameters, such as sperm concentration, motility, viability and morphology, a significant overlap of these parameters has been reported in fertile and infertile men. Moreover, conventional semen parameters do not reveal the cellular or molecular mechanisms of sperm dysfunctions leading to infertility. Therefore, sperm functional parameters, including sperm chromatin integrity, are evaluated to provide information on subtle sperm defects that are not routinely identified. Incomplete or defective sperm chromatin condensation increases the susceptibility of the sperm DNA to oxidative damage or other factors. To evaluate sperm chromatin integrity, different methods with varying degrees of diagnostic and prognostic capabilities are available. Among these assays, SCSA, TUNEL and SCD assays are most commonly used. While these assays rather evaluate the DNA directly for damages, the aniline blue and chromomycin A3 stains test for the quality of chromatin condensation. Thus, this review discusses and compares different methods used to evaluate sperm chromatin integrity and condensation, and their inclusion in the routine evaluation of the male infertility.     

Ultrastructural and DNA fragmentation analyses in swim-up selected human sperm

Seventeen sperm samples were evaluated by transmission electron microscopy (TEM) before and after swim-up separation. DNA-fragmentation was tested by terminal d-UTP nick end labeling (TUNEL) in unselected and selected semen samples, and the results were analyzed in relation to sperm ultrastructural characteristics detected by TEM. A significant improvement in mean numbers and percentages of structurally normal sperm was observed after swim-up selection, corresponding to a significant decrease in the percentage of necrotic and apoptotic sperm, while the percentage of sperm with immature nuclei did not change significantly. TUNEL indicated a significant decrease in chromatin-fragmented sperm after swim-up. Swim up selection based on sperm motility excludes many sperm with ultrastructural evidence of necrosis (absent or reacted acrosome, disrupted chromatin, broken plasma membrane) and apoptosis (misshapen nuclei with marginated chromatin), as confirmed by TUNEL analysis. Nevertheless, immature sperm with elliptical or roundish nuclei, misshapen acrosomes and uncondensed chromatin remain part of fertilizing pool.     

Nuclear chromatin decondensation of human sperm assessed by DNA measurement

A sperm nuclear decondensation ability test using 1% SDS + 6 mM EDTA was used to determine an objective method of quantification of the decondensation process by comparing two different methods of quantification: the usual histological method, after staining by the method of Shoor and classification of the sperm heads into normal heads, doubtful heads, swollen heads, and very swollen heads, and the measurement of the removal of DNA from the extremely swollen spermatozoa. After 5 min of exposure to SDS/EDTA, the percentage of removed DNA was correlated (negatively) to the percentage of normal heads, which assessed the two methods and confirmed that all decondensation results obtained after long periods of treatment with SDS/EDTA can be discarded since spermatozoa can be extremely swollen and disappear.     

精子染色质扩散实验检测精索静脉曲张及不明原因不育患者精子DNA碎片

目的了解精索静脉曲张(VC)及不明原因不育患者精子DNA碎片的发生比例。方法改进的精子染色质扩散(SCD)实验分析精子DNA碎片。检测VC不育患者39例,不明原因不育患者57例。以生育健康成年男性32例为对照组。结果VC不育患者SCD小光晕和无光晕精子(精子DNA碎片)比值平均为(36.6±18.9)%,VC不育组明显高于对照组(12.1±5.2)%(P<0.001),而大光晕和中光晕精子比值VC不育组明显低于对照组(P<0.01);不明原因不育患者精子DNA碎片比值平均为(26.8±10.2)%,与对照组[(12.1±5.2)%]比较有显著性差异(P<0.001)。结论SCD实验表明,VC及不明原因不育患者精子DNA碎片比值增高。     

Evaluation of sperm nuclear morphology in specimens with abnormal sperm chromatin structural assays

Two recent tests have claimed to identify the subfertile male even when other semen parameters were normal: the sperm chromatin structure assay (SCSA) and abnormal sperm nuclear morphology using much higher magnification. The present study attempted to determine if having a high (> 30%) DNA fragmentation index (DFI), thus resulting in an abnormal SCSA test, is associated with a greater likelihood of sperm with abnormal nuclei. Four males with high DFI scores (57.6%, 65.4%, 31.0%, and 35.3%) had their nuclei evaluated by a complex microscope set-up that magnifies the sperm at least 6000x. The corresponding % of normal nuclei was 0%, 20.0%, 23.7% and 40.0%. The mean and median % of normal nuclei was 20.9+/-16.43 and 21.8, respectively. More studies of similarly matched refractory in vitro fertilization cases, where males have normal DFI scores, are needed to determine if having a high DFI index is associated with a lower percentage of normal nuclei.     

