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Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome
MALE FACTOR
FERTILITY AND STERILITYt VOL. 72, NO. 1, JULY 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome Liliana T. Colombero, M.D., June J. Hariprashad, B.A., Ming C. Tsai, M.D., Zev Rosenwaks, M.D., and Gianpiero D. Palermo, M.D. The Center for Reproductive Medicine and Infertility, New York Presbyterian Hospital-Weill Medical College of Cornell University, New York, New York
Received November 20, 1998; revised and accepted January 21, 1999. Supported by The Center for Reproductive Medicine and Infertility, New York Presbyterian Hospital-Weill Medical College of Cornell University, New York, New York. Presented at the 16th World Congress on Fertility and Sterility and the 54th Annual Meeting of the American Society for Reproductive Medicine, San Francisco, October 4 –9, 1998. Reprint requests: Gianpiero D. Palermo, M.D., The Center for Reproductive Medicine and Infertility, New York Presbyterian Hospital-Weill Medical College of Cornell University, 505 East 70th Street HT-336, New York, New York 10021 (FAX: 212-746-4778; E-mail: [email protected] .cornell.edu). 0015-0282/99/$20.00 PII S0015-0282(99)00158-2
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Objective: To evaluate the incidence of sperm aneuploidy in men screened for infertility and identify any eventual relation with assisted reproductive outcome. Design: Controlled prospective study. Setting: University hospital– based IVF program. Patient(s): Infertile couples who were screened for sperm aneuploidy and evaluated for IVF treatment. Intervention(s): Fluorescence in situ hybridization was used to identify chromosomes 18, 21, X, and Y. The assisted reproductive techniques of IVF and intracytoplasmic sperm injection were used for infertility treatment. Main Outcome Measure(s): The incidence of sperm aneuploidy, semen parameters, fertilization rate, pregnancy characteristics, and rate of neonatal malformations were determined. Result(s): Oligozoospermic and teratozoospermic men had a significantly higher incidence of chromosomal abnormalities than men with normal semen parameters (2.7% vs. 1.8%). The increased frequency of sperm aneuploidy did not appear to affect pregnancy losses or the occurrence of neonatal malformations. Conclusion(s): Suboptimal semen samples had a higher incidence of aneuploidy. In this study, the increased frequency of chromosomal abnormalities did not have a direct effect on the fertilization rate, pregnancy characteristics, or neonatal outcome. (Fertil Sterilt 1999;72:90 – 6. ©1999 by American Society for Reproductive Medicine.) Key Words: Male factor infertility, sperm aneuploidy, fluorescence in situ hybridization, nondisjunction, semen parameters, spermatozoa
The use of assisted reproductive techniques has allowed couples with severe infertility to reproduce. However, approximately 20% of the pregnancies so achieved result in miscarriages, and the cause often is identified as aneuploidy, most commonly a trisomy. It has been demonstrated that most autosomic trisomies are due to meiotic nondisjunction during oogenesis (1), whereas most sex chromosome aneuploidies seem to be meiotic abnormalities that reside in the spermatozoon (2).
ited to germ cells (4) and associated only with a tendency toward nondisjunction during spermatogenesis (5). However, data on the incidence of aneuploidy in the spermatozoa of men with compromised semen parameters are limited and controversial, ranging from no differences (6) to a significant increase in this regard (5). A significantly higher frequency of sperm aneuploidy suggests that the patient is at an elevated risk of producing offspring with numerical chromosomal abnormalities (7).
Compared with the general male population, men with abnormal semen parameters appear to have an increased frequency of constitutional chromosomal abnormalities (3). Even men with a normal peripheral karyotype may have a chromosomal abnormality that is lim-
Intracytoplasmic sperm injection (ICSI) (8), the most effective assisted fertilization technique, has revolutionized the treatment of male infertility. It allows the use of spermatozoa from men with severely compromised semen parameters and currently is used even in some
cases of azoospermia where only immature spermatozoa can be obtained from the epididymis or testis. Although ICSI thus far has demonstrated its safety in many large programs worldwide (9), concerns have been raised that this treatment potentially may transmit genetic abnormalities (10), including sex chromosomal abnormalities (11). However, these findings have not been supported by subsequent larger series; sex chromosomal anomalies have been identified in no more than 1% of the pregnancies conceived by ICSI (9). The use of spermatozoa from oligozoospermic, asthenozoospermic, and teratozoospermic men, as well as surgically retrieved sperm, has raised additional worries about possible genetic risks. Even though the results of a standard semen analysis appear unrelated to the outcome of ICSI (12, 13), there may be a need for genetic screening of the spermatozoon. An ideal method involves the preparation of a metaphase spread followed by complete karyotyping of the sperm; however, this can be performed only after injection of the sperm into an oocyte—an approach that is time-consuming and allows the analysis of only a few spermatozoa (14). On the other hand, the fluorescence in situ hybridization (FISH) technique provides a rapid and reliable source of data, allowing for testing of the most frequently involved chromosomes in large numbers of cells in a short time. By now, almost all the chromosomes have been assessed by FISH in healthy fertile donors, in whom chromosomes 21, X, and Y appear to be more susceptible to nondisjunction than all the other chromosomes (15). Thus far, only a few reports have evidenced a higher incidence of aneuploidy for chromosome 21 in men with abnormal semen parameters (16). Further, the literature on the association of sperm head dysmorphism with chromosome defects is limited (17). The aim of this study was to uncover the possible relationship between particular semen characteristics and sperm aneuploidies. To assess the effect of sperm chromosomal abnormalities on the delivery of healthy offspring, we reviewed the assisted reproductive outcome of men with normal or compromised semen parameters.
MATERIALS AND METHODS Patients Fresh semen samples were obtained from 47 men (mean [6SD] age, 39.1 6 6 years), all of whom were patients at our infertility center and were randomly selected at the time of their first screening evaluation in the andrology laboratory. Semen samples were evaluated according to the criteria of the World Health Organization (18), and strict criteria were used for assessment of morphology (19). Semen parameters considered normal were a concentration of 20 3 106 spermatozoa per milliliter, a progressive motility of 40%, and a rate of normal morphology of 4%. This study was approved by the internal review board of New York Presbyterian Hospital-Weill Medical College of Cornell University (ProFERTILITY & STERILITYt
tocol No. 0696-389), and all patients gave informed consent to participate in the study.
