Abstract
Sperm are highly specialized cells, evolved to function as vehicles for the transport of the paternal genome to the oocyte. The sperm cell is characterized by a distinct head, mid-piece and tail, structured for a streamlined function. The sperm head consists of the haploid paternal genome (23 chromosomes), packed in a specific tight manner with the help of specialized proteins called protamines. The mid-piece consists of the centrosome and mitochondria, organelles that provide energy for sperm propulsion from the tail. The unique sperm structure, complimented with its motility, helps the sperm to swim through the male and female reproductive tract and penetrate the egg. Therefore, the primary function of the sperm is to successfully deliver the paternal genome to the oocyte.
21.1 Introduction
Sperm are highly specialized cells, evolved to function as vehicles for the transport of the paternal genome to the oocyte. The sperm cell is characterized by a distinct head, mid-piece and tail, structured for a streamlined function. The sperm head consists of the haploid paternal genome (23 chromosomes), packed in a specific tight manner with the help of specialized proteins called protamines. The mid-piece consists of the centrosome and mitochondria, organelles that provide energy for sperm propulsion from the tail. The unique sperm structure, complimented with its motility, helps the sperm to swim through the male and female reproductive tract and penetrate the egg. Therefore, the primary function of the sperm is to successfully deliver the paternal genome to the oocyte.
Abnormalities in the form of DNA strand breaks, Y chromosome microdeletions, alterations in chromosome number, and distorted epigenetic regulation have been reported in mature sperm and such sperm carrying the abnormal sperm chromatin are known to be delivered to the oocyte [1]. Hence, in recent years the characterization of such sperm chromatin abnormalities has gained importance in an effort to establish a molecular association to male reproductive health. A number of research groups worldwide have studied sperm DNA fragmentation, and DNA strand breaks are considered as the common property of sperm and are widely associated with male reproductive health and assisted reproductive treatment success [2]. There are a variety of tests available to measure the level of DNA strand breaks in sperm and the most commonly used tests are the Comet assay, Terminal deoxyuridine Nick End Labelling (TUNEL) assay, Sperm Chromatin Structure Assay (SCSA), and the Sperm Chromatin Dispersion (SCD) Assay or Halo test. These tests could be broadly classified into direct methods (Comet and TUNEL assays), and indirect methods (SCSA and SCD assays) of sperm DNA strand breaks measurement [3]. Each of these assays determines different aspects of sperm DNA strand breaks. In this chapter, we discuss in detail the Comet assay and its clinical importance.
21.2 The Principle of the Comet Assay
The principle of the assay is based on the separation of broken DNA strands under the influence of an electric field facilitated by the charge and size of the broken strands [4]. Following separation, the large strands of intact DNA remain in the comet’s head, whereas single- and double-stranded broken DNA fragments migrate into the comet’s tail. Therefore, the sperm with high levels of DNA strand breaks show an intense comet tail and increased comet tail length [5]. Additional parameters have been used to increase the efficiency of the test such as the diameter of the nucleus, olive tail moment, and comet tail length [5].
Unlike in somatic cells, the sperm chromatin is organized in a specific manner with the help of protamines and such compact structure prevents the complete access to the DNA strand breaks.
In order to expose the broken DNA strands from the sperm nucleus, the Comet assay involves the following steps: sperm cells are mixed with agarose and layered on a microscopic slide. Detergents and a high salt concentration are used to lyse their cell membranes, and remove nuclear proteins (protamines and histones), which relax the DNA into a supercoiled nucleoid structure. The slides are then incubated in a neutral or an alkaline buffer: in the neutral buffer only DNA with double-strand damage is measured due to the lack of the separation of double-strand DNA, while in the alkaline buffer, single- and double-strand breaks are detectable due to the unwinding of the strands [5]. The nucleoids are then subjected to an electrophoretic field resulting in the migration of the broken strands of DNA through the agarose, resulting in a comet like structure when visualized using a fluorescent DNA binding dye. The non-fragmented DNA remains in the comet’s head, whereas the fragmented DNA migrates, forming the comet’s tail [5].
21.3 Advantages and Disadvantages of the Comet Assay
The Comet assay is inexpensive and on average it costs about one USD in chemicals and supplies to analyze a sample. It is considered as one of the most sensitive techniques available to measure sperm DNA damage. The alkaline Comet assay can be used in various cell types including sperm. Unlike other assays, the Comet assay requires only a few cells, approximately <6 × 104 cells per slide. The results of each comet analyzed correspond to the level of DNA damage from individual cells [5].
In human sperm, the DNA is packed with specialized proteins called protamines. Complete decondensation or the removal of protamines is required to expose the fragmented DNA strands and such a step could be obtained only using Comet assay. The significance of the Comet assay in assessing male infertility and the clinical association with assisted reproductive treatment outcomes are discussed in Section 21.7 of this chapter.
