MALE FACTOR. Baylor College of Medicine, Houston, Texas

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1 FERTILITY AND STERILITY VOL. 76, NO. 5, NOVEMBER 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. MALE FACTOR Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for intracytoplasmic sperm injection Hyun-Mee Ryu, M.D., a,d William W. Lin, M.D., b Dolores J. Lamb, Ph.D., b,c Weber Chuang, M.D., b Larry I. Lipshultz, M.D., b and Farideh Z. Bischoff, Ph.D. a Baylor College of Medicine, Houston, Texas Received January 12, 2001; revised and accepted May 17, Supported by grant P01HD from the National Institutes of Health,Bethesda, Maryland (D.J.L., L.I.L., and F.Z.B.). Reprint requests: Farideh Z. Bischoff, Ph.D., Department of Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin Street, Suite 832, Houston, Texas (FAX: ; bischoff@bcm. tmc.edu). a Department of Obstetrics and Gynecology, Baylor College of Medicine. b Department of Urology, Baylor College of Medicine. c Department of Cell Biology, Baylor College of Medicine. d Department of Obstetrics and Gynecology, Samsung Cheil Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea /01/$20.00 PII S (01) Objective: To determine the incidence of nondisjunction for chromosomes X, Y, and 18 using fluorescence in situ hybridization (FISH) on morphologically normal sperm from infertile men who are candidates for ICSI. Design: After standard hematoxylin staining, sperm with normal morphology were identified using Kruger s strict morphology criteria. The location of each normal-appearing sperm was recorded using an electronic microstage locator. Slides were subsequently subjected to FISH for detection of chromosomes X, Y, and 18 (control probe). Nuclei were relocated and analyzed under the fluorescent microscope. Setting: University-affiliated IVF and intracytoplasmic sperm injection program. Patient(s): Men classified as infertile on the basis of abnormal strict morphology ( 4% by Kruger s criteria). For controls, normal fertile men (n 6) were also analyzed. Intervention(s): Semen smears were obtained retrospectively from infertile (n 8) and fertile (n 6) men. Main Outcome Measure(s): Ploidy of each cell was determined according to the number of signals detected for each probe. Result(s): Approximately morphologically normal sperm were identified and located in each case. Subsequent FISH analysis of these normal sperm showed aneuploidy to range from 1.8% to 5.5% in the infertile group as compared with 0 to 2.6% among the control fertile group. Statistically significant differences in the incidence of aneuploidy for the sex chromosomes as well as for all three (X, Y, and 18) chromosomes was observed. Conclusion(s): Although 95% to 98% of the sperm were found to be normal for X, Y, and 18, our findings show that infertile couples undergoing ICSI are likely to be at an increased risk for having a genetically abnormal conceptus as compared with the fertile controls. These results demonstrate that normal morphology is not an absolute indicator for the selection of genetically normal sperm. Hence, observed pregnancy failures among ICSI patients may in part be due to the selection of aneuploid sperm. (Fertil Steril 2001;76: by American Society for Reproductive Medicine.) Key Words: Fluorescence in situ hybridization, sperm aneuploidy, ICSI, strict morphology In the United States, about 10% of the population is infertile. Male-factor abnormalities are the predominant cause in 30% of cases and are contributory in 50%. Although some cases of infertility are due to a constitutional chromosomal abnormality, such as reciprocal translocation, robertsonian translocation, or a 47,XXY karyotype (1 3), most infertile couples are chromosomally normal. Clearly, normal individuals produce abnormal germ cells, as evidenced by observations on human sperm chromosomes obtained by the hamster egg fusion technique (4, 5) and by studies using fluorescence in situ hybridization (FISH) with chromosome-specific probes on human spermatozoa (6 9). Most cases of male infertility are a consequence of altered spermatogenesis giving rise to an inadequate quantity or quality of sperm; however, the etiology of impaired spermatogenesis is frequently idiopathic, given that the factors contributing to this problem are often undefined. 879

