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ORIGINAL RESEARCH |
From the Departments of Obstetrics and Gynecology, Surgery (Urology), and Physiology, University of Utah School of Medicine, Salt Lake City, Utah; and The Center for Reproductive Medicine, Minneapolis, Minnesota.
Address reprint requests to: Douglas T. Carrell, PhD, University of Utah School of Medicine, Division of Urology, 50 North Medical Drive, Salt Lake City, UT 84132; E-mail: dcarrell{at}mmsscc.utah.edu.
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ABSTRACT |
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METHODS: Semen quality parameters and sperm chromosome aneuploidy for chromosomes X, Y, 13, 18, and 21 were evaluated in the recurrent pregnancy loss patients, fertile controls, and a control group of men from the general population.
RESULTS: The mean aneuploidy rate in the recurrent pregnancy loss group was 2.77 ± 0.22, significantly higher (P < .005) than in either the general population (1.48 ± 0.12) or in fertile (1.19 ± 0.11) control groups. In the recurrent pregnancy loss patients, the percentage of aneuploid sperm was correlated to the percentage of apoptotic sperm (r = .62, P < .001). Normal morphology was diminished in the patient group, compared with the general population group (P < .01) and the donor group (P < .001).
CONCLUSION: These data indicate that some recurrent pregnancy loss patients have a significant increase of sperm chromosome aneuploidy, apoptosis, and abnormal sperm morphology. This study demonstrates a new possible cause of recurrent pregnancy loss.
Recurrent pregnancy loss, defined as a couple having three or more pregnancy losses, affects approximately 0.5–1.0% of couples.1 Recurrent pregnancy loss is a particularly difficult emotional hardship, compounded by the fact that even after a comprehensive evaluation, the etiology of recurrent pregnancy loss remains unexplained in more than half of those affected.2,3 Recent reports have indicated new possible causes of recurrent pregnancy loss. First, studies have shown that nonrandom X chromosome inactivation occurs in approximately 14% of women with unexplained recurrent pregnancy loss and may indicate a role for lethal, recessive X chromosome mutations as a cause of recurrent pregnancy loss.4,5 Second, studies have indicated that sperm defects may be associated with recurrent pregnancy loss.6–10
Evaluations of the relationship between sperm quality and spontaneous abortion have come to varying conclusions, perhaps partly owing to the lack of strict criteria and controls in some studies. Homonnai et al evaluated semen quality and the fate of 534 pregnancies and reported no link between semen quality and miscarriages.9 Hill et al studied semen quality in couples with recurrent pregnancy loss and found no increase in abnormal morphology or other semen quality parameters but did report an increase in CD4 and CD8 T lymphocytes in the husbands’ semen.7,8 A subsequent study confirmed that no correlation was observed between sperm morphology and pregnancy loss but did find a relationship between morphology and the probability of fertility after recurrent pregnancy loss.10 A recent study, however, has reported a link between increased sperm nuclear vacuoles or abnormal chromatin condensation and recurrent pregnancy loss.6
In 1999, two studies reported a link between unexplained recurrent pregnancy loss and sperm chromosome aneuploidy. Rubio et al reported that the sperm of some husbands with a history of recurrent pregnancy loss had increased disomy and diploidy.11 Their initial findings were later confirmed in a follow-up study.12 Our laboratory reported the preliminary results of a study in which we evaluated sperm morphology and chromosome aneuploidy in the semen of couples with recurrent pregnancy loss, donors of known fertility, and men from the general population (Lowy LJ, Hatasaka HH, Peterson CM, Jones KP, Udoff LC, Wilcox AL, et al. Evaluation of semen and sperm chromosome aneuploidy rates in couples with unexplained recurrent pregnancy loss [abstract number O-133]. In: Abstracts of the Conjoint Annual Meeting of the American Society for Reproductive Medicine and Canadian Fertility and Andrology Society. Birmingham, Alabama: American Society for Reproductive Medicine, 1999:S53). Those data demonstrated a significant increase in sperm chromosome aneuploidy in the recurrent pregnancy loss group compared with both control groups. The mean chromosome aneuploidy rate was increased in the group as a whole; the aneuploidy rates were increased in approximately 15% of the unexplained recurrent pregnancy loss patients.
