18.1 Introduction

Gametogenesis is a central biological process for sexual reproduction. In this process, both haploid male and female gametes are needed for successful fertilization. Male gamete or sperm are produced during spermatogenesis; a complex, unique, and tightly regulated process, which includes a series of physiological, biochemical and morphological events. During this process, round diploid spermatogonia are differentiated into haploid spermatozoa with an acrosome, flagellum, and condensed nucleus. Condensation of nucleus or chromatin compaction play a paramount role in formation of morphology, especially the size of sperm. Therefore, during spermiogenesis, nucleo-histones are replaced by protamines. To achieve this aim, histones become hyper-acetylate to reduce their binding affinity to DNA. Subsequently, hyper-acetylated histones are replaced by transition proteins and next by protamines. According to the literature, in the human, 85–95 percent of the histones are replace with protamines while 5–15 percent of histones remain bounded to mature sperm DNA in humans [1, 2]. Therefore, through this process, sperm chromatin becomes six-fold more condensed than chromatin of other cells. This condensation will protect sperm chromatin from chemical and mechanical damages, bacterial infections, detrimental molecules such as oxidants, and also damaging molecules within the female reproductive system [2]. Alterations in the sperm histone/protamine ratio due to reduction of hyper-acetylation of histones or excessive histone retention are associated with abnormal chromatin packaging which can increase susceptibility to DNA damage, and eventually result in male infertility [1]. Therefore, normal chromatin packaging in the spermatogenesis process is essential for maintaining genomic integrity and accomplishment of fertility. In this regard, numerous studies have shown that there is a significant positive correlation between proper sperm chromatin condensation with clinical outcomes of infertile couples being candidates for assisted reproductive technology (ART) [3]. In addition, a significant positive association has been reported between sperm abnormal chromatin packaging with recurrent pregnancy loss [4]. Therefore, numerous studies suggested that the evaluation of “sperm chromatin condensation” may have a prognostic value in the assessment of male infertility. For this aim, several analytical methods based on cyto-chemical or fluorescent dyes have been proposed for the assessment of sperm nuclear compaction such as acidic aniline blue (for direct detection of excessive presence of histones), toluidine blue staining and chromomycin A3 (for indirect assessment of protamine deficiency), sperm chromatin structure assay (SCSA) or acridin orange test (for indirect assessment of sperm chromatin DNA stability based on chromatin compaction termed “high DNA stability structure”). Toluidine blue (TB) staining is one of the procedures used for evaluation of chromatin structure. Therefore, this chapter, provides detailed practical advice on TB staining, optimization, and interpretation of results in the field of medically assisted reproduction.

18.2 Protocol of Toluidine Blue Staining

The below protocol is a modification of the previous protocols [7, 9–11].

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   1. Allow the ejaculate to liquefy at 37°C for 30 minutes

   
   
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   2. Centrifuge the liquefied sample at 250×g for 10 minutes

   
   
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   3. Remove the supernatant and re-suspend pellets in sperm washing media containing 5 percent BSA

   
   
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   4. Prepare smears on pre-cleaned defatted slides

   
   
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   5. Allow the smears to dry for 30 minutes, and then fix the slides with freshly prepared 96 percent ethanol: acetone (1:1) at 4°C for 30 minutes. Allow the fixed slides to dry.

   
   
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   6. After 12 hours, hydrolysis the slides with 0.1 M HCl at 4°C for five minutes

   
   
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   7. Wash slides with distilled water, three times for two minutes each time

   
   
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   8. Prepare TB working solution (0.05 percent of TB in 50 percent McIlvain’s citrate phosphate buffer at pH=3.5–4)

   
   
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   9. Cover the slides for 5–10 minutes with TB working solution

   
   
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    10. Wash slides in distilled water

    
    
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    11. Dehydrate slides in tertiary butanol at 37°C (2×3 minutes) or ethanol (70 percent, 96 percent and 100 percent)

    
    
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    12. Mount slides with xylene at room temperature (two to three minutes)

    
    
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    13. Evaluate the slide for positive- or negative-TB spermatozoa using oil immersion on light microscope

    
    
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    14. Count of 200–500 sperm per sample

    
    
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    15. Report percentage of sperm with dark blue stain of TB as abnormal chromatin packaging (Figure 18.1)

    
    
    
    
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      ➤ Fixed slides can be stored before staining in the dark box for up to one week at room temperature and for up to two weeks in an exicator in the cold room. This duration will not affect the results

      
      
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      ➤ The TB working solution can be prepared monthly from 1 percent TB with distilled water and stored at 4°C.

