The semen samples were obtained from 400 men, having been referred to our institute. Semen collection was conducted by masturbation after 3–4 days of sexual abstinence. After liquefaction, routine semen analysis, such as sperm motility, concentration, and morphology, was carried out using light microscopy according to the recommendations of the World Health Organization (WHO, 2010)10 and then, each sample was categorized into 4 experimental groups such as normospermia (n=100), oligospermia (n=100), asthenospermia (n=100), and teratospermia (n=100). For evaluating morphological abnormalities, at least 200 sperm were examined per slide (10). Sperm concentration was assessed by a Makler counting chamber. For evaluating the sperm viability, eosin–nigrosin staining technique was carried out. The amount of 10μl of prepared sperm was mixed with 10μl of eosin–nigrosin stain on a glass slide and assayed using a light microscope to determine the percentage of live sperm. All analyses were performed by an experienced laboratory technician blinded to the study. This prospective study was approved by the ethical and scientific committee of …. Reproductive Sciences Institute….

Chromomycin A3 (CMA3) was applied as one of the verified sperm DNA integrity assessments to assess sperm protamine deficiency. Briefly, all semen samples were washed in phosphate-buffered saline (PBS) free of Ca2+ and Mg2+ and fixed in Carnoy's solution (methanol/glacial acetic acid 3:1 (Merck, Darmstadt, Germany)) at 4°C for 5min. Following the preparation of smears, each slide was treated with 100μl of CMA3 solution (0.25mg/ml in McIlvaine buffer (7ml citric acid 0.1M+32.9ml Na2HPO47H2O 0.2M, pH 7.0, containing 10mmol/l MgCl2)) (Sigma, St Louis, MO, USA) for 20min. The slides were then rinsed in PBS buffer and mounted with buffered glycerol (1:1). Microscopic analysis was performed by fluorescent Nikon Eclipse 600 microscope (Tokyo, Japan), with the appropriate filters (460–470nm). In each slide, 200 spermatozoa were assessed and scored as positive (CMA3+ with bright yellow) or negative (CMA3− with dull yellow) for CMA3, according to their brightness (Fig. 1).11

Figure 1.

CMA3 staining for detection of human sperm protamine deficiency. Bright yellow sperm heads (CMA3+) show abnormal DNA and yellowish green sperm heads showed DNA damage (magnification 100×).

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The results were analyzed using the SPSS software version 20 for Windows (SPSS Inc., Chicago, IL, USA). ANOVA and Student's t-test were applied to compare the groups, and the term ‘statistically significant’ was used to denote a two-sided p value<0.05 for sperm parameters and the protamine deficiency test. The correlation between CMA3+ and each experimental group was performed using Pearson coefficient of correlation.

Almost all of our findings in sperm analyses corresponded to WHO 2010 criteria as expected in the current study. For instance, the sperm progressive motility and the sperm count showed a significant decrease in asthenospermia and oligospermia than others respectively (Table 1). In accordance with our results, Rahiminia et al. (2018) assessed the semen from 32 OAT and 32 normal fertile men. They demonstrated significantly lower sperm concentration, progressive motility, and normal morphology in OAT patients than in the normozoospermic controls.14 One study by Dehghanpour et al. reported the similar observation of significantly lower percentages of sperm concentration, motility, and normal morphology in the tapered-head spermatozoa when they were compared to normozoospermic samples.15 Moreover, the data showed significant correlations between viability of abnormal spermatozoa and their motility status, as an indicator of spermatozoa vitality in the groups with defined sperm abnormalities. In contrast, we did not observe considerable correlations between normal morphology and other sperm characteristics as well. The study aimed to compare sperm characteristics among patients with male factor infertility (MFI) etiology (n=166), patients undergoing infertility evaluation (n=406), and fertile men (Proven fertile men or normal donors (n=147)), and showed similarities between normal morphology rates (%) in most of the patients and almost half of the fertile men with abnormal morphology.16 Therefore, they concluded that sperm motility and concentration can prepare more valued data than morphology during an infertility evaluation.

Regardless of the asthenospermia group having a large proportion of sperm cells with excessive histones and protamine deficiency, we predicted a significantly elevated sperm DNA fragmentation and the data showed no significant correlations between CMA3 (+) spermatozoa of abnormal spermatozoa and its normal morphology in experimental groups with defined sperm abnormalities.

Having a methodology like that of ours, we found one study (2014) that evaluated sperm DNA damage in a large cohort of infertile men with isolated sperm defects grouped as oligozoospermia, asthenozoospermia, and teratozoospermia.16 However, they focused on evaluating their % sperm DNA fragmentation (%SDF) by using flow cytometry-based Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling assay.17 They stated that the proportion of men with high %SDF was remarkably superior in the asthenospheric samples compared to the oligospermia and teratospermia groups (31% vs. 18% and 19%, respectively, p<0.0001).17

There was a significant decrease in the proportion of spermatozoa with normal chromatin condensation in men with OAT as well as teratospermic patients, as has been previously reported.14,15,18 Moreover, it should be considered that the abnormal replacement of histones by protamine can increase sperm DNA fragmentation, and there is a clear relation between sperm chromatin quality and DNA damage.19

However, the nature of the relationship between sperm DNA damage and sperm motility has not been fully characterized; our data, in line with those of Belloc et al. (2014) indicated that poor motility is the sperm parameter abnormality most closely related to both sperm DNA and chromatin deficiency.17 A possible explanation for this relationship is that the formation of a mature, compacted sperm nucleus and the development of the sperm flagellum, both originate during spermiogenesis.20,21 Furthermore, experimental research studies reported that disruption of nuclear chromatin compaction is associated with the development of an abnormal flagellum and defective motility.7,21 By working on both total and partial globozoospermic men two recent studies with similar results stated that the sperm chromatin/DNA anomalies may be considered as the main etiology of ART failure in globozoospermic patients as well.22,23

Also, we observed significant negative correlations between sperm concentration and progressive motility and spermatozoa with positive CMA3, in men with oligospermia, representing high proportion of abnormal chromatin packaging in ejaculated sperm of oligospermic men. Staining by CMA3 has shown to be one of the SDF testing methods with vigorous correlation with other SDF assays. It demonstrates a DNA protamination status associated with quality of sperm chromatin packaging.24 Regarding promising outcomes in the use of testicular sperm for ICSI as a treatment plan for high SDF, it has been suggested that the use of testicular sperm rather than ejaculated sperm may be beneficial in men with oligozoospermia due to the three- to fivefold lower SDF detected in testicular sperm rather than ejaculated spermatozoa.25,26

In spite of as evaluating the sperm parameters and chromatin condensation in a large group of patients with different types of sperm abnormalities, we could not perform several standard assays to demonstrate different disorders of sperm chromatin and DNA integrity among these groups. This is the main weakness of the current study.

The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).

The authors declare that they have followed the protocols of their work center on the publication of patient data.

The authors have obtained the written informed consent of the patients or subjects mentioned in the article. The corresponding author is in possession of this document.