Abstract
Several sperm DNA integrity and chromatin quality assays have been developed and used for the detection of sperm DNA fragmentation and chromosomal aberrations [1, 2]. The Comet assay is one of the most commonly used single cell gel electrophoresis methods first developed by Östling and Johanson [3]. Damaged or fragmented sperm DNA is separated from undamaged DNA by electrophoresis. Fluorescent in situ hybridization (FISH) is a hybridization method that provides specific identification of selected DNA sequences and is frequently used for cytogenetic analysis of spermatozoa. These two methods were combined as the Comet-FISH technique first by Santos and colleagues in 1997 [4]. The Comet-FISH technique is a combination of two well-known methods, the Comet assay and FISH, and is a useful detection tool for screening of whole and region-specific DNA damage [5, 6]. In addition, the analysis of labeled DNA sequences and whole chromosomes of interest and identification of region-specific DNA and overall damage and repair in single cells is possible [7, 8]. Therefore, the modification of these assays interpolates hybridization with specific fluorescent-labeled probes to selected DNA sequences of interest after unwinding and electrophoresis [6] and developed a standardized Comet assay for human sperm with additional information for telomeres or special DNA sequences of interest. Thus, specific gene sequences can be detected using the Comet-FISH technique [9].
23.1 Fluorescent In-Situ Hybridization
Several sperm DNA integrity and chromatin quality assays have been developed and used for the detection of sperm DNA fragmentation and chromosomal aberrations [1, 2]. The Comet assay is one of the most commonly used single cell gel electrophoresis methods first developed by Östling and Johanson [3]. Damaged or fragmented sperm DNA is separated from undamaged DNA by electrophoresis. Fluorescent in situ hybridization (FISH) is a hybridization method that provides specific identification of selected DNA sequences and is frequently used for cytogenetic analysis of spermatozoa. These two methods were combined as the Comet-FISH technique first by Santos and colleagues in 1997 [4]. The Comet-FISH technique is a combination of two well-known methods, the Comet assay and FISH, and is a useful detection tool for screening of whole and region-specific DNA damage [5, 6]. In addition, the analysis of labeled DNA sequences and whole chromosomes of interest and identification of region-specific DNA and overall damage and repair in single cells is possible [7, 8]. Therefore, the modification of these assays interpolates hybridization with specific fluorescent-labeled probes to selected DNA sequences of interest after unwinding and electrophoresis [6] and developed a standardized Comet assay for human sperm with additional information for telomeres or special DNA sequences of interest. Thus, specific gene sequences can be detected using the Comet-FISH technique [9].
23.1.1 Sperm Aneuploidy Screening with Fluorescent In-Situ Hybridization
Aneuploidies are numerical chromosomal aberrations of a cell/organism and these aneuploidies are the major causes of early pregnancy loss, mental retardation, developmental disorders, and infertility [10]. Aneuploidies arise from non-disjunction of sister chromatids and anaphase lagging (Figure 23.1). FISH is a molecular cytogenetic assay, which is used for detecting and measuring of sperm aneuploidy and based on binding of fluorescently labeled primers specifically to each chromosome in the sperm sample and visualization of the samples under a fluorescent microscope. A decrease or increase in the fluorescent signals indicates aneuploidy [11].
Figure 23.1 Sperm aneuploidy mechanisms and consequences.
Infertile men with aneuploidy in their somatic cells usually have impaired spermatogenesis. Therefore, these chromosomal aberrations could also be observed as sperm chromosome abnormalities [12]. Although a full detection of sperm aneuploidy should comprise all autosome and sex chromosomes, non-lethal chromosomes as well as chromosomes compatible with survival may be sufficient for sperm aneuploidy detection. These chromosomes are 13, 18, 21, X and Y [13].
Previously, sperm aneuploidy was detected using karyotype analysis. However, nowadays, it is detected using the FISH technique in sperm nuclei or using sequencing technology such as next-generation sequencing assays [14]. Sarrate and Anton [15] have described the sperm FISH assay in five steps. These steps are sample (epididymal, ejaculated or testicular sperm) processing with fixation of the cells, decondensation, annealing, post-annealing washes and visualization. Briefly, sperm chromosomes are fixed and decondensate on the slide. Fluorescently labeled DNA probes specific for the chromosomes that shall be analyzed are hybridized. After the hybridization, slides are washed and finally analyzed by means of fluorescent microscopy.