      
      
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      ➤ One percent TB can be stored at 4°C for up to one year.

Figure 18.1 Assessment of sperm chromatin packaging by toluidine blue (TB) staining. Sperm with dark blue stain of TB dye were considered as abnormal chromatin packaging while sperm with light blue stain of TB dye were considered as normal chromatin packaging.

18.3 Advantages and Disadvantages of Toluidine Blue Staining

Several advantages are mentioned for the assessment of sperm chromatin structure by the TB staining through light microscopy as it is a simple, easy to perform, fast, and inexpensive method. The disadvantages of this test are that only a limited number of sperm can be assessed as compared to flow cytometry where a minimum of 5000–10,000 sperm per sample are assessed. In addition, the technique is labor-intensive.

18.4 Clinical Interpretation of the Results of Toluidine Blue Staining

The sperm chromatin integrity is essential for fertilization, early embryonic development and even development to term. Recently, researchers suggested that besides standard semen parameters, sperm chromatin integrity should be evaluated in parallel with semen analysis [12]. Sperm chromatin integrity is influenced by both external and internal factors such as lifestyle, age, overproduction of oxidative stress, apoptosis, and protamine deficiency [13]. In this regard, several studies demonstrated the mean values of sperm DNA fragmentation and abnormal chromatin packaging were significantly higher in different groups of infertile men such as men with varicocele, globozoospermia, and abnormal sperm parameters, compared to fertile men or individuals with normal semen parameters (14–16).

Evenson, the pioneer of SCSA, believes that a high level of DNA damage in a semen sample could be considered as a predictive parameter for “subfertility and infertility”. On the other hand, since fertility is a complex orchestrated process and is influenced by numerous factors, a high level of DNA integrity in a semen sample could not be considered as a direct predictor of “fertility” [17]. Indeed, during natural fertilization and even in the process of in vitro fertilization (IVF), numerous barriers exist that help to select the most competent sperm and thereby, through these selection barriers, the chance of sperm with damaged DNA or chromatin anomalies to participate in the process of fertilization is reduced. However, in intracytoplasmic sperm injection (ICSI), all these barriers are bypassed and the chance of sperm with reduced chromatin integrity to be inseminated is increased. In this context, Avendaño et al. (2010) believe that in the case of male infertility there is a likelihood of oocytes being inseminated with sperm with normal morphology and reduced chromatin integrity and damaged DNA [18]. Clinical outcomes in the field of infertility treatment revealed significant negative correlations between sperm DNA damage with fertilization, embryo quality and pregnancy rates [18–20] while there are studies that reported no influence of sperm DNA damage on reproductive outcome [21]. When systematic review and meta-analysis studies are checked, it was suggested that sperm DNA damage could have adverse effects on ART outcome, especially as it decreases the likelihood of formation of a good‐quality embryo [19, 20].

DNA repair mechanisms in male germ cells are active during mitotic and meiotic stages of spermatogenesis. However, at the time when the excess cytoplasm becomes redundant and the sperm nucleus becomes highly compacted, the ability of DNA repair machinery is nearly completely reduced [22]. Therefore, if sperm are exposed to oxidants, the chance of DNA damage is increased. In this state, fertilization of sperm with highly damaged DNA can result in the formation of embryos with retarded development, which may eventually arrest and result in implantation failure or pregnancy loss [18–20]. Although it has been stated the DNA repair machinery of oocytes could repair sperm DNA damages, this process is highly dependent on the degree of sperm DNA damage and the age of the female [23]. In light of these considerations, assessment of chromatin structure by TB, which has a high affinity to chromatin DNA phosphate residues, could be considered as one of the approaches for assessment of DNA integrity.

Although several direct and indirect methods are introduced for the assessment of DNA integrity and chromatin packaging in sperm, recently, it has been suggested that the TUNEL assay and the SCSA are the most reliable tests for clinical assessment of chromatin integrity and cut-off values of 30–35 percent and 10–20 percent, respectively, have been proposed for SCSA and TUNEL assay [20]. In this regard, Erenpreiss et al. (2004) demonstrated a strong significant correlation between the results of TB with the SCSA and TUNEL assay [r=0.84; r=0.8; p<0.001, respectively] (24). In addition, Tsarev et al. (2009) observed a significant difference in TB-positive spermatozoa or abnormal sperm chromatin structure between fertile and infertile men [7]. They presented a cut-off value of 45 percent for this test and concluded that the results of the TB test can be considered as a predictor for “infertility” and not “fertility” with high specificity (92 percent) and low sensitivity (42 percent).

