Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection*

Rita S. Raman1,3, Philip J. Chan1,2, Johannah U. Corselli1, William C. Patton1, John D. Jacobson1, Steven R. Chan1 and Alan King1

1 Departments of Gynecology and Obstetrics and 2 Physiology and Pharmacology, Loma Linda University School of Medicine, California, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This paper postulates that in the ovary, the close association between the cumulus cells and the oocytes permits the fertilizing ability of the oocytes to be determined indirectly through cumulus cell DNA status. The objective was to use a modified comet assay to analyse cumulus cell DNA and relate the data to oocyte fertilization after intracytoplasmic sperm injection (ICSI) procedures. Oocytes were retrieved (n = 15 cases) and free-floating cumulus cells were pooled and smeared on clear glass slides to dry. Meanwhile, the denuded oocytes were injected with spermatozoa and fertilization was assessed, followed by embryo transfer. The fixed cumulus cells were stained in acridine orange, coated with a mini-gel agarose layer, lysed in alkaline buffer and electrophoresis performed. Analyses of fluorescent cell images (n = 449) showed that the tail moment was positively correlated to the percentage of fertilization after ICSI (r = 0.567, P < 0.05). In contrast, there was no correlation between tail moment and number of oocytes retrieved, total ampoules used, endometrial thickness and age of patient. The results suggested that the competence of the oocytes was associated with the cumulus cell DNA status. A unique feature here was the comet assay for archived material with obvious advantages.

Key words: alkaline comet assay/apoptosis/cumulus oophorus/intracytoplasmic sperm injection/single cell gel electrophoresis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In 1984, Östling and Johanson first introduced the single cell gel electrophoresis (SCGE) for the analysis of DNA damage in an individual cell (Östling and Johanson, 1984Go). The broken pieces of DNA loops unwinding from the relaxed supercoiled nucleus gave the appearance of a comet tail, hence giving it the name `comet assay'. They used neutral electrophoresis buffer conditions to show that the migration of double-stranded DNA loops from the damaged cell nucleus was proportional to the extent of the damage inflicted on the cell. The comet assay was modified by Singh and colleagues (Singh et al., 1988Go) using alkaline electrophoresis buffers to expose alkali-labile sites (Singh et al., 1989Go) on the DNA which increased the sensitivity of the assay to detect both single and double-stranded DNA breaks. Whereas single-stranded DNA breaks could be repaired by the cell, double stranded breaks were considered lethal or mutagenic lesions and much more damaging than single stranded breaks (Collins et al., 1997Go). The comet assay has been modified several times since its introduction and this has been reviewed (McKelvey-Martin et al., 1993Go; Fairbairn et al., 1995Go; Kizilian et al., 1999Go).