Standardized evaluation criteria must be used for correct assessment of sperm aneuploidy. Every normal spermatozoon must give one blue, green and red signal corresponding to chromosome 18, X and Y, respectively. In addition, green and red signals must be differentiated for chromosomes 13 and 21, respectively, in each normal spermatozoa as well [15]. To avoid variation of the results, sperm heads without a well-defined boundary or overlapped spermatozoa must not be counted. In the evaluation of disomic and diploidic spermatozoa, the intensity of the signal should be separated and be equal [16].
Sperm FISH analysis gives a predication of the frequency of sperm chromosomal aberrations including disomy, nullisomy, and diploidy. However, there is no concurrence on statistical analysis of sperm FISH results. Recently, a study suggested a non-parametric Wilcoxon Rank Sum test for the statistical analysis of sperm FISH results including disomy, nullisomy, diploidy and a number of 13 chromosomes (1, 2, 9, 13, 15, 16, 17, 18, 19, 21, 22, X, and Y) in human sperm nuclei from 14 fertile males using automatized FISH. In this manner, fertile males have been classified as normal or altered regarding the sperm aneuploidy with application of this approach [17]. The main limitation of this analysis is the difficulty of evaluation of all chromosomes of spermatozoa. Evaluation of clinically relevant chromosomes (13, 18, 21, X and Y) is sufficient to reveal the presence of meiotic defects [18].
Nonetheless, sperm DNA FISH analysis is recommended for men whose partners have a history of recurrent pregnancy loss (RPL) or IVF failures [11]. Hence, sperm with sex chromosome, chromosome 13 or 21 and chromosome 18 aneuploidies were found to be 2.7-, 3.3- and 6-times more frequent in men whose partners have a history of RPL compared with fertile men. In total, 40 percent of normozoospermic controls showed sperm aneuploidy in all evaluated chromosomes. Sex chromosome and sperm aneuploidy were found to be higher in men with abnormal strict morphology compare with those of men with normal morphology (>4 percent) (57 percent versus 28 percent). Additionally, no association between sperm DNA fragmentation and sperm aneuploidy studied group has been reported [19].
23.1.1.1 Indications for the Fluorescent In-Situ Hybridization Analysis in Spermatozoa
Before performing sperm FISH analysis, it is useful to select, which patients should undergo the analysis. Sperm FISH analysis is indicated in men with normal semen parameters whose partners suffer from RPL [19].
Several studies revealed a significant relation between sperm aneuploidy and decreased sperm count, motility and morphology. Thus, FISH evaluation of spermatozoa for infertile men with low sperm count [18, 20] and abnormal sperm morphology [19] have been suggested as useful methods. Rubio et al. [20] investigated the association between semen parameter and numerical chromosomal abnormalities in 63 men [20]. The rate of disomy and diploidy of chromosomes 18 and 21 and sex chromosomes (average 0.28 percent) have been found to be significantly higher in patients with oligoasthenoteratozoospermia (OAT) (<20 × 106 spermatozoa/mL [WHO 1999]) compared to normozoospermic men (0.1 percent of diploidy). Results were found to be more prominent in patients with severe oligozoospermia (<5 × 106 spermatozoa/mL) (0.45 percent). Similarly, a small study including nine severe OAT patients and four proven fertile donors showed higher autosomal disomy (0–5.4 percent versus 0.05–0.2 percent), disomy of the sex chromosome (1.6–4.9 percent versus 0.15 percent) and also diploidy (0.4–9.6 percent versus 0.04 percent) than the controls [22].
The most frequent chromosome aberration of spermatozoa of infertile males is diploidy of sex chromosomes, 24,XY and 24,YY. Therefore, a second group of candidates for FISH analysis of spermatozoa are men with 47,XXY and 47,XYY karyotypes. Men with 47,XXY and 47,XYY karyotypes produce more diploidy of sex chromosomes compared to men with normal karyotype [23].
Since spermatozoa have to be obtained from the epididymis or testicle, men with non-obstructive azoospermia (NOA) are also candidates for sperm FISH analysis. Several studies have found higher frequency of aneuploidy in testicular spermatozoa compared with those of ejaculated spermatozoa from normal controls [24, 25, 26, 27]. However, the results were found to be contradictory regarding aneuploidy frequency in spermatozoa retrieved from testicle or epididymis. The aneuploidy rate of chromosomes 18, 21,X and Y was found to be significantly (p<0.0001) higher in testicular spermatozoa of men with NOA (11.4 percent) than that observed in epididymal sperm of men with obstructive azoospermia (1.8 percent) and in ejaculated spermatozoa of healthy men (1.5 percent), respectively [27]. Similarly, Rodrigo et al. [28] observed no difference in the aneuploidy rate between the epididymal and ejaculated spermatozoa.