Semen Collection and Preparation Ejaculates were centrifuged after 1:1 dilution in human tubal fluid medium buffered with HEPES (H-HTF; Irvine Scientific, Santa Ana, CA) at 300 3 g for 20 minutes on a three-layer (90%/70%/50%) density gradient to select progressive motile spermatozoa. Spermatozoa retrieved in the 90% fraction were rinsed in H-HTF and centrifuged for 5 minutes at 500 3 g. In some samples (n 5 9) with a concentration of 1 3 106 spermatozoa per milliliter or with a complete absence of progressive motility, the semen simply was centrifuged twice at 1,800 3 g for 5 minutes with H-HTF. After the final wash, the sample was resuspended, preferably to a concentration of approximately 10 3 106 spermatozoa per milliliter. Ten microliters of washed semen was smeared on glass slides (precleaned in 99% ethanol for at least 8 hours) and allowed to dry.
Preparation of Spermatozoa for FISH Two different protocols were used. Twenty-eight semen samples were processed for chromosomes 18, X, and Y. For these, a simple fixation/permeabilization without decondensation of the spermatozoa was performed. In another series, 19 samples were assessed for chromosomes 21, X, and Y. For these, nuclear decondensation of the sperm chromatin was required. Sperm fixation/permeabilization was performed by placing slides in methanol plus glacial acetic acid (3:1 vol/vol) for 1 hour, after which they were air dried and stored at 220°C until further processing or immediate analysis by FISH. For the analysis of chromosome 21, fixation/permeabilization alone was insufficient and sperm decondensation was required to visualize the signal. Slides, processed according to the method of Martin and Ko (20), were placed in a Coplin jar containing 0.01M dithiothreitol (Sigma Chemical Co., St. Louis, MO) in 0.1M tris(hydroxymethyl)aminomethane (Trizma HCl; Sigma Chemical Co.), pH 8, for 30 minutes at room temperature. Subsequently, they were transferred to another jar containing 0.01M 3,5-diiodosalicylic acid, a lithium salt (Sigma Chemical Co.), and 0.001M dithiothreitol in 0.1M tris(hydroxymethyl)aminomethane, pH 8, for 30 minutes, and then washed for 2 minutes in 23 standard saline citrate (Vysis, Downers Grove, IL), pH 7. As observed in the phase-contrast microscope, partial decondensation of the sperm nuclei was considered to be adequate when most of the cells were decondensed but the shape of the nuclei was maintained and the tails were still evident. Such preparations were air dried and either assessed immediately or stored in airtight boxes at 220°C for later processing. Directly labeled DNA probes were alpha-satellite repeat clusters in the centromeric region of the 18 and X chromosomes, and the satellite-III DNA on the long arm of the Y chromosome (Vysis). Chromosome 18 was labeled directly 91
FIGURE 1
FIGURE 2
Fluorescence in situ hybridization of human spermatozoa using probes for chromosomes 18 (green), X (yellow), and Y (red). The sperm chromatin is stained with 49,6-diamino-2phenylindole and appears blue. Normal haploid sperm nuclei are seen that exhibit one signal for chromosome 18 and one for chromosome X or Y.
with a green chromosome enumeration probe (CEP Spectrum Green; Vysis); the X probe was a 1:1 mixture of probes labeled with red and green fluorochromes (CEP Spectrum Green and CEP Spectrum Orange; Vysis); and the Y-specific probe was labeled with a red fluorochrome (CEP Spectrum Orange; Vysis) (Fig. 1). The hybridization solution was prepared by mixing 7 mL of Spectrum CEP hybridization buffer, 1 mL of Y Spectrum Orange, 1 mL of 18 Spectrum Green, 0.5 mL of X Spectrum Orange, and 0.5 mL of X Spectrum Green. The mixture was vortexed thoroughly, centrifuged for 1–3 seconds, and left at room temperature for a short time. When chromosomes 21, X, and Y were assessed, the Y chromosome was labeled green and the X chromosome was labeled yellow (Fig. 2). The chromosome 21 probe, a locusspecific identifier (Vysis), was labeled with a red fluorochrome. The hybridization solution consisted of 7 mL of Spectrum locus-specific identifier hybridization buffer, 1 mL of Y Spectrum Green, 1 mL of 21 Spectrum Red, 0.5 mL of X Spectrum Orange, and 0.5 mL of X Spectrum Green. When the slides were stored at 220°C, they were allowed to equilibrate at room temperature. The hybridization mixture (10 mL) was transferred to the slides and a 22-mm2 coverglass was placed over it. After the hybridization mix92
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Fluorescence in situ hybridization of human spermatozoa using probes for chromosomes 21 (red), X (yellow), and Y (green). The sperm chromatin is stained with 49,6-diamino-2phenylindole and appears blue. Two normal haploid sperm nuclei are seen that exhibit one signal for chromosome 21 and one for chromosome X or Y.
ture had spread evenly, the edges of the coverglass were marked with a tungsten-carbide pencil, allowing the identification of the processed area on its removal. Rubber cement was applied to seal the coverglasses on the slides, and they were air-dried for 5 minutes. After denaturation in a dark, preheated slide warmer for 10 minutes at 80°C, the preparations were allowed to hybridize in a moist, dark chamber at 37°C for at least 6 hours. On removal from the chamber, the rubber cement was peeled carefully from around the coverglass. Stringency was performed by plunging the slides in 50% formamide/standard saline citrate (pH 7) at 42°C for 15 minutes followed by two 15-minute washes in phosphate-Nonidet buffer (PN buffer, 0.1M sodium phosphate buffer, pH 8; Sigma Chemical Co., and 0.1% Nonidet-P40, Sigma Chemical Co.) at room temperature. Sperm nuclei were counterstained with 15 mL of 49,6diamino-2-phenylindole in antifade solution (0.5 mg/mL; Vysis), covered with a coverglass, and assessed at 31,000 magnification with an epifluorescence microscope (Olympus B Max 60; New York/New Jersey Scientific, Middlebush, NJ) equipped with a triple bandpass filter (Olympus UC83103; New York/New Jersey Scientific). This system allowed the observation of sperm nuclei in blue together
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TABLE 1 Comparison of chromosomal abnormalities in normal and abnormal semen. No. (%) of spermatozoa with indicated type of chromosomal abnormality Type of semen
No. of samples
No. of cells
Nullisomy
Sex chromosome disomy
Autosomal disomy
Diploidy
24 23 47
39,202 41,851 81,053
198 (0.51) 125 (0.30) 323 (0.40)
251 (0.64) 192 (0.46) 443 (0.55)
328 (0.84) 265 (0.63) 593 (0.73)
295 (0.75) 170 (0.241) 465 (0.57)
Oligoasthenoteratozoospermic Normozoospermic Total
Note: Oligoasthenoteratozoospermic 5 ,20 3 106 spermatozoa per milliliter, ,40% motility (World Health Organization criteria), and ,4% normal morphology (Kruger’s strict criteria). Pearson x2 test, 2 3 2, 1 df; influence of semen parameters on: P,.001 (oligoasthenoteratozoospermic vs. normozoospermic) for all types of chromosomal abnormalities.
