We are witnesses of a dramatic increase in the extent of knowledge of human gamete biology and reproductive medicine. Andrological investigations still rely on a thorough history and physical examination of the male partner. Standard semen analysis is routinely used to evaluate the male infertility; however, some researchers believe that sperm functional test should also be used with standard semen analysis for evaluation of male fertility. The success rate of fertilization is greatly affected by the quality of sperm used for insemination [18
One of the central events of spermiogenesis is substituation of the chromatin proteins (especially histones) by protamines, allowing a different structural organization to take place in the sperm nucleus. This type of organization provides sperm with a highly condensed nucleus and also protects sperm DNA against the enzymatic attack of nucleases and polymerases. Due to the tight packaging afforded by the protamines, any modification or absence of these proteins lead to an anomaly in the packaging process of sperm nucleus and influence sperm quality (morphology) and fertilizing capacity. Abnormalities such as loosely packaged chromatin and damaged DNA have already been observed in poor quality semen samples [10
]. Pervious studies have shown that protamine deficiency measured by CMA3 staining independently effect fertilization [1
]. However abnormality in sperm chromatin can take place at several levels including 1) histone/protamine replacement, 2) absence of protamine, 3) epididymal maturation, and 4) chromatin stability during ejaculation. Therefore during this study we observed that number of CMA3 and AB positive spermatozoa are significantly higher in patients with spontaneous recurrent abortion compared to fertile men. As these two tests reflects the quality of sperm chromatin, this can be concluded that semen form men whose partners suffer from spontaneous recurrent abortion has low chromatin quality especially protamine deficiency. We also found that progressive sperm motility (%) and abnormal sperm morphology are strongly correlated to percentage of positive CMA3 and AB positive spermatozoa in a negative fashion. This shows that abnormal sperm chromatin packaging will result in destructed semen parameters. This result is in concordance with many others authors, who have concentrated their attention on the predictive value of morphology, using WHO standards or strict criteria [21
], and it is felt that normal morphology is indicative of normal sperm function while high level of abnormal spermatozoa in ejaculation is associated with reduce fertilization potential. Even though the role of sperm morphology in prediction of pregnancy and in vitro fertilization outcome is well established in literatures [13
], some authors question the sole predictive role of morphology in prediction of male fertility potential [24
]. Also, when studying the role of sperm morphology in relation to fertilization rate in ICSI, literature shows that there is no clear relation between percentage of spermatozoa with normal form and fertilization, cleavage, and percentage outcome of ICSI [25
Positive CMA3 staining has been shown to be increased in the sperm cells of the infertile patients [9
]. In some studies, CMA3 fluorochrome has been used to evaluate the sperm quality and fertilization capacity. Sakkas et al. [27
] found that men who had a high percentage of anomalies in their chromatin, i.e. >30% CMA3 fluorescence and >10% nicks, had more than double the number of unfertilized oocytes containing spermatozoa that had remained condensed. Bianchi et al. [2
] and Lolis et al. [13
] showed 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. On the other hands, Oliva [3
] found that the percentage of protamines in the fertile men was the same as that in infertile patients with normal seminal parameters, but it differed in the patients with abnormal seminal parameter. It seems likely that poor chromatin packaging may contribute to failure in the decongestion process in specific cases [15
]. In our study, although all the semen parameters were normal according to WHO criteria in both groups, patients with spontaneous recurrent abortion had significantly higher percentage of abnormal morphology and lower percentage of progressive motility. Higher percentage of CMA3 and AB positive spermatozoa was observed in case group while all semen parameters were normal.
AB staining detects the presence of histones and, therefore, indirectly infers the presence of lower amounts of protamines in the sperm nucleus [28
]. Some studies have reported a correlation between staining of spermatozoa with AB and decreased percentage of normal spermatozoa [28
]. Franken et al. [16
] showed that there was significantly higher percentage of AB positive spermatozoa among men with teratozoospermia when compared with normozoospermic (51% vs. 26%). Despite normal semen analysis in this study, the mean percentage of positive AB staining was 31.6% in the case group and 14.1% in the control group (p<0.001
Among the assays that have been developed to assess DNA damage, the most frequently used in published clinical studies is the Acridine orange staining, which measures the stability of sperm chromatin in acid media with acridine orange. The dye gives rise to green fluorescence when bound to intact DNA and red when bound to fragmented DNA. The proportion of sperm with fragmented DNA is determined by flow cytometric analysis. Using light microscopy for this test is one of the limitations of this study. In some studies, AO test has been used for evaluation of sperm quality. Angelopoulos et al. [29
] believed that AO staining does not predict fertilization efficiency or pregnancy outcome in IVF cycles. Oppositely, Virant-Klun et al. [30
] found that sperm single-stranded DNA, detected by AO staining, reduces fertilization and quality of ICSI-derived embryos. In our study the percentage of AO positive spermatozoa did not differ between patients and controls that indicates that sperm denaturation may not play a role in spontaneous recurrent abortion.
To the best of our knowledge, it is the first study using CMA3, AB and AO test to evaluate the male factor in couples suffering from spontaneous recurrent abortion. This study sheds light on the pathogenesis of spontaneous recurrent abortion. However, the results of this study indicate that low sperm chromatin quality is one of the common etiologies of spontaneous recurrent abortion. So assisted reproduction techniques including ICSI and IVF using CMA3 and AB negative spermatozoa could be a novel treatment option in these patients.
In conclusion, CMA3 and AB staining are useful tools for evaluating of the sperm’s chromatin quality in couples with spontaneous recurrent abortion, because we found that there is a higher percentage of CMA3 and AB positive spermatozoa between the patients and controls. This study also showed that abnormal sperm parameters including progressive motility and abnormal morphology are correlated with protamines deficiency detected by both CMA3 and AB staining.