In this study, we report the results of our evaluation of the role of sperm quality on recurrent pregnancy loss. In addition to analyzing the incidence of sperm chromosome aneuploidy, we have also evaluated standard semen quality parameters. Recurrent pregnancy loss patients were strictly screened before inclusion in the study. Sperm from a control group consisting of men of known fertility and a second control group of men from the general population were also analyzed. Some patients and controls in this study were previously analyzed for sperm deoxyribonucleic acid (DNA) fragmentation, a measure of cellular apoptosis, as detected by the terminal deoxynucleotidyl transferase–mediated dUTP-biotin end-labeling assay.13 For those patients, we have explored the relationship between DNA fragmentation and chromosome aneuploidy.
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MATERIALS AND METHODS |
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The patients were selected from a chart review of patients presenting at clinic for evaluation of recurrent pregnancy loss within the previous 5 years. Patients meeting the criteria listed above were solicited by phone. Nearly all couples agreed to participate, with a few couples unable to participate owing to inaccessibility. Two men included in the study had a child with a previous spouse. Most of the recurrent pregnancy loss patients in this study were included in the previously reported studies (Lowy LJ, et al. Abstracts of the Conjoint Annual Meeting of the American Society for Reproductive Medicine and Canadian Fertility and Andrology Society, 1999:S53).13
Donors of known fertility were randomly selected from a semen donation program in which donors were rigorously screened for inclusion into the program. Screening included semen analysis, sperm penetration assay results, medical and genetic background screening, karyotyping, and sexually transmitted disease screening. Additionally, all donors had a history of a pregnancy within the past 2 years. The general population group consisted of respondents to a newspaper advertisement soliciting semen donors for medical research. No screening was performed other than age (18–45 years). All general population donors were admitted to the study within a 2-week period, and consecutive donors were used in this study.
Semen samples were collected by masturbation after 2–5 days of sexual abstinence. The semen was allowed to undergo liquefaction, then was evaluated for sperm motility, concentration, viability, morphology, and hypoosmotic solution reactivity according to World Health Organization criteria and techniques.14 Briefly, sperm were classified as tapered if elongated (greater than 25% elongation compared with normal sperm) or if the sperm were narrower at one third of the length from the base of the head than at two thirds the length from the base of the head. Amorphous sperm did not fall within any other classification and generally include diverse shapes of the head. Two semen smears were made on clean, frosted microscope slides for nuclear decondensation and evaluation of sperm chromosome aneuploidy.
Sperm chromosome aneuploidy was analyzed as previously described.15 The technician performing the analysis was blind to the identity and patient or control status. Unless otherwise noted, all chemicals were obtained from Sigma Chemical (St. Louis, MO). Briefly, semen smears were fixed in 100% methanol then incubated for 15 minutes in 10 mmol/L dithiothreitol in 0.1 mol/L Tris buffer, then 90 minutes in Tris buffer containing 1 mmol/L dithiothreitol and 10 mmol/L 3,5 diiodosalicylic acid, both at room temperature, to decondense the chromatin sufficiently to allow hybridization with chromosome probes. The slides were then rinsed in 2x standard saline citrate twice at room temperature. The dried slides were stored in the dark. Hybridization was performed by adding 3 µL of probe solution (Vysis, Downers Grove, IL), then sealing a coverslip over the reaction mixture. The sealed slides were incubated at 76C for 5 minutes then incubated at 37C for 4 hours using the HyBrite apparatus (Vysis Inc., Downers Grove, IL). The coverslip was removed and the slide incubated for 5 minutes at 76C in 0.4 x standard saline citrate containing 0.3% Nonidet P40, then 1 minute at room temperature in 2x standard saline citrate containing 0.1% Nonidet P40. The slide was dried vertically in the dark, then coverslipped with 3 µL of antifade solution (phosphate-buffered saline, glycerol).
At least 5000 sperm were analyzed for each chromosome probe. Sperm heads were not counted if they were in contact with other sperm. Signals were only counted if they had a high intensity of fluorescence, consistent with the intensity of control slides. Signals were counted as disomic if they were separated by more than one domain and if both signals were of high and equal intensity. The data were reported as the percentage of sperm with an aneuploidy for each chromosome and the total percentage of sperm with any measured aneuploidy.