with red and green signals. Yellow signals also could be observed with this filter set because these were a mixture of green and red fluorochromes. A dual bandpass filter (Olympus U-C83102; New York/New Jersey Scientific) was used for the observation of the specific chromosome signals. During analysis, chromosome 18 appeared green, chromosome X appeared yellow, and chromosome Y appeared red. For the samples assessed for 21, X, and Y, chromosome X appeared yellow, chromosome Y appeared green, and chromosome 21 appeared red. Single bandpass filters for fluorescein isothiocyanate (FITC, Olympus U-C83490; New York/New Jersey Scientific) and Texas red (Olympus UC83570; New York/New Jersey Scientific) also were used to detect overlapping signals. In addition, the use of an FITC filter allowed the differentiation of debris that usually stained red from “true” red signals; real hybridization signals disappeared when visualized with this filter, whereas debris appeared yellow.
Scoring Criteria Normal haploid sperm nuclei carried one signal for a sex chromosome and one signal for an autosome. Sperm missing one of the two signals were nullisomic for the corresponding chromosome, and sperm with an extra signal were disomic for the corresponding chromosome. The simultaneous scoring of one autosome and the two gonosomes allowed the distinction between nullisomy and hybridization failure (no signals), and between disomy and diploidy (two signals for autosomes and two signals for gonosomes). A spermatozoon was considered disomic for a specific chromosome when two fluorescent domains of the same color were clearly positioned within the sperm head, comparable in brightness and size, and at least one domain apart. One domain was considered to be the diameter of the signal. Diploid cells with a clearly defined round shape and without a tail were considered spermatogenetic or other cells and were not scored. Overlapping nuclei for which it was not possible to assign a signal to a given nucleus, disrupted nuclei with indistinct margins, and large nuclei (more than twice the size of a nondecondensed sperm head) with diffused chromatin as a result of excessive decondensation FERTILITY & STERILITYt
were eliminated from scoring. Scoring was done blindly on coded samples whose origins were unknown to the individuals involved in the scoring.
Assisted Reproduction Assisted reproduction was performed by standard IVF (n 5 11) or ICSI (n 5 31), and the details of these procedures have been described previously (13). The mean (6SD) maternal age was 35.7 6 5 years.
Statistical Analysis
The customary Pearson x2 test was used for discrete univariate and bivariate data, except where test assumptions were violated, necessitating the use of Fisher’s exact test. Continuous variables were analyzed using the two-sample independent t-test with the Fisher-Behrens correction as needed. Statistical significance was defined as P,.05 for discrete and continuous analysis (21). All statistical computations were conducted using the Statistical Analysis System (SAS Institute, Cary, NC).
RESULTS In 23 men with a mean (6SD) age of 38.5 6 6 years, the semen parameters were normal; in the remaining 24 men with a mean (6SD) age of 38.2 6 7 years, at least one semen parameter was compromised. A mean of 1,725 spermatozoa (range, 1,006 –3,661 spermatozoa) were analyzed per sample. Of the total 81,053 spermatozoa counted, 38,560 (48%) were X-bearing and 39,465 (49%) were Y-bearing. The rate of technical FISH failure was estimated to be as high as 2% for intact sperm nuclei and 1% for decondensed sperm nuclei. To determine any potential influence of the sperm fixation/decondensation method, aliquots of five ejaculates were processed by both methods and analyzed for chromosomes 18, 21, X, and Y. Whereas analysis of chromosomes 18, X, and Y was possible with both methods, identification of chromosome 21 was possible only after decondensation of the nucleus. The readability of the signals, rate of FISH failure, and incidence of abnormalities were unaffected for the three chromosomes that could be analyzed by the two different fixation methods. 93
FIGURE 3 Examples of spermatozoa that carry chromosomal abnormalities. (A), A sperm head carrying a sex chromosome disomy (XX,21). (B), A sperm nucleus displaying an autosomal disomy (Y,2121). (C), A spermatozoon considered diploid (XX,1818).
The incidence of total abnormalities (nullisomy, autosomal disomy, and sex chromosome disomy and diploidy) was 3.3% in oligozoospermic (,20 3 106/mL) men (n 5 13) and 2% in normozoospermic ($20 3 106/mL) men (n 5 34) (P,.001). In the 24 men with at least one abnormal semen parameter, the incidence of chromosomal abnormalities was 2.7%, which was significantly higher than in the 23 men with normal semen parameters (1.8%; P,.001). Patients with abnormal semen parameters had significantly higher (P5.0001) incidences of chromosome 18 (2.7%) and chromosome 21 (3.6%) abnormalities compared with samples from putatively healthy men. Patients were grouped according to a combination of all semen parameters (Table 1). In all groups, rates of sex chromosome abnormalities (Fig. 3A), autosomal disomy (Fig. 3B), and diploidy (Fig. 3C) were consistently higher in 94
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patients with compromised semen parameters than in healthy men (Table 1). Of the individual abnormalities, autosomal disomy appeared to be the most predominant. The incidence of disomy was higher for chromosome 21 (1.2%) than for chromosome 18 (0.6%) (P5.001) in samples with compromised semen parameters. For all samples, the incidence of gonosomal disomy increased from 0.5% to 0.6% in abnormal semen (Table 1). To eliminate any effect of the sperm selection process on our results, an analysis was performed that included only those semen samples that were processed by density gradient centrifugation. A statistically significant difference in the incidence of chromosomal abnormalities was maintained between patients with normal and abnormal semen parameters (1.6% vs. 2.7%; P,.01).
