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
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Abstract |
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Key words: chromatin structure/human fertility/in-vitro fertiliza- tion/intracytoplasmic sperm injection/SCSA
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Introduction |
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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., 1996). Some studies indicate that fertilization, embryo cleavage and pregnancy following ICSI are independent of conventional sperm parameters (Hammadeh et al., 1996
; Oehninger, 1996
). 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., 1998
), poor blastocyst quality (Janny and Ménézo, 1994
) and high abortion rates (Sanchez et al., 1996
). 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., 1995), lower resistance to sodium dodecyl sulphate (SDS)-induced decondensation (Colleu et al., 1988
), poor DNA packing quality (Hofmann and Hilscher, 1991
; Bianchi et al., 1996
; Filatov et al., 1999
), increased DNA strand breaks (Lopes et al., 1998
; Irvine et al., 2000
) and susceptibility to acid-induced DNA denaturation in situ (Evenson et al., 1980
; Evenson, 1999
; Evenson et al., 1999
; Spano et al., 2000
). 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, 1999
). 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, 1999
).
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., 1991). In addition, SCSA parameters are correlated with DNA strand breaks (Sailer et al., 1995
; Aravindan et al., 1997
) and decreased fertility in vivo (Evenson et al., 1980
, 1999
). However, SCSA parameters are not strongly correlated with World Health Organization (WHO) parameters including concentration, motility and morphology (Evenson et al., 1991
). 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.
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Materials and methods |
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Sperm chromatin structure assay
Following the procedure(s) of Evenson and Jost (Evenson and Jost, 1994), the SCSA evaluated chromatin integrity of spermatozoa in neat and washed semen. 200 µl of sperm samples (12x106spermatozoa/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 (515530 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., 1975
).
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 t [red/(red+green) fluorescence] distribution and associated parameters for each sample. Sperm populations with normal chromatin structure have a small mean
t (X
t), SD
t, and percentage of cells outside the main population (COMP
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, 1983
).
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 ~1618 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., 1993).
Fertilization assessment and embryo culture
Oocytes were assessed for fertilization 1618 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
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.
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
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 II
) by means of the SAS program (SAS, 1988).
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Results |
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Discussion |
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There was no relationship between the susceptibility to DNA denaturation, assessed by the SCSA, and normal fertilization and early embryo development (Table IV); however there was a trend for increased abnormal fertilization when DNA denaturation exceeded the established threshold. It was reported (Twigg et al., 1998
) 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, 1988
), and spermatozoa with genetic-based defects fertilized normally (Engel et al., 1996
). 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., 1997; Larson et al., 1999
). 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., 1994
; De Vos et al., 1997
). 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
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., 1999
) 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.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 5, 1999; accepted on April 26, 2000.