Dual color fluorescence in situ hybridization to investigate aneuploidy in sperm from 33 normal males and a man with a t(2;4;8)(q23;q27; p21)*

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1 FERTILITY AND STERILITY Copyright" 1994 The American Fertility Society Printed on acid-free paper in U. S. A. Dual color fluorescence in situ hybridization to investigate aneuploidy in sperm from 33 normal males and a man with a t(2;4;8)(q23;q27; p21)* Peter Y. Lu, M.D.t Diane G. Hammitt, Ph.D.t:!: Alan R. Zinsmeister, Ph.D. Gordon W. Dewald, Ph.D.:j:II~ Mayo Clinic and Mayo Foundation, Rochester, Minnesota Objective: To establish the relative frequency of aneuploidy in sperm from normal and abnormal subjects using dual color fluorescence in situ hybridization and probes for six different chromosomes. Design: Semen from 33 normal males and a patient with a translocation was studied using dual color fluorescence in situ hybridization with probes for chromosomes 4, 7, 8,12,18, X and Y. The frequency of aneuploidy for each chromosome is compared with one another and with the patient who had a t(2;4;8)(q23;q27;p21). Setting: Specimens were obtained from patients at the Mayo Clinic, Rochester, Minnesota. Results: The percentage of sperm with disomy or nullisomy in normal subjects ranged from 0.2% to 0.6% for each of the chromosomes studied. No statistically significant differences were observed between these chromosomes. The frequency of aneuploidy in sperm from a patient with a t(2;4;8) was 3.3% and 4.8% for chromosomes 4 and 8, respectively. Conclusion: Fluorescence in situ hybridization was useful to establish the normal range of nullisomic and disomic sperm for six different chromosomes and to study a patient with a clinically significant chromosome abnormality. In normal males, no difference in the frequency of meiotic nondisjunction was observed among the chromosomes studied. Fertil Steril 1994;62:394-9 Key Words: Human sperm, fluorescence in situ hybridization, sperm disomy, sperm nullisomy, sex chromosomes, chromosome 4, chromosome 7, chromosome 8, chromosome 12, chromosome 18, sperm nondisjunction Aneuploidy is associated with 8% of human conceptions, 50% of first trimester spontaneous abortions, and 0.7% of liveborns (1, 2). Trisomy of al- Received January 13, 1994; revised and accepted March 25, * Presented at the 41st Annual Scientific Meeting of the Society for Gynecologic Investigation, Chicago, Illinois, March 23 to 25,1994. t Division of Reproductive Endocrinology. * Division of Laboratory Genetics. Section of Biostatistics. II G.W.D. is supported by a grant from Imagenetics, Framingham, MA. 'If Reprint requests: Gordon W. Dewald, Ph.D., Mayo Clinic, 200 First Street, SW, Rochester, Minnesota (FAX: ). most every chromosome has been reported, but 45,X and trisomy 15, 16, and 22 are common in abortuses (3). Among liveborns, trisomy 13, 18, 21, and sex chromosome aneuploidy are common whereas aneuploidy of other chromosomes are rare (4). Therefore, different trisomies are not equally viable or the frequency of nondisjunction is not the same for different chromosomes. Aneuploidy in human sperm and oocytes has been investigated. However, because human 00- cytes are not easily accessible, most research has focused on the incidence of aneuploidy in sperm. The examination of sperm morphology alone has not proven to be reliable to determine the chromosomal constitution (5). Quinacrine staining of the Y 394 Lu et al. Investigation of aneuploidy in sperm Fertility and Sterility