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TABLE 2 Assisted reproductive outcome in patients with normal and abnormal semen parameters. Semen parameter Variable No. of couples No. of cycles Mean (6SD) sperm concentration (3 106/mL) Mean (6SD) progressive motility (%) Mean (6SD) morphology (%) Spermatozoa with chromosomal abnormality/spermatozoa with signal (%) 2 pronuclei/oocytes inseminated (%) No. of clinical pregnancies (%)
Normal
Abnormal
13 14 74.3 6 38.0 59.0 6 10.0 7.8 6 3.0
19 28 25.3 6 32.0 42.8 6 21.0 1.3 6 1.0
535/25,292 (2.1) 96/137 (70.1) 9 (64.3)
815/25,557 (3.2) 137/222 (61.7) 12 (42.9)
P value — — ,.01 .01 ,.01 .0001 NS NS
Test — — t-test t-test t-test Pearson x2 Pearson x2 Fisher’s exact
Note: NS 5 not significant.
Of the patients who were treated by assisted reproductive techniques, 19 couples with abnormal semen parameters had a fertilization rate of 61.7% and a clinical pregnancy rate of 42.9%. Among the 13 couples with normal semen parameters, the fertilization rate was 70.1% and the clinical pregnancy rate was 64.3% (Table 2). One patient did not undergo embryo replacement because all the zygotes were triploid. There was no difference in the rate of pregnancy loss or the incidence of neonatal malformations between the two groups. One miscarriage occurred in the abnormal semen group, whereas one ectopic pregnancy was diagnosed in the normal semen group; no neonatal malformations were observed in either group. In couples treated by assisted reproductive techniques, the difference in the incidence of chromosomal abnormalities was confirmed, as was the relation with semen parameters (Table 2). The age of the female partners who were inseminated with semen that had normal parameters was similar to that of the female partners who were inseminated with semen that had compromised parameters (35.6 6 6 years vs. 35.8 6 5 years). In spite of a significant difference in chromosomal abnormalities, fertilization and pregnancy rates were unaffected. Among the couples with normal semen parameters, 8 patients were delivered of 10 healthy infants. In the group with abnormal semen parameters, 11 patients were delivered of 17 healthy infants.
DISCUSSION Several studies recently have raised concern about the safety of ICSI (12). The results that we obtained from FISH analysis demonstrated a significantly increased frequency of aneuploidy in the spermatozoa of men with abnormal semen parameters. These results were most striking in men with oligozoospermia. All types of chromosomal abnormalities appeared to be increased, including nullisomy, gonosomal disomy, autosomal disomy, and diploidy. Autosomal disFERTILITY & STERILITYt
omy, however, was the most predominant abnormality when chromosome 21 was assessed. Previous reports on the incidence of aneuploidy in the spermatozoa of men with abnormal semen parameters have been controversial. Although some studies recorded comparable levels of chromosomal abnormalities in fertile and infertile patients (6), others reported an increased frequency of numerical chromosomal abnormalities in the infertile population (5). There was a higher frequency of nondisjunction in chromosomes 21, X, and Y than in other chromosomes in fertile men with normal semen parameters (16). In addition, we observed a higher incidence of gonosomal disomy and disomy 21 in men with abnormal semen parameters. Our figures, however, were higher than those previously reported, possibly because of the subnormal semen status of these patients. The origin of trisomy 18 and trisomy 21 in offspring has been reported to be maternal in approximately 91% (22) and 95% (1), of the cases, respectively. Our findings could signify that paternal disomy might be responsible for these aberrations when pregnancies are established by men with subnormal semen parameters. Our present findings support the report by Martin and Rademaker (23) that sex chromosomes appear to be involved frequently in aneuploidy among men with abnormal semen parameters. Men with abnormal semen parameters have an increased frequency of pairing disruptions resulting in meiotic arrest (24). The sex chromosome bivalent is particularly susceptible to pairing abnormalities because there generally is only one crossover in the pseudoautosomal region. Thus, men with abnormal semen parameters may have decreased recombination and pairing leading to both meiotic arrest (oligozoospermia) and nondisjunction of the sex chromosomes (20); the immediate consequence of this observation is a higher incidence of trisomies and gonosomal aneu95
ploidies in the offspring of men with abnormal semen parameters. Aneuploidies of maternal origin can be identified by polar body biopsy (25). However, it is not possible to assess the ploidy of a particular spermatozoon before it is used for ICSI. Because spermatogenetic arrest resulting from chromosomal abnormalities is inconsistent, the genetic constitution of the gamete is equally unpredictable. Therefore, at the present time, preimplantation diagnosis is the only means of determining the karyotype of the embryo before transfer. The information provided by FISH, however, may be helpful in counseling candidates for assisted reproductive techniques regarding their chances for transmitting chromosomal abnormalities of paternal origin. Although it is still low in absolute terms, the spermatozoa of men with abnormal semen parameters have a slightly higher incidence of both autosomal and gonosomal anomalies. The higher incidence of disomy 21 compared with disomy 18 confirms the higher frequency of nondisjunction for this autosome, as evidenced in men with abnormal semen parameters. Some specimens (n 5 9) were not processed by density gradient centrifugation because of the extremely low number of spermatozoa present in the retrieved sample. Although the preparation method could be a source of potential bias, the analysis of samples processed by density gradient centrifugation confirmed the association between a higher incidence of chromosomal abnormalities and poor semen characteristics. We decided to assess a relatively small number of spermatozoa (approximately 1,725 per sample) because, in our preliminary experiments, this number was sufficient to provide reliable information on the karyotypic distribution of the entire sample and allowed the assessment of normozoospermic and oligozoospermic samples in an equitable fashion. Although the lower number of spermatozoa counted could be inadequate, the incidence of chromosomal abnormalities observed in this study is comparable to that in previously published work (5). This study indicates that men with suboptimal semen quality have an overall higher incidence of chromosomal abnormalities that appears to be inversely related to each individual semen parameter. However, the patient group with the higher frequency of sperm chromosomal abnormalities had fertilization and pregnancy rates comparable to those of the control group and did not have higher rates of pregnancy loss or neonatal malformations. Although concern related to the use of suboptimal spermatozoa still remains, sperm chromosomal abnormalities at this rate do not appear to compromise the ultimate reproductive outcome.
Acknowledgments: The authors thank the clinical and scientific staff of The Center for Reproductive Medicine and Infertility, J. Michael Bedford,
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Ph.D., D.V.M., for his critical review of the manuscript, Ms. Miriam Feliciano, B.Sc., and Ms. Maria Oquendo, B.Sc., for technical assistance, and Ms. Queenie V. Neri, B.Sc., for editorial assistance.