Recombinant human FSH reduces sperm DNA fragmentation in men with idiopathic oligoasthenoteratozoospermia

A prospective randomized controlled study was designed to evaluate the effects of recombinant human follicle-stimulating hormone (rFSH) treatment on sperm DNA fragmentation in men with idiopathic oligoasthenoteratozoospermia (iOAT). One hundred twenty-nine men with sperm count less than 10 × 10(6) spermatozoa/mL and forward motility <25% were included; normal serum levels of FSH, luteinizing hormone (LH), and testosterone, and no other causes of infertility were enrolled. The patients were randomized into 2 groups: 65 men were treated on alternate days for 90 days with injections of 150 IU rFSH, and 64 subjects received nonantioxidant vitamin supplements. Main outcome measures were serum levels of FSH, LH, testosterone, and inhibin B and DNA fragmentation index (DFI) at baseline and after 90 days. No significant differences were observed between the 2 groups with regard to sperm parameters and hormone values. The DFI was similar between the 2 groups at the time of the enrollment but reduced significantly (P < .05) after rFSH therapy in study group, whereas no significant variation occurred in the control group. In the subgroup of patients with high basal DFI values (>15%), rFSH treatment significantly increased DFI (P < .01), whereas no significant variation occurred after 90 days of vitamin supplements. We conclude that rFSH administration improves sperm DNA integrity in iOAT men with increased DFI values. The degree of sperm DFI might be useful to identify those iOAT patients in which rFSH treatment can be advantageous.     

Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis

The aim of this study was to evaluate conventional semen parameters (density, morphology, and progressive motility) and the flow-cytometric parameters of DNA fragmentation, mitochondrial membrane potential, phosphatidylserine externalization, and chromatin compactness in patients with varicocele before and after varicocelectomy. Thirty men (26.5 ± 3.2 years old, range 20-32 years) with oligoasthenoteratozoospermia and grade 3 left varicocele were selected (without other causes of male infertility). Each of them underwent sperm analysis and flow cytometric evaluation before and 4 months after subinguinal microsurgical varicocelectomy (SMV). After varicocelectomy, men had significantly higher sperm density, progressive motility, and normal forms compared with baseline. They also had a significantly lower percentage of spermatozoa with low mitochondrial membrane potential. After SMV, they showed a significantly lower percentage of spermatozoa with phosphatidylserine externalization, an early sign of apoptosis. Significantly decreased percentages of spermatozoa with abnormal chromatin compactness and spermatozoa with DNA fragmentation were found after SMV compared with baseline. Subinguinal microsurgical varicocelectomy improves sperm function in oligoasthenoteratozoospermia secondary to grade 3 left varicocele. Improvements are seen in conventional parameters and biofunctional parameters not routinely evaluated.     

Aneuploidy and DNA fragmentation in morphologically abnormal sperm

In light of the relative success of ICSI in the treatment of male infertility, much importance has been made to the selection of morphologically viable sperm. However, correlation between specific sperm morphology and chromosomal abnormalities is still relatively limited and less is known about the connection between sperm morphology and DNA integrity. Sperm obtained from isolated teratozoospermic men ( n  = 10) and control men ( n  = 9) were analysed using FISH (for chromosomes 13, 18, 21, X and Y) and TUNEL assays to determine the level of aneuploidy and DNA fragmentation. Sperm morphology was evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. Sperm from teratozoospermic men, compared with fertile men, had higher rates of total chromosomal abnormality ( p  < 0.05), total aneuploidy ( p  < 0.01) and chromosome 13 disomy ( p  < 0.01). Associations between particular types of sperm morphology and chromosomal abnormalities were observed in both control (tapered heads) and teratozoospermic (amorphous heads and tail abnormalities) samples. Levels of DNA fragmented sperm were higher in teratozoospermic men than in the control men (60.28 ± 21.40% vs. 32.40 ± 17.20%, p  < 0.05) and positively correlated to sperm with bent necks in control samples and round heads in teratozoospermic samples ( p  < 0.05). Sperm of isolated teratozoospermic men have higher rates of chromosomal abnormalities and DNA fragmentation than that of the fertile controls. Specific abnormal sperm morphology can be correlated to chromosomal abnormalities and level of DNA fragmentation in sperm.     

Application of erythrocyte lysing buffer (ELB) has detrimental effects on human sperm quality parameters,DNA fragmentation and chromatin structure

Erythrocyte lysing buffer (ELB) facilitates the search for spermatozoa by eliminating erythrocytes in testicular suspension used during the ICSI procedure. This study investigates the effects of ELB on sperm quality parameters, sperm chromatin and sperm DNA fragmentation. Normal ejaculations were used as the model for testicular spermatozoa in this study. After swim-up, the sperm pellets were divided into two parts. Part I, the control (Group A), was diluted with culture media; and Part II, the intervention group (Group B), was diluted with ELB for 10 min. After centrifugation in both groups, the sperm pellets were re-suspended with culture media. The samples were immediately evaluated (A0 and B0) and then evaluated again after 1 hr (A1 and B1). The results indicated ELB decreased the progressive motility (81.60 ± 8.69 vs. 64.69 ± 19.08) and viability (97.62 ± 3.02 vs. 85.91 ± 11.46), in Group A and B, respectively, both immediately and 1 hr after preparation. Also, ELB engendered a significant increase in the DNA fragmentation index both immediately (9.68 ± 3.55 vs. 14.38 ± 6.52) and after 1 hr (10.37 ± 5.03 vs. 19.38 ± 6.39). In conclusion, ELB may damage sperm cells, shown by a decreased motility and viability, and it increased DNA fragmentation. Therefore, the use of ELB in testicular semen handling should be discouraged.     