Statistical evaluation was performed using Statview software (Abacus, Cary, NC) for Macintosh. Differences in means were evaluated using analysis of variance. Previously reported data on the rate of apoptosis in 16 recurrent pregnancy loss patients was reevaluated to determine the relationship between the total aneuploidy rate, DNA fragmentation, and semen quality parameters.13 Those data were evaluated by regression analysis and determination of the correlation coefficient.
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RESULTS |
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). The percentage of progressively motile sperm was decreased (P < .001) in the general population group compared with both the recurrent pregnancy loss patients and fertile donors. The percentage of morphologically normal sperm was decreased in the recurrent pregnancy loss patients compared with both the general population control group (P < .01) and the donors (P < .001). Tapered sperm were increased in the recurrent pregnancy loss group (P < .005). No differences were observed between the recurrent pregnancy loss group and general population for the percentage of amorphous sperm, hypoosmotic solution reactivity, or viability, but were observed between the recurrent pregnancy loss group and the donors of known fertility (Table 1).
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shows the percentage of sperm aneuploidy for each chromosome. Significant differences (P <.005) were noted between the recurrent pregnancy loss group and both control groups for all chromosomes except chromosome 18, which was only significantly different between the recurrent pregnancy loss group and fertile donors (P < .005). The greatest observed difference in the aneuploidy rate between the patients and general population group was for chromosome 13 (P < .005). Aneuploidy rates were not increased in either male from the recurrent pregnancy loss group who had a child with a prior spouse.
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DISCUSSION |
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Studies have shown that men with abnormal semen quality generally have an increased rate of sperm chromosome aneuploidy.16 Those data are especially pronounced in men undergoing intracytoplasmic sperm injection because of severely decreased sperm quality.17 Prenatal analysis of intracytoplasmic sperm injection–derived concepti has shown an increased incidence of paternally derived aneuploidies.18 Additionally, we have recently reported a case in which chromosome 15 aneuploidy was markedly higher in the sperm of a male whose wife miscarried a fetus with trisomy 15.19 Chromosome defects are a frequent cause of spontaneous abortions, and it is therefore likely that increased gamete aneuploidy could be a cause of some cases of recurrent pregnancy loss.
This study indicates a possible link between sperm chromosome aneuploidy and spontaneous abortion. The incidence of aneuploidy in the recurrent pregnancy loss group was increased compared with the control group but still relatively low. The clinical relevance of the increased aneuploidy rate can only be determined as further data are obtained, including the incidence of aneuploidy of other chromosomes. The chromosomes analyzed in this study are not necessarily the most likely to be aneuploid in recurrent pregnancy loss patients.20 We are now evaluating the aneuploidy rate for other chromosomes.
We have previously shown that sperm DNA fragmentation is increased in some of the recurrent pregnancy loss patients analyzed in this study. Deoxyribonucleic acid fragmentation is a hallmark of apoptosis in human sperm and may be caused by abnormal endonuclease activity and mediated by the plasma membrane protein Fas.21 Previous studies have demonstrated that the apoptosis is increased in the semen of infertile males22–25 and is increased in samples with abnormal sperm morphology or other pathologies.26,27 Deoxyribonucleic acid fragmentation is also inversely correlated with in vitro fertilization rates.28
The strong correlation between aneuploidy rates and DNA fragmentation in the recurrent pregnancy loss group is intriguing. During spermatogenesis, meiotic checkpoints regulate the continuation of meiosis and spermatogenesis by signaling induction of cellular apoptosis when meiotic errors occur.29,30 This mechanism is similar to mitotic checkpoints and is probably not present, or is less efficient, during oogenesis.31 Handel et al have shown that abnormal kinetochore function, which results in abnormal chromosome alignment and segregation during metaphase-anaphase I, is associated with increased levels of aneuploidy and apoptosis.30 Such a mechanism would likely result in cellular death before spermiogenesis, not in mature spermatids as seen in this study. However, other studies have reported links between aneuploidy and apoptosis.32,33 Therefore, it is possible that the aneuploid sperm are the result of a leaky or defective checkpoint mechanism employing cellular apoptosis, or a later checkpoint after meiosis or spermiogenesis are nearly complete.