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FERTILITY AND STERILITYt VOL. 72, NO. 1, JULY 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome Liliana T. Colombero, M.D., June J. Hariprashad, B.A., Ming C. Tsai, M.D., Zev Rosenwaks, M.D., and Gianpiero D. Palermo, M.D. The Center for Reproductive Medicine and Infertility, New York Presbyterian Hospital-Weill Medical College of Cornell University, New York, New York
Received November 20, 1998; revised and accepted January 21, 1999. Supported by The Center for Reproductive Medicine and Infertility, New York Presbyterian Hospital-Weill Medical College of Cornell University, New York, New York. Presented at the 16th World Congress on Fertility and Sterility and the 54th Annual Meeting of the American Society for Reproductive Medicine, San Francisco, October 4 –9, 1998. Reprint requests: Gianpiero D. Palermo, M.D., The Center for Reproductive Medicine and Infertility, New York Presbyterian Hospital-Weill Medical College of Cornell University, 505 East 70th Street HT-336, New York, New York 10021 (FAX: 212-746-4778; E-mail: [email protected] .cornell.edu). 0015-0282/99/$20.00 PII S0015-0282(99)00158-2
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Objective: To evaluate the incidence of sperm aneuploidy in men screened for infertility and identify any eventual relation with assisted reproductive outcome. Design: Controlled prospective study. Setting: University hospital– based IVF program. Patient(s): Infertile couples who were screened for sperm aneuploidy and evaluated for IVF treatment. Intervention(s): Fluorescence in situ hybridization was used to identify chromosomes 18, 21, X, and Y. The assisted reproductive techniques of IVF and intracytoplasmic sperm injection were used for infertility treatment. Main Outcome Measure(s): The incidence of sperm aneuploidy, semen parameters, fertilization rate, pregnancy characteristics, and rate of neonatal malformations were determined. Result(s): Oligozoospermic and teratozoospermic men had a significantly higher incidence of chromosomal abnormalities than men with normal semen parameters (2.7% vs. 1.8%). The increased frequency of sperm aneuploidy did not appear to affect pregnancy losses or the occurrence of neonatal malformations. Conclusion(s): Suboptimal semen samples had a higher incidence of aneuploidy. In this study, the increased frequency of chromosomal abnormalities did not have a direct effect on the fertilization rate, pregnancy characteristics, or neonatal outcome. (Fertil Sterilt 1999;72:90 – 6. ©1999 by American Society for Reproductive Medicine.) Key Words: Male factor infertility, sperm aneuploidy, fluorescence in situ hybridization, nondisjunction, semen parameters, spermatozoa
The use of assisted reproductive techniques has allowed couples with severe infertility to reproduce. However, approximately 20% of the pregnancies so achieved result in miscarriages, and the cause often is identified as aneuploidy, most commonly a trisomy. It has been demonstrated that most autosomic trisomies are due to meiotic nondisjunction during oogenesis (1), whereas most sex chromosome aneuploidies seem to be meiotic abnormalities that reside in the spermatozoon (2).
ited to germ cells (4) and associated only with a tendency toward nondisjunction during spermatogenesis (5). However, data on the incidence of aneuploidy in the spermatozoa of men with compromised semen parameters are limited and controversial, ranging from no differences (6) to a significant increase in this regard (5). A significantly higher frequency of sperm aneuploidy suggests that the patient is at an elevated risk of producing offspring with numerical chromosomal abnormalities (7).
Compared with the general male population, men with abnormal semen parameters appear to have an increased frequency of constitutional chromosomal abnormalities (3). Even men with a normal peripheral karyotype may have a chromosomal abnormality that is lim-
Intracytoplasmic sperm injection (ICSI) (8), the most effective assisted fertilization technique, has revolutionized the treatment of male infertility. It allows the use of spermatozoa from men with severely compromised semen parameters and currently is used even in some
cases of azoospermia where only immature spermatozoa can be obtained from the epididymis or testis. Although ICSI thus far has demonstrated its safety in many large programs worldwide (9), concerns have been raised that this treatment potentially may transmit genetic abnormalities (10), including sex chromosomal abnormalities (11). However, these findings have not been supported by subsequent larger series; sex chromosomal anomalies have been identified in no more than 1% of the pregnancies conceived by ICSI (9). The use of spermatozoa from oligozoospermic, asthenozoospermic, and teratozoospermic men, as well as surgically retrieved sperm, has raised additional worries about possible genetic risks. Even though the results of a standard semen analysis appear unrelated to the outcome of ICSI (12, 13), there may be a need for genetic screening of the spermatozoon. An ideal method involves the preparation of a metaphase spread followed by complete karyotyping of the sperm; however, this can be performed only after injection of the sperm into an oocyte—an approach that is time-consuming and allows the analysis of only a few spermatozoa (14). On the other hand, the fluorescence in situ hybridization (FISH) technique provides a rapid and reliable source of data, allowing for testing of the most frequently involved chromosomes in large numbers of cells in a short time. By now, almost all the chromosomes have been assessed by FISH in healthy fertile donors, in whom chromosomes 21, X, and Y appear to be more susceptible to nondisjunction than all the other chromosomes (15). Thus far, only a few reports have evidenced a higher incidence of aneuploidy for chromosome 21 in men with abnormal semen parameters (16). Further, the literature on the association of sperm head dysmorphism with chromosome defects is limited (17). The aim of this study was to uncover the possible relationship between particular semen characteristics and sperm aneuploidies. To assess the effect of sperm chromosomal abnormalities on the delivery of healthy offspring, we reviewed the assisted reproductive outcome of men with normal or compromised semen parameters.
MATERIALS AND METHODS Patients Fresh semen samples were obtained from 47 men (mean [6SD] age, 39.1 6 6 years), all of whom were patients at our infertility center and were randomly selected at the time of their first screening evaluation in the andrology laboratory. Semen samples were evaluated according to the criteria of the World Health Organization (18), and strict criteria were used for assessment of morphology (19). Semen parameters considered normal were a concentration of 20 3 106 spermatozoa per milliliter, a progressive motility of 40%, and a rate of normal morphology of 4%. This study was approved by the internal review board of New York Presbyterian Hospital-Weill Medical College of Cornell University (ProFERTILITY & STERILITYt
tocol No. 0696-389), and all patients gave informed consent to participate in the study.