There are a few disadvantages of the Comet assay. The assay lacks standardized protocols, which makes it difficult to fully understand and relate the results of different laboratories. The assay condition is known to damage the alkaline-labile sites, making it difficult to discriminate between endogenous and induced DNA breaks. Unlike sperm, somatic cells contain excess levels of RNA, in such case DNA damage can be overestimated by the Comet assay due to the presence of residual RNA which can increase the background intensity during analysis [6]. The assay is also criticized for underestimation of DNA damage due to entangling of DNA strands. In scenarios where the chromatin decondensation of sperm is incomplete, then the level of DNA damage will be underestimated. Overlapping comet tails decrease the accuracy of the assay, small tail fragments may be lost, and excessively small fragments are difficult to visualize [7]. The assay is laborious, can have a high level of inter-laboratory variation, and hence it is not commonly used under clinical settings [8].
21.4 Methodology of the Comet Assay
This is a modified and improved protocol commonly used [9]. All steps are performed at room temperature unless otherwise specified.
1. Prepare normal melting point (NMP) agarose gel (0.5 percent gel) in phosphate-buffered saline (PBS) and low melting point (LMP) agarose gel (0.25 percent gel) in PBS. Heat flasks in the microwave or burner to completely melt the agarose. Place the NMP gel in the 45°C water bath and the LMP gel in the 37°C water bath, until use.
2. Carefully pipette 200 μL of NMP agarose gel onto the fully frosted surface of the glass slide and immediately cover with a 24 × 50 mm coverslip. Leave the slides on the bench for 15 minutes to allow the agarose to solidify. The function of the NMP agarose layer is to hold the LMP agarose (containing sperm) firmly to the slide surface.
3. Adjust the concentration of sperm to 6 × 106/mL using PBS. Place, 10 μL of the sperm sample into a 0.5 mL Eppendorf tube. After the NMP agarose is solidified, add 75 μL of LMP agarose to the sperm in tube, mix gently using a pipette, add dropwise on top of the layer of NMP agarose gel and immediately cover with a coverslip. Allow the gel to solidify on the bench for 15 minutes. The LMP agarose acts as a platform to hold the sperm cells during the experimental process for electrophoresis.
4. Incubate the agarose slides without coverslip in lysis buffer (2.5 M NaCl, 100 mM Na2EDTA, and 10 mM Tris–HCl, pH 10) with 1 percent Triton X-100 for one hour at 4°C. The function of the lysis buffer is to remove the cell wall of the sperm.
5. After lysis, remove the slides out of the jar, add dithiothreitol (DTT) to obtain a final concentration of 0.5 mM/mL, mix well and incubate for 30 minutes at 4°C. Following incubation with DTT, remove the slides out of the jar, add lithium diiodosalicyclate (LIS) to obtain a final concentration of 0.2 mM/mL, mix well and incubate for 90 minutes. Decondensation of sperm chromatin takes place with the help of DTT and LIS. During this process, the sperm nuclear proteins are denatured facilitating the decondensation process.
6. Remove the slides from the jar and place them in a horizontal gel electrophoresis tank with the gel surface facing upwards. For the alkaline Comet assay, add freshly prepared alkaline electrophoresis buffer (300mM NaOH and 1mM EDTA, pH 13.0) and incubate the slides for 20 minutes. The high pH conditions help to unwind the double-strand DNA facilitating the exposure of single- and double-strand DNA breaks.
The neutral Comet assay is performed with neutral buffer (300 mM NaOAc and 100 mM Tris-HCl, pH 8.3), during which only double-strand DNA strand breaks are accessed due to the lack of the separation of DNA strands.
7. Electrophoresis is performed with alkaline buffer or neutral buffer corresponding to the type of assays. The electrophoresis is performed for 10 minutes by applying a current at 25 V (0.714 V/cm) adjusted to 300 mA by adding or removing (±1–20 mL) buffer in the tank. During this step, the broken DNA strands migrate towards the anode based on their size. Smaller fragments move faster than the larger fragments through the LMP agarose.
8. Following electrophoresis, drain the slides of any electrophoresis buffer, place them on a tray and flood them with three changes of neutralization buffer (0.4M Tris; pH 7.5) for five minutes each. This step reduces the pH of the gel towards neutral conditions in order to facilitate the binding of ethidium bromide (EtBr) stain to DNA fragments.
9. Drain the slides thoroughly to remove the neutralization buffer. Add 50 μL of fresh EtBr solution (20 μg/mL) to each slide and cover with a coverslip. View the slides using a fluorescence microscope with appropriate Comet software. Analyze 50–100 comets per slide.
The intact DNA stays in the Comet’s head and the broken DNA fragments migrate into the Comet’s tail, which can be visualized based on the intensity of fluorescence emitted by the Comets.