Intracytoplasmic sperm injection (ICSI) of an oocyte is frequently used in assisted reproductive technology procedures. However, for ICSI to be successful, the maturity (nuclear and cytoplasmic) of the oocyte must be considered in enabling pronuclei development and completion of the fertilization event (Eppig et al., 1996Go). Assessment of oocyte maturity based on the expansion of the corona–cumulus cell complex or follicular fluid hormonal status has proven to be less than reliable (Rattanachaiyanont et al., 1999Go). In the developing ovarian follicle, the cumulus cells are in close gap junctional communication with the oocyte. After ovulation, the cumulus cells are shed and undergo apoptosis or programmed cell death marked by DNA fragmentation (Kerr et al., 1972Go). In this study, the null hypothesis was that the close association between the cumulus cells and the oocytes permits the maturity of the oocytes to be determined through cumulus cell DNA status. The objective was to use a modified fixed cell comet assay to analyse the DNA status of the cumulus cells and relate the data to oocyte fertilization after ICSI procedures. The information from this study will help scientists and clinicians establish the simple modified comet assay for the evaluation of cumulus cells which is linked to the health of the oocyte. Furthermore, the quality of the ovulation induction regimen can be evaluated and appropriate adjustments made for the following cycle of ovulation induction.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Assisted reproductive technologies
The IVF procedure was the same as the established protocols (Edwards and Brody, 1996Go). Briefly, female patients and their partners (n = 15) scheduled for the IVF procedure were given routine examinations at the Center for Fertility and In Vitro Fertilization at Loma Linda University. The reasons for infertility ranged from male factors (n = 9) to female factors such as endometriosis (n = 2), tubal (n = 2) and unexplained factors (n = 2). For the female patient, ovulation induction was achieved after several days of leuprolide acetate suppression and initiated using recombinant follicle stimulating hormone (FSH) (average of 4 ampoules in split doses daily, 75 IU per ampoule) for about 9–12 days followed by 10 000 units of human chorionic gonadotrophin (HCG) generally administered on the 10th day where the oestradiol was over 500 pg/ml and the largest follicular diameter was over 18 mm. The thickness of the endometrial lining was also measured using ultrasound techniques on several occasions including on the day of HCG administration. Patients on leuprolide acetate cycles followed a similar induction protocol with the exception that 0.5 mg leuprolide acetate was administered beginning on day 21 of the previous cycle and ending on the last day of stimulation (Edwards and Brody, 1996Go).

At about 34–36 h after HCG administration, the oocytes were retrieved from the ovarian follicles by transvaginal ultrasound techniques and the washed isolated oocytes incubated for 4–6 h to complete maturation in vitro. The culture medium used for all protocols was human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) supplemented with synthetic serum substitute (Irvine Scientific). The percentage of oocytes with cumulus expansion was noted for each case. At the time of the ICSI procedure, the sperm cells were washed by standard wash procedures and ICSI was performed on denuded oocytes with the cumulus cells removed by hyaluronidase (Type III, Sigma Chemical Co., St Louis, MO, USA). The cumulus cells were derived from small loose clumps of cells and pooled from several large follicles. After dispersing the cumulus cells with a pipette, the cells were smeared on glass slides and air-dried. The air-dried smears were fixed in 50% methanol solution (fixative solution, Diff-Quik®; Dade Behring Inc., Newark, DE, USA) for 15 s, air-dried and stored at room temperature (23°C) in a dark drawer. The slides were batched and analysed after 2 days to 2 weeks as described below. A pilot study to demonstrate DNA damage was also carried out using the remaining cumulus cells. These cells were incubated in the presence of 100 µmol/l hydrogen peroxide for 60 min at 37°C, air-dried, fixed and processed as described below.

In the meantime, each oocyte was microscopically analysed for evidence of fertilization (two or more pronuclei in the ooplasm) after about 24 h of incubation (37°C, 5% CO2 in air) and transferred to fresh culture medium. Day 3 embryos were transcervically returned to the uterine lumen of the patients. Excess embryos were cryopreserved using standard procedures appropriate for the cell stage. Each patient was monitored for evidence of clinical pregnancy. The criteria for clinical pregnancy were both an elevated serum HCG concentration as well as the ultrasound detection of uterine sac(s) each with a beating heart.

Modified comet assay of fixed cumulus cells
The comet assay procedure was carried out with the room lights off or in diffuse lighting. The cumulus cell smears on the slides were stained for 5 min in freshly prepared acridine orange solution (Tejada et al., 1984Go). This solution was prepared by dissolving 0.2 mg acridine orange (Catalog No. 10390, United States Biochemical Corp., Cleveland, OH, USA) in 1.0 ml purified water. It was important to carry out the acridine orange staining procedure in dark or diffuse room lighting conditions to ensure that the stained cells did not become bleached.