2 --4 chromosome has been helpful to study nondisjunction of the Y chromosome, but this technique is unreliable because it stains both the Y chromosome and the nuclear background. This makes it difficult to consistently identify the Y chromosome (6, 7). Direct analysis of sperm chromosomes in the male pronucleus is possible after fertilization of hamster oocytes (8,9). This technique has been used to determine the frequency of aneuploidy for both autosomes and sex chromosomes (10). Although this method is accurate, it is an expensive and labor intensive technique that is not widely used today. Furthermore, it limits the analysis only to sperm that are capable of fertilization. The recent development of chromosome-specific probes now permits the use of fluorescence in situ hybridization to study cells in interphase. Fluorescence in situ hybridization appears to be a reliable, sensitive, and rapid method to establish the frequency of aneuploidy for specific chromosomes in sperm (11) and has been used to determine the frequency of aneuploidy in sperm for chromosomes 1, 12, 15, 16, 17, X and Y (11-13). The purpose of the present study was to [1] determine the normal range of aneuploidy for chromosomes 4, 7, 8, 12, 18, X and Y in spermatozoa from males with normal semen, [2] study aneuploidy in sperm of a man with a chromosome translocation, and [3] determine whether paternal nondisjunction contributes to the origin of certain clinically recognized trisomies. MATERIALS AND METHODS Sperm Preparation Fresh semen samples were collected from 33 normal males and a man with a complex translocation. The investigators were not aware of the origin of any specific semen sample until all results were collected. Preparations were made by placing 10 ~L of liquefied semen on each of three glass slides. This was done using an automatic slide spreading device. After air drying, slides were fixed with methanol:glacial acetic acid (3:1) for 1 hour and then air dried. Fluorescence in Situ Hybridization Fluorescence in situ hybridization was performed using chromosome specific probes for X and Y chromosomes and chromosomes 4, 7, 8, 12, and 18 (Imagenetics, Framingham, MA) using a modified in situ hybridization method developed in our laboratory (14). The probes for the X chromosome and chromosomes 4,7,8,12, and 18 were centromerespecific alpha satellite DNA that was directly labeled with a fluorescent stain. The X chromosome and chromosomes 4 and 18 were labeled with SpectrumOrange, and chromosomes 7, 8, and 12 were labeled with SpectrumGreen. The Y chromosome probe was labeled with Spectrum Green and hybridized to most of Yq11.2 and all of Yq12. For each slide, we studied two different chromosomes simultaneously using probes with different signal colors (green and orange). The hybridization solution was prepared at room temperature and consisted of 1 ul of each probe, 1 ~L of distilled water, and 7 ~L of a modified hybridization mix (Imagenetics). The hybridization solution was placed on the sperm preparation and covered with a 22 X 22 mm coverslip. The coverslip was sealed in place with rubber cement. Denaturation and hybridization of probe DNA and sperm DNA occurred in a preprogrammed prototype Accel oven (Imagenetics, Naperville, IL) over a 60-minute cycle. Then, the coverslips were removed and the slides were placed in a series of 5-minute washes at 45 C as follows: 50% formamide solution for three washes, sodium chloride and sodium citrate solution (2X SSC), and then a 2X SSCjO.1 % NP40 solution. The slides were air dried, and a counterstain with 16 ~L of 4'6'-damino-2-phenylindole (DAPI; Imagenetics, Framingham, MA) was applied. Then, the preparation was again covered with a 22 X 22 mm coverslip. With this method, normal haploid sperm showed one fluorescence signal for each chromosome. A disomic sperm showed two signals for one chromosome and a single signal for the other chromosome. A diploid sperm showed four signals, two for each target chromosome. Sperm without any fluorescence signals were considered technical artifacts and were not scored. For the normal samples, fluorescence in situ hybridization was performed using the following combination pairs of probes: [1] chromosome 4 orange and chromosome 7 green and [2] chromosome 8 green and chromosome 18 orange. To study the sex chromosomes, a probe mixture of chromosome X orange, chromosome Y green, and chromosome 12 green was used. Although the Y chromosome and chromosome 12 probes were the same color, their signal size was significantly different to distinguish between them. The slides were examined at 100X magnification with a microscope equipped for fluorescence. We Lu et al. Investigation of aneuploidy in sperm 395