Recent studies have reported that chromosomal aberrations of human spermatozoa in older men are more often structural rearrangements than aneuploidies [29]. Indeed, the analysis of sperm from men older than 50 years demonstrated significantly higher percentages of sperm with damaged DNA, elevated global aneuploidy rates, and a significantly (p≤0.05) increased number of embryos with trisomy in IVF/ICSI cycles [30, 31]. Moreover, paternal aging not only leads to several alterations in the male endocrine system including lower androgen and higher follicle stimulating hormone (FSH) levels, but also to variations in testicular structure and volume leading to changes in sperm production and quality, elevated sperm DNA fragmentation [29, 31] ]. Consequently, several studies showed that men with NOA, severe OAT, severe teratozoospermia [32, 33] are good candidates for FISH analysis. Similarly, sperm samples retrieved from the testes are reported to be prone to aneuploidy compared to those retrieved from the epididymis [27, 28]. Nevertheless, men from female partners with unexplained RPL or recurrent implantation failure (RIF) may benefit from sperm aneuploidy screening [17]. However, data on the reproductive outcome after performing this examination are still missing [23].
23.1.2 Genetic Counseling in Men with Sperm Aneuploidies
The progress of assisted reproductive technologies including IVF and ICSI helps infertile men with abnormal semen parameters to father a child. Gametes from these men demonstrate increased rates of chromosomal aberrations compared with fertile men. Furthermore, higher rates of aneuploidy have been reported after preimplantation genetic diagnosis (PGD) in embryos obtained after IVF/ICSI with sperm from men with OAT and higher chromosomal alterations [18].
Genetic counseling is necessary to help these couples to understand the nature and consequences of sperm chromosomal aberrations. Likewise, genetic counseling for these patients may provide appropriate treatment and management options to increase their chances to fertilize oocytes and decrease the risk of recurrent miscarriages.
A family history of infertility, RPL, birth defects, intellectual disability, possible consanguinity and ethnic information is obtained by drawing a three-generation pedigree. Genetic counselors advise couples that the general frequency of birth defects and intellectual disabilities is about 3 percent in all pregnancies regardless of maternal age, ethnicity, or family history. Subsequently, couples are counseled for the risk of trisomy 21 (Down syndrome) and other aneuploidies related to maternal age, reproductive risks related to family history and ethnic background [11].
An inverse correlation was found between sperm aneuploidy frequency and sperm quality [34]. Sperm morphology aberrations such as multinucleate, macrocephalic, and multi-flagellate sperm were found to be related with increased sperm disomy, diploidy and polyploidy [34, 35, 36].
Sperm FISH evaluation reveals aneuploidy of a single chromosome or a global increase of aneuploidy in all evaluated chromosomes. Balanced chromosomal translocations lead to an increase in aneuploidy of a single chromosome and are known causes of infertility, repeated miscarriages and IVF failure [11]. Patients carrying balanced chromosomal translocations, including both reciprocal and Robertsonian translocations, may show variable sperm production phenotypes ranging from normozoospermia to azoospermia. Reciprocal translocations are rearrangements between two non-homologous chromosomes. Likewise, Robertsonian translocations are centric fusions of two acrocentric chromosomes. Diagnostically, PGD is recommended for men with both reciprocal and Robertsonian chromosomal translocations. Several studies demonstrated that the live birth rate might increase from 4.9 percent to more than 80 percent if PGD is used [37, 38].
Males with numerical karyotype abnormalities including non-mosaic Klinefelter’s patients [39] and Y-chromosome microdeletions are prone to develop spermatozoa with sex chromosome aneuploidy [40]. Likewise, fertile men who have fathered a child with paternally derived aneuploidy, men whose partner suffers from RPL, infertile oligozoospermic men, and even men with normal karyotypes are also at elevated risk of producing chromosomally abnormal spermatozoa [41]. Nicopoullos et al. [42] investigated the aneuploidy rates of chromosomes 13, 18, 21, X/Y in infertile men who achieved unsuccessful and successful ICSI outcome using FISH. In this study, the total aneuploidy rate (2.37 percent versus. 1.18 percent, p=0.01; unsuccessful and successful ICSI) and aneuploidy rate of chromosomes 18, X/Y and 18 + X/Y (1.48 percent versus 0.67 percent, p=0.005; unsuccessful and successful ICSI) have been found to be significantly higher in infertile men who achieve unsuccessful ICSI compared with successful ones.
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