The slides were washed with water to remove background staining and air-dried. Self-adhesive sticker labels (Catalog No. 43–540, Preaply®; Dennison Co., Framingham, MA, USA) were cut and pasted to form a rectangular frame around the stained cells on each glass slide. The stickers served to anchor the mini-agarose gel layer. The 0.8 % agarose was prepared by mixing 0.8 g agarose (Catalog No. 15510–019, Life Technologies, Gibco BRL, Grand Island, NY, USA) in 100 ml of the 1xTBE solution consisting of 13.5 g Tris-HCl pH 7.5 (Life Technologies, Gibco BRL), 6.8 g boric acid (Sigma Chemical Co.), 5 ml of 0.5 mol/l EDTA (Sigma Chemical Co.) in 1250 ml purified water, heating until dissolved and cooling to 45°C. The agarose was pipetted on to each glass slide surface placed at a 20° angle and excess agarose allowed to run off the edges. The agarose gel layer should be made as thin to allow the use of high-powered objective lens on the microscope. A coverslip was not used. Each slide was placed horizontally and once the agarose had solidified, the slides were placed in 4°C alkaline lysis buffer consisting of 1% N-Lauroylsarcosine (Sigma Chemical Co.), 1.0 mol/l TrisHCl pH 7.5 (Gibco BRL Life Technologies), 0.5 mol/l EDTA (Sigma Chemical Co.) and 0.3 mol/l mercaptoethanol (Sigma Chemical Co.) pH adjusted to >10 with sodium hydroxide pellets. The slides were in lysis buffer for 1 h to release and unwind the DNA in the cells. Then the slides were rinsed with purified water and placed submerged (1–2 mm from the surface) in 1xTBE solution (pH 10) in the electrophoresis chamber for 10 min to equilibrate. Constant voltage at 50 volts (0.02 ampoules) was applied for 40 min. Excess solution was blotted from each slide and the slide was immediately examined using an ultra-violet fluorescent microscope at x500 magnification. Over 25 cells were analysed for each patient (sample size based on Singh et al., 1988). The images (Figure 1Go) were captured by placing an inexpensive QuickCam Pro® camera (Logitech Inc., Fremont, CA, USA) over one of the microscope eyepieces and saving the 640x400 pixels images to computer hard disk. The colour of the images and background were inverted using the Windows 98 Microsoft Paint program and the pixels (red density) analysed using Paint Shop Pro 6® software (Jasc Software Inc., Eden Prairie, MN, USA). Each data point representing the pixel density was the mean of multiple points on the comet tail, computed using the averaging function on the software. For accuracy, each cell image was corrected for differences in background intensity. The tail moment of each cell was manually calculated from the mean length of the tail (from the centre of the nucleus to tail edge) multiplied by the mean density of the tail (Kent et al., 1995Go). The tail moment parameter rather than the percentage of cells with damaged DNA was measured because it has been demonstrated that the tail moment parameter was more sensitive and resolved subpopulations with subtle variations (Fairbairn et al., 1995Go; Kent et al., 1995Go).



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Figure 1. The modified comet assay for archived cumulus cells. The dried cumulus cell smear was fixed, stained in acridine orange, layered over with a mini-agarose gel, lysed and electrophoresis was performed. Analyses of the fluorescent images showed degenerating cumulus cell nuclei with (A) DNA fragmentation in the form of fan-like tails unwinding from a diminished nuclear scaffolding versus (B) little or no DNA fragmentation with darker nuclei in intact cumulus cells.

 
Statistical analysis
The number of patients to analyse was determined from power analysis (Epistat computer statistical program) based on accepting limits of 95% and 90% type I and II errors respectively. The pixel data as well as associated ICSI parameters were stored in a Microsoft Excel spreadsheet and analysed on the computer by linear regression statistics and expressed as correlation coefficients. The significance of the correlation was tested using Students' t-test statistic. A value of P < 0.05 was considered a significant correlation or relationship.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Analyses of the fluorescent images showed cumulus nuclei with DNA fragmentation in the form of fan-like tails unwinding from a diminished nuclear scaffolding (Figure 1AGo). The tail moment parameter, calculated by multiplying the DNA tail length with the density, showed a positive correlation (Figure 2Go) with the percentage of fertilization following the ICSI procedure (r = 0.567, P < 0.05). In contrast, there was no significant correlation between the percentage of oocytes that exhibited cumulus expansion at the time of retrieval and the tail moment (Table IGo). Similarly, the remaining parameters such as the number of oocytes retrieved, total ampoules used, endometrial thickness, day 3 FSH and oestradiol and age of patient were not associated with the tail moment of the cumulus cells.