Semen Collection and Preparation Ejaculates were centrifuged after 1:1 dilution in human tubal fluid medium buffered with HEPES (H-HTF; Irvine Scientific, Santa Ana, CA) at 300 3 g for 20 minutes on a three-layer (90%/70%/50%) density gradient to select progressive motile spermatozoa. Spermatozoa retrieved in the 90% fraction were rinsed in H-HTF and centrifuged for 5 minutes at 500 3 g. In some samples (n 5 9) with a concentration of 1 3 106 spermatozoa per milliliter or with a complete absence of progressive motility, the semen simply was centrifuged twice at 1,800 3 g for 5 minutes with H-HTF. After the final wash, the sample was resuspended, preferably to a concentration of approximately 10 3 106 spermatozoa per milliliter. Ten microliters of washed semen was smeared on glass slides (precleaned in 99% ethanol for at least 8 hours) and allowed to dry.
Preparation of Spermatozoa for FISH Two different protocols were used. Twenty-eight semen samples were processed for chromosomes 18, X, and Y. For these, a simple fixation/permeabilization without decondensation of the spermatozoa was performed. In another series, 19 samples were assessed for chromosomes 21, X, and Y. For these, nuclear decondensation of the sperm chromatin was required. Sperm fixation/permeabilization was performed by placing slides in methanol plus glacial acetic acid (3:1 vol/vol) for 1 hour, after which they were air dried and stored at 220°C until further processing or immediate analysis by FISH. For the analysis of chromosome 21, fixation/permeabilization alone was insufficient and sperm decondensation was required to visualize the signal. Slides, processed according to the method of Martin and Ko (20), were placed in a Coplin jar containing 0.01M dithiothreitol (Sigma Chemical Co., St. Louis, MO) in 0.1M tris(hydroxymethyl)aminomethane (Trizma HCl; Sigma Chemical Co.), pH 8, for 30 minutes at room temperature. Subsequently, they were transferred to another jar containing 0.01M 3,5-diiodosalicylic acid, a lithium salt (Sigma Chemical Co.), and 0.001M dithiothreitol in 0.1M tris(hydroxymethyl)aminomethane, pH 8, for 30 minutes, and then washed for 2 minutes in 23 standard saline citrate (Vysis, Downers Grove, IL), pH 7. As observed in the phase-contrast microscope, partial decondensation of the sperm nuclei was considered to be adequate when most of the cells were decondensed but the shape of the nuclei was maintained and the tails were still evident. Such preparations were air dried and either assessed immediately or stored in airtight boxes at 220°C for later processing. Directly labeled DNA probes were alpha-satellite repeat clusters in the centromeric region of the 18 and X chromosomes, and the satellite-III DNA on the long arm of the Y chromosome (Vysis). Chromosome 18 was labeled directly 91
FIGURE 1
FIGURE 2
Fluorescence in situ hybridization of human spermatozoa using probes for chromosomes 18 (green), X (yellow), and Y (red). The sperm chromatin is stained with 49,6-diamino-2phenylindole and appears blue. Normal haploid sperm nuclei are seen that exhibit one signal for chromosome 18 and one for chromosome X or Y.
with a green chromosome enumeration probe (CEP Spectrum Green; Vysis); the X probe was a 1:1 mixture of probes labeled with red and green fluorochromes (CEP Spectrum Green and CEP Spectrum Orange; Vysis); and the Y-specific probe was labeled with a red fluorochrome (CEP Spectrum Orange; Vysis) (Fig. 1). The hybridization solution was prepared by mixing 7 mL of Spectrum CEP hybridization buffer, 1 mL of Y Spectrum Orange, 1 mL of 18 Spectrum Green, 0.5 mL of X Spectrum Orange, and 0.5 mL of X Spectrum Green. The mixture was vortexed thoroughly, centrifuged for 1–3 seconds, and left at room temperature for a short time. When chromosomes 21, X, and Y were assessed, the Y chromosome was labeled green and the X chromosome was labeled yellow (Fig. 2). The chromosome 21 probe, a locusspecific identifier (Vysis), was labeled with a red fluorochrome. The hybridization solution consisted of 7 mL of Spectrum locus-specific identifier hybridization buffer, 1 mL of Y Spectrum Green, 1 mL of 21 Spectrum Red, 0.5 mL of X Spectrum Orange, and 0.5 mL of X Spectrum Green. When the slides were stored at 220°C, they were allowed to equilibrate at room temperature. The hybridization mixture (10 mL) was transferred to the slides and a 22-mm2 coverglass was placed over it. After the hybridization mix92
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Fluorescence in situ hybridization of human spermatozoa using probes for chromosomes 21 (red), X (yellow), and Y (green). The sperm chromatin is stained with 49,6-diamino-2phenylindole and appears blue. Two normal haploid sperm nuclei are seen that exhibit one signal for chromosome 21 and one for chromosome X or Y.
ture had spread evenly, the edges of the coverglass were marked with a tungsten-carbide pencil, allowing the identification of the processed area on its removal. Rubber cement was applied to seal the coverglasses on the slides, and they were air-dried for 5 minutes. After denaturation in a dark, preheated slide warmer for 10 minutes at 80°C, the preparations were allowed to hybridize in a moist, dark chamber at 37°C for at least 6 hours. On removal from the chamber, the rubber cement was peeled carefully from around the coverglass. Stringency was performed by plunging the slides in 50% formamide/standard saline citrate (pH 7) at 42°C for 15 minutes followed by two 15-minute washes in phosphate-Nonidet buffer (PN buffer, 0.1M sodium phosphate buffer, pH 8; Sigma Chemical Co., and 0.1% Nonidet-P40, Sigma Chemical Co.) at room temperature. Sperm nuclei were counterstained with 15 mL of 49,6diamino-2-phenylindole in antifade solution (0.5 mg/mL; Vysis), covered with a coverglass, and assessed at 31,000 magnification with an epifluorescence microscope (Olympus B Max 60; New York/New Jersey Scientific, Middlebush, NJ) equipped with a triple bandpass filter (Olympus UC83103; New York/New Jersey Scientific). This system allowed the observation of sperm nuclei in blue together
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TABLE 1 Comparison of chromosomal abnormalities in normal and abnormal semen. No. (%) of spermatozoa with indicated type of chromosomal abnormality Type of semen
No. of samples
No. of cells
Nullisomy
Sex chromosome disomy
Autosomal disomy
Diploidy
24 23 47
39,202 41,851 81,053
198 (0.51) 125 (0.30) 323 (0.40)
251 (0.64) 192 (0.46) 443 (0.55)
328 (0.84) 265 (0.63) 593 (0.73)
295 (0.75) 170 (0.241) 465 (0.57)
Oligoasthenoteratozoospermic Normozoospermic Total
Note: Oligoasthenoteratozoospermic 5 ,20 3 106 spermatozoa per milliliter, ,40% motility (World Health Organization criteria), and ,4% normal morphology (Kruger’s strict criteria). Pearson x2 test, 2 3 2, 1 df; influence of semen parameters on: P,.001 (oligoasthenoteratozoospermic vs. normozoospermic) for all types of chromosomal abnormalities.
