Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques

K.L. Larson1,4, C.J. DeJonge2,3, A.M. Barnes3, L.K. Jost1 and D.P. Evenson1

1 Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, 2 Department of Obstetrics and Gynecology, University of Minnesota, Minneapolis, MN 55454 and 3 Department of Obstetrics and Gynecology, University of Nebraska Medical Center (UNMC), Omaha, NE, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 ± 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 ± 3.2). No pregnancies resulted if >=27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.

Key words: chromatin structure/human fertility/in-vitro fertiliza- tion/intracytoplasmic sperm injection/SCSA


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Identification of sperm parameters that predict the outcome of assisted reproductive techniques would allow physicians to counsel patients on their chance of pregnancy following IVF and intracytoplasmic sperm injection (ICSI). The value of this information has been the impetus for numerous studies designed to identify a single parameter or group of parameters that have significant predictive value for assisted reproductive techniques' (ART) outcomes. Sperm strict morphology criteria have been used effectively to develop thresholds that are predictive of fertilization and pregnancy rates following IVF (Kruger et al., 1986Go; Vawda et al., 1996Go). In addition, better embryo quality has been associated with normal sperm morphology (Van der Zwalmen et al., 1991; Parinaud et al., 1993Go; Janny and Ménézo, 1994Go). Many investigators (Claassens et al., 1992Go; Sukcharoen et al., 1995Go; Hammadeh et al., 1997Go; Parinaud et al., 1997Go) have shown the importance of optimizing conventional sperm parameters, including morphology for IVF success. However, the importance of sperm morphology for ART's success has become increasingly ambiguous with the advent of ICSI which overrides many inherent boundaries for natural selection (Hoshi et al., 1996Go).

The ability of ICSI to bypass oocyte-associated boundaries has facilitated the successful treatment of males with severe oligozoospermia, teratozoospermia, azoospermia and immotile sperm cells (Engel et al., 1996Go). Some studies indicate that fertilization, embryo cleavage and pregnancy following ICSI are independent of conventional sperm parameters (Hammadeh et al., 1996Go; Oehninger, 1996Go). However, other studies have reported that impaired sperm quality, characteristic of ICSI patients, leads to a lower percentage of embryos that form blastocysts (Shoukir et al., 1998Go), poor blastocyst quality (Janny and Ménézo, 1994Go) and high abortion rates (Sanchez et al., 1996Go). Janny and Ménézo (1994) showed the strong paternal influence in preimplantation embryo development, reporting that both the overall number of blastocysts obtained and the number of patients having at least one blastocyst were reduced in patients with severely impaired sperm quality.

The influence(s) of sub-optimal sperm chromatin integrity on post-embryonic development is the subject of intense investigation. Spermatozoa from infertile men have a higher frequency of chromosomal abnormalities (Moosani et al., 1995Go), lower resistance to sodium dodecyl sulphate (SDS)-induced decondensation (Colleu et al., 1988Go), poor DNA packing quality (Hofmann and Hilscher, 1991Go; Bianchi et al., 1996Go; Filatov et al., 1999Go), increased DNA strand breaks (Lopes et al., 1998Go; Irvine et al., 2000Go) and susceptibility to acid-induced DNA denaturation in situ (Evenson et al., 1980Go; Evenson, 1999Go; Evenson et al., 1999Go; Spano et al., 2000Go). It is unclear if ART are effective in compensating for poor chromatin packaging and/or DNA damage or if sub-optimal chromatin integrity may contribute to the poor implantation rate (<20%) in the majority of ART patients (Edwards and Beard, 1999Go). Understanding of the effect of the paternal genome becomes more critical as we become less discriminatory with the maturity and quality of paternal nuclear material which is introduced into the oocyte (Sakkas, 1999Go).