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Figure 2. The tail moment of cumulus cells measured by the modified comet assay is positively correlated (r = 0.567, P < 0.05) with the percentage of fertilized oocytes by intracytoplasmic sperm injection (ICSI).

 

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Table I. Comet assay analyses of cumulus cells derived from aspirated oocytes after recombinant FSH stimulation (n = 15 cases) and relationship to percentage fertilization after intracytoplasmic sperm injection (ICSI)
 
When the age of the patient was used to divide the data into two categories, 29–34 and 35–44 years, only the total number of ampoules used per cycle was different (Table IIGo). As expected, patients in the age 35–44 group used significantly (P < 0.05) more ampoules of recombinant FSH for the ovulation induction treatment. The mean percentage of ICSI fertilization in the age 35–44 group was numerically higher when compared with the age 29–34 group due to two data points approaching outlier criterion. Although the sample size was small, statistical analysis was adequately robust to show a lack of significant difference in percentage ICSI fertilization between these two groups.


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Table II. Basic parameters of assisted reproductive technology cases (n = 15) involving ICSI categorized by age group
 
In contrast to the total ampoules parameter, the remaining parameters including comet tail moment of the cumulus cell were not different between the two age groups. The data from both groups were pooled for the correlation analysis and presented in Figure 2Go.

There were five clinical (two were from the age 35–44 group) and one biochemical pregnancies (40%) among the 15 patients. The mean tail moment of cumulus cells from patients who were clinically pregnant was numerically lower (2235.4 ± 269.8 squared pixel units; mean ± SEM) but was not significantly different from non-pregnant patients (2412.2 ± 257.8 squared pixel units).

A pilot study to assess cumulus cell DNA damage by peroxides showed significant DNA fragmentation (P < 0.05) with the 100 µmol/l hydrogen peroxide treatment (tail moment 1924.1 ± 155.9 versus 1642.9 ± 126.0 squared pixels; mean ± SEM in the control).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The comet assay is based on the principle that alterations in nuclear DNA such as fragmentation, unwinding, or strand breakages during apoptosis or necrosis will result in the appearance of a trailing tail of DNA loops from the nucleus after electrophoresis (Östling and Johanson, 1984Go; Singh et al., 1988Go). In this study, the oocytes that became fertilized after the ICSI procedure were positively associated with cumulus cells that had increased fragmented DNA. This suggested that the fertilizing ability of the oocyte was associated with the cumulus cell DNA status. At first glance, this might appear unusual but actually the analyses were based on shed cumulus cells that were once in contact with the oocyte (Eppig et al., 1996Go). These apoptotic cumulus cells (Hakuno et al., 1996Go) that fall away from the oocyte would be the signal that the oocyte has attained competence for fertilization, similar to the removal of scaffolding from a finished building. In contrast, the immature oocytes would be accompanied by cumulus cells that were still growing with intact nuclear DNA. Note that granulosa cells were not examined in this study: the response might have been different (Tilly 1996Go; Nakahara et al., 1997Go). The fertilizing ability of the oocyte appeared to be related to nuclear maturity and not cytoplasmic maturity as shown by the lack of correlation to expanded cumulus assessments (Rattanachaiyanont et al., 1999Go).

The age of the patient did not influence the degree of DNA fragmentation, in contrast to animal studies (Perez and Tilly, 1997Go). Although there is controversy on whether or not the cell cycle stage affected the comet assay results (Olive and Banath, 1993Go; Salagovic et al., 1997Go), it was not a factor here when analysing cumulus cells that have stopped proliferating. However, it was not known if the luteinization process affected the comet assay outcome.