with red and green signals. Yellow signals also could be observed with this filter set because these were a mixture of green and red fluorochromes. A dual bandpass filter (Olympus U-C83102; New York/New Jersey Scientific) was used for the observation of the specific chromosome signals. During analysis, chromosome 18 appeared green, chromosome X appeared yellow, and chromosome Y appeared red. For the samples assessed for 21, X, and Y, chromosome X appeared yellow, chromosome Y appeared green, and chromosome 21 appeared red. Single bandpass filters for fluorescein isothiocyanate (FITC, Olympus U-C83490; New York/New Jersey Scientific) and Texas red (Olympus UC83570; New York/New Jersey Scientific) also were used to detect overlapping signals. In addition, the use of an FITC filter allowed the differentiation of debris that usually stained red from “true” red signals; real hybridization signals disappeared when visualized with this filter, whereas debris appeared yellow.
Scoring Criteria Normal haploid sperm nuclei carried one signal for a sex chromosome and one signal for an autosome. Sperm missing one of the two signals were nullisomic for the corresponding chromosome, and sperm with an extra signal were disomic for the corresponding chromosome. The simultaneous scoring of one autosome and the two gonosomes allowed the distinction between nullisomy and hybridization failure (no signals), and between disomy and diploidy (two signals for autosomes and two signals for gonosomes). A spermatozoon was considered disomic for a specific chromosome when two fluorescent domains of the same color were clearly positioned within the sperm head, comparable in brightness and size, and at least one domain apart. One domain was considered to be the diameter of the signal. Diploid cells with a clearly defined round shape and without a tail were considered spermatogenetic or other cells and were not scored. Overlapping nuclei for which it was not possible to assign a signal to a given nucleus, disrupted nuclei with indistinct margins, and large nuclei (more than twice the size of a nondecondensed sperm head) with diffused chromatin as a result of excessive decondensation FERTILITY & STERILITYt
were eliminated from scoring. Scoring was done blindly on coded samples whose origins were unknown to the individuals involved in the scoring.
Assisted Reproduction Assisted reproduction was performed by standard IVF (n 5 11) or ICSI (n 5 31), and the details of these procedures have been described previously (13). The mean (6SD) maternal age was 35.7 6 5 years.
Statistical Analysis
The customary Pearson x2 test was used for discrete univariate and bivariate data, except where test assumptions were violated, necessitating the use of Fisher’s exact test. Continuous variables were analyzed using the two-sample independent t-test with the Fisher-Behrens correction as needed. Statistical significance was defined as P,.05 for discrete and continuous analysis (21). All statistical computations were conducted using the Statistical Analysis System (SAS Institute, Cary, NC).
RESULTS In 23 men with a mean (6SD) age of 38.5 6 6 years, the semen parameters were normal; in the remaining 24 men with a mean (6SD) age of 38.2 6 7 years, at least one semen parameter was compromised. A mean of 1,725 spermatozoa (range, 1,006 –3,661 spermatozoa) were analyzed per sample. Of the total 81,053 spermatozoa counted, 38,560 (48%) were X-bearing and 39,465 (49%) were Y-bearing. The rate of technical FISH failure was estimated to be as high as 2% for intact sperm nuclei and 1% for decondensed sperm nuclei. To determine any potential influence of the sperm fixation/decondensation method, aliquots of five ejaculates were processed by both methods and analyzed for chromosomes 18, 21, X, and Y. Whereas analysis of chromosomes 18, X, and Y was possible with both methods, identification of chromosome 21 was possible only after decondensation of the nucleus. The readability of the signals, rate of FISH failure, and incidence of abnormalities were unaffected for the three chromosomes that could be analyzed by the two different fixation methods. 93
FIGURE 3 Examples of spermatozoa that carry chromosomal abnormalities. (A), A sperm head carrying a sex chromosome disomy (XX,21). (B), A sperm nucleus displaying an autosomal disomy (Y,2121). (C), A spermatozoon considered diploid (XX,1818).
The incidence of total abnormalities (nullisomy, autosomal disomy, and sex chromosome disomy and diploidy) was 3.3% in oligozoospermic (,20 3 106/mL) men (n 5 13) and 2% in normozoospermic ($20 3 106/mL) men (n 5 34) (P,.001). In the 24 men with at least one abnormal semen parameter, the incidence of chromosomal abnormalities was 2.7%, which was significantly higher than in the 23 men with normal semen parameters (1.8%; P,.001). Patients with abnormal semen parameters had significantly higher (P5.0001) incidences of chromosome 18 (2.7%) and chromosome 21 (3.6%) abnormalities compared with samples from putatively healthy men. Patients were grouped according to a combination of all semen parameters (Table 1). In all groups, rates of sex chromosome abnormalities (Fig. 3A), autosomal disomy (Fig. 3B), and diploidy (Fig. 3C) were consistently higher in 94
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patients with compromised semen parameters than in healthy men (Table 1). Of the individual abnormalities, autosomal disomy appeared to be the most predominant. The incidence of disomy was higher for chromosome 21 (1.2%) than for chromosome 18 (0.6%) (P5.001) in samples with compromised semen parameters. For all samples, the incidence of gonosomal disomy increased from 0.5% to 0.6% in abnormal semen (Table 1). To eliminate any effect of the sperm selection process on our results, an analysis was performed that included only those semen samples that were processed by density gradient centrifugation. A statistically significant difference in the incidence of chromosomal abnormalities was maintained between patients with normal and abnormal semen parameters (1.6% vs. 2.7%; P,.01).