In addition, Ajina et al. (2016) showed that the mean of abnormal sperm chromatin structure by TB and DNA denaturation by acridine orange were significantly higher in infertile men with astheno-terato-zoospermia than in fertile groups. They also observed significant correlations between these two parameters with sperm morphology and viability [10].

In support of these results, Alves et al. (2018) concomitantly assessed the sperm head area, sperm DNA fragmentation with acridine orange and the sperm head area with chromatin compaction by TB in caput, corpus and caudal regions of the cat epididymis. These authors showed that the percentage of sperm DNA fragmentation as well as major and minor defects of sperm morphology were reduced, while the percentage of DNA compaction and DNA integrity were increased as sperm move from caput to cauda. In addition, they showed that the head area of TB-stained sperm decrease as sperm move from caput toward caudal region. Therefore, the sperm head size and TB stainability could predict the quality of chromatin condensation [25]. From this study we conclude that in addition to semen analysis, TB staining could provide better information regarding chromatin integrity for the assessment of hidden anomalies in the sperm chromatin packaging.

A number of studies suggested that a major contributor of DNA damage in sperm is oxidative stress and that this phenomenon could lead to compromised sperm quality and decreased fertility outcome with advanced age [26]. In this regard, a significant negative correlation was observed between male age and reproductive outcomes in ART [27]. When Kim et al. (2013) used the TB test for assessment of sperm chromatin structure, they did not find any significant correlation between percentage of sperm abnormal chromatin structure with age, while significant correlations were observed between sperm chromatin structure with abnormal sperm chromatin condensation (r=0.594, p=0.000) and strict morphology (r=-0.219, p=0.029) [28]. These controversies could be related to sample size, evaluation method, type of sample, and age range of individuals, but one rational conclusion is that TB staining does not appear to be able to detect oxidative stress-induced DNA damage.

Sperm processing is an inevitable step for separation of normal viable spermatozoa from plasma and also from heterogeneous population, including abnormal sperm and other cells in semen samples in an ART setting. To achieve this goal, swim-up and density gradient centrifugation (DGC) are two commonly used procedures for semen preparation in the andrology laboratory. A number of studies show that these procedures can reduce the number of spermatozoa with abnormal chromatin packaging and DNA fragmentation, while some studies demonstrated centrifugation force could aggravate production of oxygen reactive species and effect DNA integrity of sperm [29, 30]. In this regard, Kim et al. (2015) assessed sperm chromatin integrity by the TB test and observed that this parameter and sperm DNA oxidation significantly increased after swim-up procedure compared to before swim-up. When they divided their participants into smoker and non-smoker groups, mean sperm DNA oxidation significantly increased after swim-up procedure compared to before in the smokers (not the non-smokers), while sperm chromatin integrity assessed by TB test was not different before and after swim-up in smoking and non-smoking men [11].

Talebi et al. (2012) assessed the sperm chromatin packaging status and DNA integrity in couples with a history of recurrent spontaneous abortion (RSA). Except sperm motility, sperm chromatin and DNA status were similar between the control and RSA group. However, the percentages of abnormal spermatozoa higher than the proposed cut-off values for various sperm chromatin tests (aniline blue>35 percent, chromomycin A3>30 percent, TB>45 percent, acridine orange>50 percent and nuclear chromatin stability assay) were significantly higher in RSA compared to the control group. Therefore, they emphasized the importance of the assessment of sperm chromatin status and DNA integrity in couples with unexplained RSA [31]. In addition, the aforementioned sperm chromatin tests were also assessed in infertile men with varicocele, and the results demonstrated that percentages of abnormal spermatozoa for each of the aforementioned tests were significantly higher in infertile men with varicocele compared to fertile men [32]. In this regard it has been shown that microsurgical varicocelectomy reduces these anomalies and significantly improves the quality of sperm chromatin, DNA integrity, and sperm parameters. Therefore, sperm function tests such as TB staining can be used to assess whether a surgical procedure like varicocelectomy has improved the quality of sperm [33].