The comet software is designed to convert the intensity of fluorescence into parameters such as percent head DNA, percent tail DNA, tail extent moment, olive tail moment, and tail length. Most studies in the existing literature have used the percent tail DNA as the classic parameter to describe the level of fragmentation in the sperm cell [2, 10, 11, 12, 13]. The level of DNA fragmentation can be expressed in a numerical continuous value by obtaining a mean across all the comet’s tail DNA of a given sample. Alternately, each comet can be converted into a binary value based on the level of DNA fragmentation. For example, each Comet can be classified into damaged sperm (if the percent tail DNA exceeds 25 percent), resulting in the expression of the percentage of damaged or normal sperm present in a given sample [2, 13, 14].
21.5 Threshold Values
The level of DNA fragmentation measured by the alkaline Comet assay is always greater than the neutral Comet assay for a given sample as the alkaline version measures both single- and double-strand DNA breaks, whereas the neutral Comet assay measures only the double-strand DNA breaks. As a result, most studies in the existing literature have used alkaline Comet assay as a test to measure sperm DNA damage compared to the neutral version. Therefore, in this chapter we will discuss the clinical importance of DNA fragmentation as measured by the alkaline Comet assay.
Studies using the alkaline Comet assay have established two critical threshold values, first a diagnostic value to determine male infertility, determined by comparing the level of DNA fragmentation in the sperm of fertile and infertile men. A threshold value of 25 percent mean DNA fragmentation has been established to diagnose male infertility [2].This study reported that 95 percent of the fertile population had sperm DNA fragmentation below 25 percent, whereas only 10 percent of the infertile group had sperm DNA fragmentation below 25 percent, while the mean DNA fragmentation of sperm from infertile men was 57.92±2.67 percent and that of donors was 12.47±1.67 percent (p<0.001) [2]. Using this threshold value of 25 percent DNA fragmentation for the diagnosis of male infertility, the OR (95 percent CI) was 117.3 (12.73–2731.83). Men with DNA fragmentation more than this threshold value had a relative risk for infertility of 8.75 (95 percent CI: 4.48–17.08), with an area under the ROC of 0.970 cm2, 63.6 percent sensitivity and 98.5 percent specificity [2].
A second threshold value was established in [14] to determine the success of assisted reproductive treatment (ART) by comparing the level of sperm DNA fragmentation in couples who were successful following ART with those who were unsuccessful. This study shows that the mean percentage of sperm DNA fragmentation was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples undergoing in vitro fertilization (IVF) in both the native semen (51.7±23.6 percent versus 39.5±17.9 percent; p=0.004) and the density gradient centrifugation (DGC) prepared sperm for clinical use (36.8±21.6 percent versus 26.9±14.6 percent; p=0.01). Using the threshold values of 56 percent for the native semen and 44 percent for the DGC sperm, the odds ratios (95 percent CI) calculated for clinical pregnancies were 4.52 (1.79–11.92) and 6.20 (1.74–26.30), respectively.
In a later study, Simon et al. [15] established another threshold value using the parameter percentage of sperm with DNA fragmentation. In this clinical study, 82 percent sperm with DNA fragmentation (or 18 percent sperm with normal DNA) was established as a threshold to have high prognostic value to determine the chances of a clinical pregnancy, in vitro. Using this threshold value of 82 percent sperm DNA fragmentation, the OR (95 percent CI) to determine a clinical pregnancy was 7.00 (3.62–13.94). Men with DNA fragmentation higher than this threshold value had a relative risk for an unsuccessful clinical pregnancy of 1.89 (95 percent CI: 1.51–2.38), with 85.3 percent sensitivity and 45.0 percent specificity. The threshold value of 82 percent of sperm with DNA fragmentation is comparable to the threshold value of 56 percent mean Comet DNA fragmentation published previously by [14].
21.6 Laboratory and Clinical Interpretation of Threshold Values
The threshold value of 82 percent sperm with DNA fragmentation and the 56 percent threshold of mean sperm DNA fragmentation in the native semen established for the alkaline Comet assay is significantly higher than the threshold values used for SCSA and TUNEL assays [14]. This can be attributed to the sensitivity of the alkaline Comet assay, where in each sperm, the assay can measure variable levels of DNA fragmentation ranging from 0 to 100 percent. In sperm absent of DNA fragmentation – 100 percent of the DNA remains in the comet’s head, whereas in sperm with extensive DNA fragmentation the entire nuclear DNA migrates to the comet’s tail, leading to absence of comet’s head [5].