The sperm chromatin structure assay (SCSA) holds promise for determining the importance of chromatin structure in ART outcomes. SCSA is an unbiased, quantitative assessment of sperm chromatin integrity defined as susceptibility of DNA to acid-induced denaturation in situ. SCSA parameters are stable within individuals over time if men are not exposed to reproductive stressors (Evenson et al., 1991Go). In addition, SCSA parameters are correlated with DNA strand breaks (Sailer et al., 1995Go; Aravindan et al., 1997Go) and decreased fertility in vivo (Evenson et al., 1980Go, 1999Go). However, SCSA parameters are not strongly correlated with World Health Organization (WHO) parameters including concentration, motility and morphology (Evenson et al., 1991Go). Therefore, SCSA parameters are independent and may have predictive value beyond WHO parameters for patient success in ART. The objective of this study was to examine the relationship between neat and washed semen sample SCSA parameters and fertilization, embryo grade and pregnancy outcome following ART.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Semen analysis
Semen samples were collected by masturbation from 24 men (mean age of 35 ± 4.8 years) after 2–7 days of abstinence and on the day of their partner's (mean age of 34 ± 5.1 years) oocyte aspiration for IVF or ICSI at the University of Nebraska Medical Center. Semen analysis was performed following WHO (1992) guidelines. Means for sperm concentration (x106/ml), motility (%), forward progression (%) and qualitative morphology (%) of neat and washed samples are shown in Table IGo. Strict morphology of neat samples was also assessed for 17 of the patients during a semen analysis before the day of IVF or ICSI (Table IGo). No individuals with varicocele, obstructive and non-obstructive azoospermia, or underlying reproductive tract pathologies were included in the study population. Of the original sample of 24 men, six suffered from male factor infertility, two of whom achieved pregnancy.


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Table I. Mean and SE of semen analysis values for neat and washed samples of men who did and did not initiate pregnancy
 
Sperm separation
A double-layered discontinuous Isolate© (Irvine Scientific, Santa Ana, CA, USA) gradient technique was used to prepare spermatozoa for IVF or ICSI. To form the gradient, 1 ml of the `lower layer' Isolate© (~90%) was pipetted into the bottom of a 15 ml conical centrifuge tube, and 1 ml of `upper layer' Isolate© (~45%) was pipetted gently over the bottom layer. Liquefied semen was placed on the top of the gradient using a sterile pipette, and the tube centrifuged at 200 g for 25 min. The supernatant was removed and discarded, and the sperm pellet suspended in 1 ml fresh HEPES-buffered human tubal fluid (HTF) containing 1% human serum albumin (h-HTF) (In Vitro Care Inc., San Diego, CA, USA). The suspension was centrifuged at 300 g for 3–5 min, and the supernatant aspirated and discarded. The sperm pellet was resuspended in h-HTF and a post-wash sperm analysis performed for concentration, motility and forward progression.

Sperm chromatin structure assay
Following the procedure(s) of Evenson and Jost (Evenson and Jost, 1994Go), the SCSA evaluated chromatin integrity of spermatozoa in neat and washed semen. 200 µl of sperm samples (1–2x106spermatozoa/ml) were treated for 30 s with 400 µl of a pH 1.2 solution containing 0.1% Triton X-100, 0.15 mol/l NaCl and 0.08 N HCl. Triton X-100 permeabilizes sperm cell membranes providing greater accessibility of acridine orange (AO) to DNA. The low pH solution partially denatures DNA in spermatozoa with abnormal chromatin structure. Spermatozoa with normal chromatin structure do not demonstrate DNA denaturation. After the 30 s acid treatment, 1.20 ml of AO staining buffer (6 µg AO/ml, 37 mmol/l citric acid, 126 mmol/l Na2HPO4, 1 mmol/l disodium EDTA, 0.15 mol/l NaCl, pH 6.0) was added to the cells before analysing by flow cytometry. AO that intercalates into double-stranded DNA (native; normal) fluoresces green (515–530 nm) while AO that associates with single-stranded (denatured) DNA fluoresces red (>=630 nm) when excited by a 488 nm light source (Darzynkiewicz et al., 1975Go).