2 The introduction of intracytoplasmic sperm injection (ICSI) in 1992 by Palermo et al. (10) has revolutionized the treatment of severe male-factor infertility. However, the ICSI pregnancy rates from IVF centers across the world remain less than optimal, ranging from 30% 50%. A major concern associated with the use of ICSI for the treatment of couples with male-factor infertility is the safety of the procedure. Reports on prenatal diagnosis and on infants born as a result of ICSI with an increased rate of sex chromosomal abnormalities have shown the importance of studying the chromosomal constitution of sperm used for ICSI (11, 12). Morphology in addition to motility is one of the main selection criteria for identifying sperm to be injected during the ICSI micromanipulation procedure. It has been documented that sperm with abnormal morphology have increased incidence of aneuploidy (13 16). However, the risk of selecting abnormal sperm with gene and/or chromosome aberrations remains unclear. In fact, there have been no studies to determine the genetic integrity of sperm with normal morphology that embryologists have confidently and routinely selected for ICSI. The genomic instability of the spermatozoa as manifested by chromosomal aneuploidy may in fact be a contributing factor to the increased incidence of sex chromosome nondisjunction in offspring conceived by ICSI. The objective of the current study is to determine the incidence of aneuploidy for chromosomes 18, X, and Y in morphologically normal sperm identified in infertile men undergoing ICSI. MATERIALS AND METHODS Samples Semen smears were obtained from eight infertile men using the following criteria: [1] each participant was identified as infertile based on abnormal strict morphology ( 4% by Kruger s criteria), [2] they were candidates for ICSI (17), and [3] there was sufficient specimen for localization of 100 normal sperm. Although many specimens were available from infertile men based on criteria 1 and 2, few had normal sperm. For use as controls, semen smears from six normal donors of proven fertility were obtained. All patients (infertile and fertile) had a normal (46, XY) karyotype. The study was performed under Baylor College of Medicine institutional review board approved guidelines, with all individuals giving written consent before enrollment. Analysis of Strict Morphology After standard hematoxylin staining, sperm with normal morphology were identified using Kruger s strict morphology criteria. The criteria for the normal spermatozoon include a smooth, oval sperm head measuring 3 m to5 m in length and 2 m to3 m in width. Normal sperm had no defects of the neck, midpiece, or tail, with a well-defined acrosome comprising 40% 70% of the sperm head. Cytoplasmic droplets no larger than half the size of the sperm head were present. During analysis, the location of normal-appearing sperm was recorded using an electronic microstage locator. Among the infertile cases, the maximum number of morphologically normal sperm found was 107 (two cases), 109 (three cases), 112 (one case), 117 (one case), and 130 (one case; Table 1). Among the normal control cases, nuclei with normal morphology were randomly selected. For comparison, approximately 100 nuclei with abnormal morphology in each case (infertile and fertile) were also located for subsequent FISH. Fluorescence In Situ Hybridization Stain and oil were removed by serially placing the slides in xylene for 5 minutes and in ethanol for 5 minutes and then air drying. The nuclei were then fixed in methanol for 15 minutes. Slides were incubated in 2 SSC (diluted from 20 SSC: 3 M NaCl, 0.3 M trisodium citrate 2H 2 0; ph 7.0) and then passed through three ethanol washes (70%, 80%, and 100%) for 2 minutes each and allowed to air dry. Slides were then incubated for 6 minutes to 8 minutes at 37 C ina freshly made solution of 25 mm dithiothreitol (Sigma, St. Louis, MO), allowing the sperm heads to decondense and swell. Slides were immediately placed in 2 SSC at room temperature for 3 minutes, then dehydrated in three ethanol washes as previously described and allowed to air dry. Three-color FISH to detect chromosomes X, Y, and 18 was performed, as described elsewhere (18), with directlabeled chromosome-specific alpha satellite probes (Vysis, Inc., Downers Grove, IL). The probe mixture was sealed onto the slide and then placed in an 80 C oven for 2 to 3 minutes to denature cellular DNA and probes simultaneously. After an overnight hybridization at 37 C, the slide was washed in 0.25 SSC (ph 7.0) at 68 C for 10 seconds and rinsed in 1 phosphate buffer detergent (PBD) (Oncor, Thornwood, NY). Nuclear counterstain, 4,6-diamidino2- phenylindole (DAPI II solution; Vysis), was applied. The morphologically normal and abnormal cells were then relocated and