The biology and structural organization of the sperm nucleus restricts the complete evaluation of sperm DNA fragmentation. The sperm nuclear DNA is crystalline in nature due to the supercoils of negatively charged DNA strands around positively charged protamines resulting in a highly compact DNA-protein complex. The SCSA and TUNEL assays permeabilize the cell wall to reach the nuclear DNA, which may not be sufficient for the evaluation of complete DNA fragmentation [13]. However, the sensitivity of the alkaline Comet assay to measure sperm DNA fragmentation can be attributed to its elaborate methodology [16]. During the Comet protocol, the sperm cells are lysed for an hour (step 4) resulting in the complete removal of cell wall. The decondensation of DNA (step 5) for two hours facilitates the removal of nuclear proteins. During this step the chemicals DTT and LIS reduce the disulphide bonds of protamines and nucleo-histones, thereby inducing relaxation of supercoiled DNA. The unwinding of the double-stranded DNA into single-strand at high pH conditions (step 6) facilitates the exposure of single-strand DNA breaks. These steps result in the complete exposure of double- and single-strand breaks throughout the entirety of relaxed chromatin, in contrast to other assays where perhaps more peripheral DNA strand breaks are determined [5].
The alkaline Comet assay has the ability to show a distinct distribution of damaged sperm populations ranging from 0 to 100 percent, which could be depicted graphically. The study by Simon et al. [5] demonstrated the distribution of sperm with DNA fragmentation by categorizing the scattering into three distinct patterns (A, B and C), based on the level of DNA fragmentation exhibited by the sperm population when depicted on a graphical plot. In type A distribution plot, the peak of sperm population lies between 0 and 25 percent, in type B distribution plot (25–75 percent) and in type C distribution plot (>75 percent). This study [5] reported that all the fertile donors exhibited type A distribution, while the infertile patient populations exhibited type A (45 percent), type B (41 percent) and type C (14 percent) distribution. Type B distribution could be further categorized into two types: B1 when the peak is between 26 and 50 percent damage and B2 when the peak is between 51 and 75 percent. The same study also showed that 66 percent of couples with type A distribution plot were successful after ART, whereas couples with type B1, B2 and C distribution plots achieved 56 percent, 44 percent and 33 percent clinical pregnancy, respectively [5]. Such distinct distribution patterns of sperm DNA fragmentation can be established only using the alkaline Comet assay as a result of its elaborated methodology to expose the DNA fragmentation present within the nucleus.
21.7 Clinical Significance of the Alkaline Comet Assay
DNA fragmentation is known to be more prevalent in the sperm of infertile men and may contribute to their declined fertility status. An increase in the level of DNA fragmentation in infertile men can be attributed to abnormal histone to protamine exchange, abnormal protamine content and ratio, and reduced antioxidant activity in the seminal plasma [17]. Dorostghoal et al. [18] reported that infertile men showed significantly higher percentage of sperm with fragmented DNA in comparison with fertile subjects using the neutral Comet assay. With the help of the alkaline Comet assay, Simon et al. [2] reported a significant difference in DNA fragmentation of sperm from infertile men compared to fertile donors. A recent meta-analysis including 28 studies drawn across all four sperm DNA fragmentation assays showed a significant increase in DNA fragmentation of sperm from infertile men [19]. This meta-analysis concluded that measurement of sperm DNA fragmentation is relevant for male infertility and higher accuracy in detecting sperm function compared to the conventional semen parameters [19].
A literature search resulted in the identification of nine studies associating sperm DNA fragmentation measured by the alkaline Comet assay with fertilization rate (Tables 21.1 and 21.2). The systematic review suggested that all studies including IVF cycles reported a significant inverse relationship between sperm DNA fragmentation and fertilization rate, but no such association was observed with studies using only ICSI cycles. The differential adverse effect of sperm DNA fragmentation on IVF and ICSI fertilization may be due to the fact that conventional IVF occurs “naturally” as a result of sperm-oocyte interaction, whereas during ICSI treatment this natural selection process is bypassed due to the manual selection and injection of morphologically normal and motile sperm by the embryologists, which may increase the probability of selecting sperm with low DNA fragmentation [20, 21]. Based on the available literature, we can conclude that sperm DNA fragmentation may be associated with IVF fertilization rate but not with ICSI fertilization rates.
Table 21.1 Summary of Studies Associating Sperm DNA Fragmentation Measured by the Alkaline Comet Assay with Fertilization Rate and Embryo Quality
| IVF | ICSI | IVF + ICSI | ||||
|---|---|---|---|---|---|---|
| Studies (n) | Cycle (n) | Studies (n) | Cycle (n) | Studies (n) | Cycle (n) | |
| Studies reporting no effect on fertilization rate | 0 | 0 | 4 | 243 | 1 | 60 |
| Studies reporting adverse effect on fertilization rate | 3 | 362 | 0 | 0 | 1 | 238 |
| Studies reporting no effect on embryo quality | 0 | 0 | 2 | 138 | 0 | 0 |
| Studies reporting adverse effect on embryo quality | 4 | 402 | 1 | 28 | 2 | 298 |
IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection
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