The extent of sperm DNA denaturation was quantified using an Ortho Diagnostic Cytofluorograf II (Becton Dickinson, Westwood, MA, USA) with a closed quartz flow cell and a 100 mW argon ion laser operated at 35 mW power that was interfaced to a Cicero unit with PC-based Cyclops Software (Cytomation, Fort Collins, CO, USA). This system measured the amount of red and green fluorescence emitted from individual sperm cells flowing at ~200/s and calculated the {alpha}t [red/(red+green) fluorescence] distribution and associated parameters for each sample. Sperm populations with normal chromatin structure have a small mean {alpha}t (X{alpha}t), SD{alpha}t, and percentage of cells outside the main population (COMP{alpha}t, i.e. percentage of cells with denatured DNA). SCSA also identifies immature sperm nuclei by the percentage of cells with high green fluorescence (HGRN), reflecting uncondensed chromatin that is more accessible to the AO stain. Mature ejaculated spermatozoa have a 5-fold lesser DNA stainability than round spermatids (Evenson and Melamed, 1983Go).

Assisted reproductive techniques
Oocyte retrieval and incubation

Oocytes were retrieved from women, <40 years of age, undergoing ovarian stimulation [leuprolide acetate, FSH, and/or FSH/LH as detailed by Roy et al. (1998)] using ultrasound-guided transvaginal follicular aspiration. Oocytes were cultured in synthetic HTF-containing 1% human serum albumin (In Vitro Care Inc.) in a 5% CO2, 37°C, humidified incubator for ~16–18 h in the presence of washed spermatozoa (50 000 to 100 000 motile spermatozoa/ml) or after ICSI. ICSI was done using established techniques and only on mature oocytes (Palmero et al., 1992; Van Steirteghem et al., 1993Go).

Fertilization assessment and embryo culture
Oocytes were assessed for fertilization 16–18 h post-insemination or ICSI. For inseminated oocytes, cumulus cells were removed by repeated pipetting through a sterile, small bore, glass pipette. For inseminated and ICSI oocytes, fertilization was considered normal if two pronuclei and two polar bodies were identified. Oocytes without visible pronuclei were considered to be unfertilized. Oocytes with a single pronucleus or more than two pronuclei were considered to be abnormally fertilized and discarded. Patients were allocated to one or more of the following fertilization categories: normal (>=45% oocytes fertilizing normally), unfertilized (100% oocytes not fertilized), abnormal (>=20% oocytes fertilizing abnormally). Fertilized oocytes were transferred by pipette to a dish containing fresh culture medium (HTF plus 5% human serum albumin) and incubated for an additional 2 days.

Embryo grading
Assessment of embryo quality was made 2 days after fertilization using a modification of Veeck (1986) and the grading system was as follows. Grade 1: pre-embryo with blastomeres of equal size; no cytoplasmic fragments. Grade 2: pre-embryo with blastomeres of equal size; minor cytoplasmic fragments or blebs. Grade 3: pre-embryo with blastomeres of distinctly unequal size; few or no cytoplasmic fragments or pre-embryo with blastomeres of equal or unequal size; significant cytoplasmic fragmentation or pre-embryo with few blastomeres of any size; severe or complete fragmentation.

Embryo transfer
Tubal embryo transfer and uterine embryo transfer procedures were performed 3 days after fertilization. In general, tubal embryo transfer was used for women with normal Fallopian tubes (n = 11) and IVF and embryo transfer for women with tubal factors (n = 10).

Pregnancy outcome
Ultrasound detection for fetal sac was used to confirm a positive clinical pregnancy. One patient with only a positive quantitative ß-human chorionic gonadotrophin (ßHCG) was also included as a positive pregnancy.

Statistical analysis
{chi}2-Analysis was used to test the significance of SCSA parameter thresholds chosen for positive pregnancy. This analysis was applied to SCSA parameters of neat and washed samples separately. Each sample was analysed by SCSA twice, the replicate run immediately following the first. {chi}2-Analysis was completed using the mean of the replicate SCSA values. Three patients who did not have embryos transferred due to abnormal fertilization and/or fertilization failure were not included in {chi}2-analysis of pregnancy outcome. Four washed samples were removed from the statistical analysis because the amount and pattern of DNA degradation indicated delayed sample freezing. Two further washed samples were not included in SCSA analysis because of insufficient volume. If a relationship was identified through exploratory data analysis, regression analysis was used to determine the amount and significance of the correlation between SCSA parameters, WHO parameters and ART outcomes (Table IIGo) by means of the SAS program (SAS, 1988).