The effect of sperm DNA fragmentation on intracytoplasmic sperm injection outcome

Our study objective was to assess the effect of various sperm DNA fragmentation levels on clinical intracytoplasmic sperm injection outcome. This retrospective study included 392 patients who underwent ICSI and performed sperm DNA fragmentation testing before the procedure. Based on sperm DNA fragmentation cut-off values, the patients were differentiated into 3 groups as <20%, 20%–30% and >30%. According to the female status, patients were differentiated into favourable group (n = 259) with female age <35 years and anti-Mullerian hormone level ≥7.1 pmol/L; and unfavourable group (n = 133) with female age ≥35 years and anti-Mullerian hormone level ≤7.1 pmol/L. The patient's medical records were reviewed, and patient's demographic, laboratory data including semen analysis, sperm DNA fragmentation determined by means of sperm chromatin dispersion, hormonal profile and data regarding intracytoplasmic sperm injection cycle were collected. This cohort reported that the clinical reproductive outcomes of intracytoplasmic sperm injection showed no statistical significance with increase sperm DNA fragmentation levels. In sperm DNA fragmentation above 30%, favourable females had significantly higher clinical pregnancy rate and live birth rate than unfavourable females, while fertilisation rate and miscarriage rate showed no significance between the subgroups. High sperm DNA fragmentation is linked to poor semen parameters.     

Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA

The primary aim of this study was to determine the effect of oral antioxidant treatment (1500 mg of l ‐Carnitine; 60 mg of vitamin C; 20 mg of coenzyme Q10; 10 mg of vitamin E; 10 mg of zinc; 200 μg of vitamin B9; 50 μg of selenium; 1 μg of vitamin B12) during a time period of 3 months upon the dynamics of sperm DNA fragmentation following varying periods of sperm storage (0 h, 2 h, 6 h, 8 h and 24 h) at 37 °C in a cohort of 20 infertile patients diagnosed with asthenoteratozoospermia. A secondary objective was to use the sperm chromatin dispersion test (SCD) to study antioxidant effects upon a specific subpopulation of highly DNA degraded sperm (DDS). Semen parameters and pregnancy rate (PR) were also determined. Results showed a significant improvement of DNA integrity at all incubation points (P < 0.01). The proportion of DDS was also significantly reduced (P < 0.05). Semen analysis data showed a significant increase in concentration, motility, vitality and morphology parameters. Our results suggest that antioxidant treatment improves sperm quality not only in terms of key seminal parameters and basal DNA damage, but also helps to maintain DNA integrity. Prior administration of antioxidants could therefore promote better outcomes following assisted reproductive techniques.     

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.     

Participation of DNA structure on sperm chromatin organization

The in vitro interaction between purified bovine liver and sperm DNA with somatic histones, to form nucleosomes, and with bovine and salmon protamines were studied. DNAse or microccocal nuclease digestion of liver DNA-histone reassociated chromatin produced the expected polynucleosome type of fragments. Electrophoretic patterns of digested sperm-DNA nucleosomes were different. Micrococcal nuclease digestion produced mainly fragments smaller than 100 bp and some nucleosome-type particles. Under DNAse activity most of the products were smaller than 100 bp, indicating an increased susceptibility of the sperm DNA-histone complexes to the hydrolytic activity of both nucleases, particularly toward DNAse I. This differential susceptibility was confirmed by sucrose gradient spectrophotometric analysis. Acridine orange (AO) staining of histone-DNA reassociated nucleosomes showed significant differences in fluorescence intensity, sperm DNA-histone complexes being almost twice as fluorescent as liver DNA-histone complexes. On the contrary, liver DNA/protamine complexes stained with AO were consistently more fluorescent than sperm DNA-protamine complexes. Finally, no differences in either fluorescence intensity or spectra were observed when liver and sperm DNA were stained with AO after interaction with salmon protamines. The data suggest that sperm DNA has important structural characteristics that differentiates it from somatic DNA. These differences seem to be species specific and must surely play an important role on the determination of the dramatic sequence of that participates sperm chromatin organization.     

Fine structure of human sperm chromatin.

The fine structure of human sperm chromatin is revealed by electron microscopy. Human sperm chromatin is characterized by fibers composed of discrete spherical organizational units not unlike somatic nucleosomes. During the decondensation process fibers composed of joined spherical units of both 400-A and 150-A diameters were observed.