With a larger sample size, the previously reported trend of abnormal morphology in the recurrent pregnancy loss group became statistically significant (Lowy LJ, et al. Abstracts of the Conjoint Annual Meeting of the American Society for Reproductive Medicine and Canadian Fertility and Andrology Society, 1999:S53). Some differences would be expected between the recurrent pregnancy loss patients and the fertile donors, because semen donors are selected for high sperm motility, morphology, and concentration. However, one would not expect diminished semen quality in the recurrent pregnancy loss group compared with the general population, especially because the couples in the recurrent pregnancy loss group have a history of at least three pregnancies. No link has yet been demonstrated between tapered sperm and aneuploidy or DNA fragmentation, although in vitro fertilization embryo quality has been shown to be decreased in samples with increased percentages of tapered sperm.34 No correlation was observed between standard semen quality parameters and the percentage of sperm with DNA fragmentation.
Sperm quality may be relevant to early embryogenesis.34–36 Sperm may adversely affect embryogenesis in several possible ways, including abnormal centrosome function,37 abnormal sperm membrane proteins,38 and abnormal DNA.5,11 One theory of a possible cause of recurrent pregnancy loss is a defect in the inhibition of implantation of "poor quality" embryos.39 This study demonstrates, in highly screened and controlled patients, that increased chromosome aneuploidy and DNA fragmentation is present and may affect early embryonic development and be responsible for some cases of recurrent pregnancy loss. The data do not answer the important question of how a moderately elevated aneuploidy rate could result in repeated pregnancy losses. The results of aneuploidy testing for other chromosomes may indicate a greater overall aneuploidy rate, but it is also possible that the increased aneuploidy is also an indicator of some other, as yet undescribed, associated meiotic defect.
In summary, this study has reported a significant increase in the percentage of sperm with increased aneuploidy, increased DNA fragmentation, and increased tapered morphology in couples with unexplained recurrent pregnancy loss. The aneuploidy and DNA fragmentation are highly correlated with each other in the recurrent pregnancy loss patients but are not correlated with other semen quality parameters. The possibility of a direct link between sperm aneuploidy and apoptosis is intriguing but not demonstrated by this study. Ongoing studies are evaluating the incidence of aneuploidy of other chromosomes and will also evaluate the proteins involved in synaptonemal and kinetochore function to evaluate the mechanism responsible for increased aneuploidy and apoptosis.
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Footnotes |
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Received August 20, 2002. Received in revised form October 22, 2002. Accepted November 7, 2002.
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REFERENCES |
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15. Carrell DT, Emery B, Liu L. Characterization of chromosome aneuploidy rates, decondensation ability, nuclear protamines, and functional ability of round-headed sperm from siblings, and implications for intracytoplasmic sperm injection (ICSI). Fertil Steril 1999;71:511–6.[Medline]
16. Shi Q, Martin RH. Aneuploidy in human spermatozoa: FISH analysis in men with constitutional chromosomal abnormalities, and in infertile men. Reproduction 2001; 121:655–66.[Abstract]
17. Pfeffer J, Pang M, Hoegerman S, Osgood C, Stacey M, Mayer J, et al. Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection. Fertil Steril 1999;72:472–8.[Medline]
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21. Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: Analogy to apoptosis of somatic cells. Exp Cell Res 1993;207:202–5.[Medline]
22. Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W. A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay. Mol Hum Reprod 1996;2:613–9.
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34. Carrell DT, Peterson CM, Hatasaka HH, Jones KP, Urry RL. Abnormal sperm morphology affects embryo quality and pregnancy rates during IVF, but not during ICSI. J Assist Reprod Technol Androl 1997;9:182–92.
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37. Marvara CS, Hewitson LC, Simerly CR, Sutovsky P, Schatten G. The implications of a paternally derived centrosome during human fertilization: Consequences for reproduction and the treatment of male factor infertility. Am J Reprod Immunol 1997;37:39–49.
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