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Table II. Parameters used in statistical analysis
 

    Results
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 Materials and methods
 Results
 Discussion
 References
 
Mean SCSA values are shown for neat and washed samples in Table IIIGo. Spermatozoa in the neat semen of the seven men initiating pregnancy were significantly less susceptible to DNA denaturation than the 14 men not initiating pregnancy following embryo transfer (Table IIIGo). SCSA thresholds (X{alpha}t = 300, P < 0.01; SD{alpha}t = 200, P = 0.01; and COMP{alpha}t =27%, P < 0.01) for the susceptibility to low pH-induced denaturation in the neat sperm suspensions were predictive of no pregnancy (Table IIIGo). Pregnancy rate per oocyte transferred was 24% in patients with <27% (COMP{alpha} t) of the spermatozoa in the neat sample showing acid-induced DNA denaturation compared to 0% pregnancy rate in patients with >=27% denaturation (Table IVGo). The percentage of immature (HGRN) spermatozoa in the neat sample also appeared to have a threshold (17%) for pregnancy success; however, the confidence level was not significant (P = 0.19). No SCSA thresholds for the neat sample were identified for normal fertilization or embryo development (data not shown). However, four of the five patients with >=20% abnormally fertilizing oocytes had SCSA parameters that exceeded the thresholds for successful pregnancy (Table IVGo). The mean percentage of spermatozoa showing dentauration (COMP{alpha}t) in these five patients was 33% compared to 23% in the 19 patients with <20% abnormal fertilization.


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Table III. Threshold values of neat samples for pregnancy outcome and mean and SE of sperm chromatin structure assay values for neat and washed samples of men who did and did not initiate pregnancy
 

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Table IV. Assisted reproductive technique outcomes related to COMP{alpha}t threshold of neat sample
 
SCSA parameters of the washed sample were not predictive of fertilization, embryo development or pregnancy outcome (Figure 1Go). No patient's washed sample exceeded the neat X{alpha}t or SD{alpha}t thresholds, and only one patient's washed sample exceeded the neat COMP{alpha}t threshold. In addition, no significant relationships between the SCSA parameters (neat or washed) and WHO parameters, fertilization or embryo development were identified by regression analysis.



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Figure 1. Cytograms (A and C) and corresponding alphat ({alpha}t) histograms (C and D) comparing denaturation of 5000 acridine orange-stained spermatozoa from the neat (A and B) and washed (C and D) samples from a patient with failed pregnancy following intracytoplasmic sperm injection. Each point on the cytograms represents the green (double-stranded DNA; y-axis) versus red (denatured single-stranded DNA; x-axis) fluorescence of a single spermatozoon while the histogram illustrates the {alpha}t [red/(red + green) fluorescence] frequency distribution of spermatozoa within the suspension. Cells outside the main population (COMP) have a larger ratio of red:green fluorescence, indicative of abnormal chromatin structure. High green (HGRN) fluorescence indicates immature spermatozoa with incomplete chromatin condensation. For this patient, the neat sample had values that exceeded sperm chromatin structure assay (SCSA) thresholds whereas the corresponding washed sample values were below the thresholds, illustrating that the SCSA values of the neat sample, not the washed sample, were predictive of negative pregnancy outcome.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The SCSA was predictive of negative pregnancy outcome when >=27% (COMP{alpha} t) of the spermatozoa in the neat sample showed acid-induced DNA denaturation. The percentage of cells with DNA damage was significantly (P = 0.01) greater in the 14 men with failed pregnancy outcome (31.1 ± 3.2) than in the seven men initiating pregnancy following IVF or ICSI (15.4 ± 4.6). Predictive thresholds for two other SCSA parameters [X{alpha}t (300), SD{alpha}t (200)] were established for negative pregnancy outcome, indicating the potential value of SCSA analysis in an assisted reproduction treatment programme. Evenson et al. (1999) reported a similar threshold of 30% damaged spermatozoa (COMP{alpha}t) for in-vivo fertility (Evenson et al., 1999Go), while Spano et al. (2000) reported that fecundity became small when >40% of the cells showed denaturation (Spano et al., 2000Go). Therefore, the SCSA parameters appear to provide an unbiased, quantitative assessment of sperm chromatin structure that is an independent predictor of in-vivo as well as in-vitro fertility potential, i.e. pregnancy. In contrast to flow cytometric SCSA results, Angelopoulis et al. (1998) reported that microscopic examination of AO fluorescence in unprocessed spermatozoa does not predict pregnancy outcomes following IVF cycles (Angelopoulis et al., 1998). The difference may be due to high intra- and interspecimen coefficients of variation reported for the microscopic AO method, making it less sensitive and less specific than the flow cytometric SCSA (Claassens et al., 1992Go).