ploidy determined according to the number of signals detected for each probe under a Zeiss Axioskop microscope (Carl Zeiss) equipped with multiband pass filters. Our overall hybridization efficiency was 97%. Scoring Criteria The chromosome-specific probes were identified by color, and the nuclei were analyzed for the presence of zero, one, two, or three or more signals for each of three probes. Nuclei containing signals that were of unexpected size or that appeared to be outside the nuclear membrane were eliminated from analysis. Signals were considered to represent a split domain if [1] the size and intensity of each of the two signals was less than that of the signal for the other homologue and [2] the distance between the two signals was less than the diameter of either of the two signals. 880 Ryu et al. Aneuploidy in normal sperm of infertile men Vol. 76, No. 5, November 2001

3 TABLE 1 Semen parameters and incidence of chromosome 18, X, and Y aneuploidy as determined by FISH. Case no. Patient age (years) % Normal morphology based on Kruger s criteria Total no. of normal nuclei No. of normal haploid nuclei detected No. of nuclei with sex chromosomal aneuploidy a (P.007) No. of nuclei with chromosome 18 aneuploidy No. of diploid cells detected % Abnormal cells a (P.004) Infertile (candidate ICSI) cases b 106 c 2 d 1 e 0 f Normal, fertile control cases a Continuous variables were analyzed using the two-sample independent t test. Mann-Whitney rank sum test was used for variables with unequal variances. Statistical significance was defined as P.05 for continuous analysis. b Total number of morphologically normal sperm located and subsequently analyzed by FISH. c Nuclei determined to be normal haploid based on the detection of one chromosome 18 signal and either one X- or Y-chromosome specific signal in each cell. d Nuclei determined to have sex chromosomal aneuploidy based on the detection of one chromosome 18 signal and either no sex chromosomes (nullisomic) or two sex chromosome (disomic; XY, XX, YY) specific signals in each cell. e Nuclei determined to have chromosome 18 aneuploidy based on the detection of either one X- or Y-chromosome-specific signal and either none or two chromosome 18 specific signals. f Nuclei determined to be diploid based on the detection of two chromosome 18 signals and two sex chromosome (X and Y) signals. Ryu. Aneuploidy in normal sperm of infertile men. Fertil Steril Statistical Calculations The distribution of FISH signals was collectively compared between the ICSI and the control groups. Continuous variables were analyzed using the two-sample independent t test. The Mann-Whitney rank sum test was used for variables with unequal variances. Statistical significance was defined as P.05 for continuous analysis. RESULTS Semen smears from eight infertile patients and six fertile sperm donors with proven fertility were analyzed. The mean ( SD) age was years for the infertile group and years for the control group. In general, the frequency of finding morphologically normal sperm in specimens obtained from these infertile patients is very low. Therefore, all of the normal sperm found were localized (coordinates recorded) and subsequently analyzed for aneuploidy using FISH. A mean of sperm identified as morphologically normal were located in each of the eight infertile case. Among the controls, a mean of morphologically normal sperm was located randomly and subsequently analyzed by FISH. Table 1 summarizes the specific number of nuclei analyzed in each case. In addition, sperm with abnormal morphology were also evaluated among the infertile cases (data not shown). The mean frequency of aneuploidy among morphologically abnormal sperm was 29% (range: 15% 35%). Among the controls, aneuploid frequency among morphologically abnormal cells was slightly lower (mean, 13%; range, 7% 15%). Fluorescence in situ hybridization analysis of the localized normal-appearing sperm showed that the overall aneuploidy rate ranged from 1.8% to 5.5% in infertile group. This observed rate was approximately twofold to threefold greater than the observed 0 to 2.6% in the fertile control group. In both groups, the majority of the abnormal nuclei contained a sex chromosome aneuploidy in which the signal for either the X or Y chromosome was absent. Presence of the control probe signal for chromosome 18 confirms successful hybridization in these nuclei. The aneuploid frequency among the infertile group was significantly greater when either considering the sex chromosomes alone (P.007) or all three of the FERTILITY & STERILITY 881