The use of the ICSI procedure bypassed the need to consider the status of the sperm DNA and focused directly on the oocyte instead. Hence, the occurrence of the fertilization event characterized by the presence of the two pronuclei in the ooplasm was dependent on the status of the oocyte, which is in synchrony with the DNA status of accompanying cumulus cells. Although the analyses of cumulus cells from individual follicles might enhance the prediction of fertilization, the analyses of pooled cumulus cells appear to be adequate to make the correlation with fertilization. This was similar to judging the time to trigger ovulation in individual patients based on an analysis of the serum oestradiol concentration derived from a mixture of follicle types.

A unique feature of this study was the development of the modified comet assay to assess archived cumulus cells that were dried and fixed on a glass slide. Previous reports utilized cells, particularly lymphocytes, suspended in a mini-agarose gel layer (McKelvey-Martin et al., 1993Go; Fairbairn et al., 1995Go). At the beginning, it was not known if the archived cells dried on glass slides would produce comet tails in the layer of agarose gel. A pilot study was carried out that exposed freshly retrieved cumulus cells to 100 µmol/l hydrogen peroxide for about 1 h before being smeared on glass slides (Figure 1Go). The results showed clear comet tails representing DNA fragmentation after hydrogen peroxide treatment thus verifying the usefulness of the archived cell comet assay.

The advantages of this assay included simplicity, low cost, elimination of cell viability testing step, an intra-assay coefficient of variation of 28.2%, a reduction of three-dimensional visual artefacts associated with suspended cells and permitting the DNA analysis to be carried out at leisure. Drawbacks of the assay included the requirement of a fluorescent microscope, computer and camera for analyses. An important difference to note was that the voltage or electrophoresis time reported for the regular comet assay protocol (Klaude et al., 1996Go; Rojas et al., 1999Go) was doubled. This adjustment was made after several pilot experiments based on the lower voltage or time failed to pull out loops of fragmented DNA from the lysed cumulus cell nucleus.

The pregnancy outcome was not associated with the cumulus cell DNA integrity. This was consistent with the finding of a lack of correlation between parameters that determine pregnancy such as age, endometrial thickness or day 3 FSH, and the cumulus cell DNA tail moment. Predicting pregnancy outcome after assisted reproductive technology treatment is complex and depends on many factors, such as hormonal events, age of the female patient, the injected spermatozoa having normal DNA, quality of embryos, ease of embryo transfer and receptive conditions in the uterus (Edwards and Brody, 1996Go). Thus, it was not surprising that an assessment of the cumulus cells linked DNA integrity of these cells to fertilization events and not pregnancy outcome.

In summary, the data suggested that the fertilizing ability of the oocytes was positively correlated to the cumulus cell DNA status. A unique feature here was the development of the comet assay for archived cumulus cells. More studies are still needed to refine the modified comet assay protocol, which include the use of sensitive stains such as Sybr Green and Sybr Gold and increasing the resolution of the images with higher quality digital cameras. Ultimately, the development of the versatile comet assay will lead to a better non-invasive method to assess oocyte competence and improve current assisted reproduction technologies.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank members of the Loma Linda University Gynecology and Obstetrics Medical Group Inc., the Loma Linda University Center for Fertility and In Vitro Fertilization and the Residency Program for their help and support in the preparation of this manuscript.


    Notes
 
3 To whom correspondence should be addressed at: Department of Gynecology and Obstetrics, 11234 Anderson St, Suite 3401,Loma Linda University School of Medicine, Loma Linda, California 92354, USA. E-mail: pchann{at}yahoo.com Back

* Presented in part at the 56th Annual Meeting of the American Society for Reproductive Medicine, San Diego, California, USA, October 21–25, 2000. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on August 18, 2000; accepted on February 6, 2001.