There was no relationship between the susceptibility to DNA denaturation, assessed by the SCSA, and normal fertilization and early embryo development (Table IVGo); however there was a trend for increased abnormal fertilization when DNA denaturation exceeded the established threshold. It was reported (Twigg et al., 1998Go) that DNA strand breaks did not affect the rate of fertilization. Similarly, spermatozoa with high amounts of DNA denaturation penetrated zona-free hamster eggs (Ibrahim and Pedersen, 1988Go), and spermatozoa with genetic-based defects fertilized normally (Engel et al., 1996Go). However, chromatin abnormalities appear to influence later embryonic development as demonstrated by the absence of clinical pregnancy in patients with SCSA parameters exceeding the thresholds established for neat samples.

SCSA parameters improved following density-gradient preparation as reported previously (Golan et al., 1997Go; Larson et al., 1999Go). However, the SCSA parameters of the prepared spermatozoa were not predictive of pregnancy outcome, indicating that elevated SCSA values in neat semen reflected chromatin abnormalities within the entire sperm population that were not eliminated by sperm preparation techniques. Other studies have shown that improved sperm concentration, motility and morphology after washing are not concomitant with increased ICSI fertilization and cleavage rates (Liu et al., 1994Go; De Vos et al., 1997Go). The current study demonstrated that ICSI leads to pregnancy in patients with poor sperm morphology and motility. For example, two couples with <=4% morphologically normal spermatozoa became pregnant following ICSI. Although the prognoses of these patients was poor based on conventional semen parameters, SCSA parameters of the neat semen were below the threshold for DNA damage (12 and 20% of spermatozoa showed DNA denaturation; i.e. COMP{alpha}t), indicating that the chromatin integrity of the sperm population was adequate to support a viable pregnancy. These patients illustrate the importance of the additional, independent information provided by SCSA analysis. Other assessments of sperm chromatin structure including chromomycin A3 and acidic aniline blue staining are significantly associated with sperm morphology (Franken et al., 1999Go) and therefore may not provide the additional, independent information of the SCSA.

If applied clinically, the SCSA may assist the clinician in providing informed consent to patients, for example if donor spermatozoa should be considered. The SCSA will provide additional information, beyond conventional semen analysis, that may identify DNA damage that will not lead to viable pregnancies following transfer even though the spermatozoa may be capable of initiating fertilization and early embryo development.


    Acknowledgments
 
The authors thank Dr Paul Evenson for assistance with the statistical analysis. This work was supported in part by EPA Grant Number R820968-01, National Science Foundation Grants EPSCoR-9720642, OSR-9452894 and the South Dakota Future Fund. This is South Dakota Agricultural Experiment Station Publication Number 3127 of the journal series.


    Notes
 
4 To whom correspondence should be addressed at: Department of Chemistry and Biochemistry, Box 2170, South Dakota State University, Brookings, SD 57007, USA.E-mail: kjerstilea{at}hotmail.com Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on November 5, 1999; accepted on April 26, 2000.