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TABLE 2 Assisted reproductive outcome in patients with normal and abnormal semen parameters. Semen parameter Variable No. of couples No. of cycles Mean (6SD) sperm concentration (3 106/mL) Mean (6SD) progressive motility (%) Mean (6SD) morphology (%) Spermatozoa with chromosomal abnormality/spermatozoa with signal (%) 2 pronuclei/oocytes inseminated (%) No. of clinical pregnancies (%)
Normal
Abnormal
13 14 74.3 6 38.0 59.0 6 10.0 7.8 6 3.0
19 28 25.3 6 32.0 42.8 6 21.0 1.3 6 1.0
535/25,292 (2.1) 96/137 (70.1) 9 (64.3)
815/25,557 (3.2) 137/222 (61.7) 12 (42.9)
P value — — ,.01 .01 ,.01 .0001 NS NS
Test — — t-test t-test t-test Pearson x2 Pearson x2 Fisher’s exact
Note: NS 5 not significant.
Of the patients who were treated by assisted reproductive techniques, 19 couples with abnormal semen parameters had a fertilization rate of 61.7% and a clinical pregnancy rate of 42.9%. Among the 13 couples with normal semen parameters, the fertilization rate was 70.1% and the clinical pregnancy rate was 64.3% (Table 2). One patient did not undergo embryo replacement because all the zygotes were triploid. There was no difference in the rate of pregnancy loss or the incidence of neonatal malformations between the two groups. One miscarriage occurred in the abnormal semen group, whereas one ectopic pregnancy was diagnosed in the normal semen group; no neonatal malformations were observed in either group. In couples treated by assisted reproductive techniques, the difference in the incidence of chromosomal abnormalities was confirmed, as was the relation with semen parameters (Table 2). The age of the female partners who were inseminated with semen that had normal parameters was similar to that of the female partners who were inseminated with semen that had compromised parameters (35.6 6 6 years vs. 35.8 6 5 years). In spite of a significant difference in chromosomal abnormalities, fertilization and pregnancy rates were unaffected. Among the couples with normal semen parameters, 8 patients were delivered of 10 healthy infants. In the group with abnormal semen parameters, 11 patients were delivered of 17 healthy infants.
DISCUSSION Several studies recently have raised concern about the safety of ICSI (12). The results that we obtained from FISH analysis demonstrated a significantly increased frequency of aneuploidy in the spermatozoa of men with abnormal semen parameters. These results were most striking in men with oligozoospermia. All types of chromosomal abnormalities appeared to be increased, including nullisomy, gonosomal disomy, autosomal disomy, and diploidy. Autosomal disFERTILITY & STERILITYt
omy, however, was the most predominant abnormality when chromosome 21 was assessed. Previous reports on the incidence of aneuploidy in the spermatozoa of men with abnormal semen parameters have been controversial. Although some studies recorded comparable levels of chromosomal abnormalities in fertile and infertile patients (6), others reported an increased frequency of numerical chromosomal abnormalities in the infertile population (5). There was a higher frequency of nondisjunction in chromosomes 21, X, and Y than in other chromosomes in fertile men with normal semen parameters (16). In addition, we observed a higher incidence of gonosomal disomy and disomy 21 in men with abnormal semen parameters. Our figures, however, were higher than those previously reported, possibly because of the subnormal semen status of these patients. The origin of trisomy 18 and trisomy 21 in offspring has been reported to be maternal in approximately 91% (22) and 95% (1), of the cases, respectively. Our findings could signify that paternal disomy might be responsible for these aberrations when pregnancies are established by men with subnormal semen parameters. Our present findings support the report by Martin and Rademaker (23) that sex chromosomes appear to be involved frequently in aneuploidy among men with abnormal semen parameters. Men with abnormal semen parameters have an increased frequency of pairing disruptions resulting in meiotic arrest (24). The sex chromosome bivalent is particularly susceptible to pairing abnormalities because there generally is only one crossover in the pseudoautosomal region. Thus, men with abnormal semen parameters may have decreased recombination and pairing leading to both meiotic arrest (oligozoospermia) and nondisjunction of the sex chromosomes (20); the immediate consequence of this observation is a higher incidence of trisomies and gonosomal aneu95
ploidies in the offspring of men with abnormal semen parameters. Aneuploidies of maternal origin can be identified by polar body biopsy (25). However, it is not possible to assess the ploidy of a particular spermatozoon before it is used for ICSI. Because spermatogenetic arrest resulting from chromosomal abnormalities is inconsistent, the genetic constitution of the gamete is equally unpredictable. Therefore, at the present time, preimplantation diagnosis is the only means of determining the karyotype of the embryo before transfer. The information provided by FISH, however, may be helpful in counseling candidates for assisted reproductive techniques regarding their chances for transmitting chromosomal abnormalities of paternal origin. Although it is still low in absolute terms, the spermatozoa of men with abnormal semen parameters have a slightly higher incidence of both autosomal and gonosomal anomalies. The higher incidence of disomy 21 compared with disomy 18 confirms the higher frequency of nondisjunction for this autosome, as evidenced in men with abnormal semen parameters. Some specimens (n 5 9) were not processed by density gradient centrifugation because of the extremely low number of spermatozoa present in the retrieved sample. Although the preparation method could be a source of potential bias, the analysis of samples processed by density gradient centrifugation confirmed the association between a higher incidence of chromosomal abnormalities and poor semen characteristics. We decided to assess a relatively small number of spermatozoa (approximately 1,725 per sample) because, in our preliminary experiments, this number was sufficient to provide reliable information on the karyotypic distribution of the entire sample and allowed the assessment of normozoospermic and oligozoospermic samples in an equitable fashion. Although the lower number of spermatozoa counted could be inadequate, the incidence of chromosomal abnormalities observed in this study is comparable to that in previously published work (5). This study indicates that men with suboptimal semen quality have an overall higher incidence of chromosomal abnormalities that appears to be inversely related to each individual semen parameter. However, the patient group with the higher frequency of sperm chromosomal abnormalities had fertilization and pregnancy rates comparable to those of the control group and did not have higher rates of pregnancy loss or neonatal malformations. Although concern related to the use of suboptimal spermatozoa still remains, sperm chromosomal abnormalities at this rate do not appear to compromise the ultimate reproductive outcome.
Acknowledgments: The authors thank the clinical and scientific staff of The Center for Reproductive Medicine and Infertility, J. Michael Bedford,
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Ph.D., D.V.M., for his critical review of the manuscript, Ms. Miriam Feliciano, B.Sc., and Ms. Maria Oquendo, B.Sc., for technical assistance, and Ms. Queenie V. Neri, B.Sc., for editorial assistance.
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