4 chromosomes (X, Y, and 18; P.004) as compared with the control group (Table 1). DISCUSSION In the current study, a significantly higher rate of chromosomal aneuploidy (1.8% 5.5%) was detected in morphologically normal sperm identified in semen samples from patients classified as infertile when compared with normal controls (0 2.6%). Therefore, based on these results, we conclude that normal morphology is not an absolute indicator for the selection of genetically normal sperm. Although 95% to 98% of the sperm were found to be normal for X, Y, and 18, our findings show that infertile couples undergoing ICSI are likely to be at an increased risk for having a genetically abnormal conceptus as compared with the fertile controls. Nevertheless, because the frequency of aneuploidy is likely to be greater in morphologically abnormal sperm, sperm selection remains an important requirement in optimizing a couple s chance of conception. However, given our results, couples may be advised to consider the option of preimplantation genetic diagnosis for aneuploidy. Previous studies have reported the aneuploid frequency for the sex chromosomes to be 1% in mature sperm (2 6). In the current study, the mean rate of sex chromosomal aneuploidy among the controls was 0.96%. This rate is similar to our previous results of 1% and 1.12% XY aneuploidy observed in two normal donors (6). Spriggs et al. (19) also reported a frequency of 1.21% sex-chromosomal aneuploidy (0.78% nullisomy and 0.43% disomy) in which 50,000 sperm nuclei from five normal donors were scored for X and Y. In the current study, an approximate twofold increase in the number of sperm aneuploid for the X and Y chromosomes only was detected in the infertile group as compared with in the control group. Moreover, the overall combined aneuploid frequency of chromosomes X, Y, and 18 was nearly threefold increased in the infertile group (3.325%), as compared with in the control group (1.325%). Information on the remaining 21 autosomes is unknown, given that these chromosomes were not screened for in this study. Clearly, future studies are warranted to examine the aneuploid rates for many of the remaining chromosomes, especially those (e.g., chromosomes 1, 7, 13, 15, 16, 21, and 22) frequently identified as aneuploid through preimplantation genetic studies of human embryos that had undergone ICSI (20). Fluorescence in situ hybridization studies of human sperm have been performed to quantify chromosome-specific aneuploidy and the effects of increasing age. An increasing age effect was shown to be involved with the incidence of XY, YY, and XX disomy (21) in men between 18 and 60 years of age. The overall incidence of XY disomy, not including nullisomy, increased from 0.122% to 0.172% in men at age years and years, respectively. However, this increase is small and not likely to contribute to the differences in aneuploidy rates that we observed among the infertile (mean age of 39.6 years) and control (mean age of 27.1 years) groups. Statistical comparison similar to those in the study by Griffin et al. (21) would not be meaningful for this study, given the low number of cases within each of the two age groups listed above. Clearly, further studies with greater numbers of cases are warranted to evaluate the effect of age on sperm aneuploidy in morphologically normal sperm from infertile men. In addition, FISH has been used to examine aneuploidy in semen from infertile men (22 26) and to correlate abnormal sperm morphology with chromosome aneuploidy (13 16). There are reports proposing that there is no identifiable relationship between chromosome content and the morphology of spermatozoa, as evidenced in both Drosophila and mouse (27 29) as well as in human (14). Others, however, have proposed that sperm phenotype abnormalities are indicative of genotype abnormalities (15, 30). As a part of the ICSI procedure, normal sperm are selected on the basis of motility and morphology. Although the incidence of aneuploidy among sperm with abnormal morphology has been evaluated, little is known regarding the genetic integrity of spermatozoa with normal morphology among infertile patients who have been evaluated by strict morphology analysis. This study determined the incidence of nondisjunction in morphologically normal ejaculated sperm of infertile men using FISH and demonstrated that sperm with normal morphology may not always have a normal complement of chromosomes. References 1. Meschede D, Horst J. 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