3 Table 1 Age and Semen Profile of 33 Normal Men and a Male whose Karyotype is 46,XY,t(2;4;8)(q23;q27;p21)* Parameter Age (years) Volume (ml) Count (Xl0 6 /ml) Motility (%) Sperm speedt Normal males 34.0 ± ± ± ± ± 0.4 t(2;4;8) Male * Values are means ± SD. t Graded subjectively on a scale of 1 (sluggish) to 4 (vigorous). only scored sperm with distinct boundaries and not overlapped with adjacent sperm. For the normal specimens, 500 consecutive sperm were scored from each slide preparation for each combination of probes. The karyotype of the patient with a chromosome abnormality selected for this study was 46,XY,t (2;4;8)(q23;q27;p21). This patient was identified during a fertility workup after he and his wife experienced three consecutive first trimester spontaneous abortions. His semen was examined with probes for [1] chromosome 4 orange and chromosome 12 green and [2] chromosome 8 orange and chromosome 12 green. This analysis was performed on two slides for each of the fluorescence probe combinations. Statistical Analysis The distribution of scores for each chromosome (X and Y, 4, 7, 8,12, and 18) were summarized over all normal semen samples. An estimate of the 95th percentile for the proportion of abnormal cells (out of approximately 500 cells for each specimen) was computed for each individual chromosome and for all chromosomes combined (X and Y, 4,7,8,12, and 18), based on the distribution of observed percentages for all 33 subjects. The associations between the proportions of nullisomy and disomy cells among the six chromosomes was assessed using a (Spearman) rank correlation. Normal Subjects RESULTS The results of semen analysis for the specimens from the 33 normal men are shown in Table 1. A total of 16,500 sperm were scored by fluorescence in situ hybridization to determine the frequency of aneuploidy for each chromosome (Table 2). Significant differences in the frequency of nullisomic and disomic sperm was not detected among chromosomes 4, 7, 8, 12, 18, or X and Y. No significant differences were observed between the frequency of disomy and nullisomy for any of the individual autosomes studied. For the sex chromosomes, the frequency of disomy was significantly greater than nullisomy. No sperm had three or more signals (trisomy) for any given probe. The mean (±SD) percentage of X- and V-bearing sperm was 50.4% ± 3.4% and 49.3% ± 3.4%, respectively. No statistical association was found between the frequency of sperm aneuploidy, paternal age, or any parameter of semen analysis (volume, count, motility, or sperm speed). Adjusting for 15 tests of pairwise associations between the six chromosomes (i.e., using an a-level of 0.003) indicated significant association of the proportions of nullisomy and disomy cells for X and Y versus 12, 18 versus 12, 18 versus X and Y, and 8 versus 18 (all P < 0.003). The results of FISH studies for chromosomes 4 and 7 and chromosomes 8 and 18 revealed two and five sperm, respectively, that had two signals of each probe. This suggests that approximately 0.01% to 0.03% of sperm may be diploid. Statistical analysis detected no significant differences between the frequency of aneuploidy for these six chromosomes. Combining these proportions over all subjects, first within each subject by computing the total number of nullisomic and disomic cells observed as a proportion of 1,500 cells scored per subject and then by summarizing the distribution of overall aneuploidy, re- Table 2 The Frequency of Nullisomic and Disomic Sperm in 33 Normal Men and a Male whose Karyotype is 46,XY,t(2;4;8)(q23;q27;p21)* 95th Chromosomet Median:j: percentile:j: 46,XY,t(2;4;8) Male XandY All studied * Values are percentages (%). t No statistically significant differences between chromosomes, P > :j: Based on 500 scored sperm per chromosome pair per subject. (A total of 16,500 sperm were scored for each chromosome in the study.) Based on 909 scored sperm. II Based on 940 scored sperm. 11 Based on 1849 scored sperm. 396 Lu et al. Investigation of aneuploidy in sperm Fertility and Sterility

4 suited in a median frequency of 0.6% with an upper bound of normal (95th percentile) of 1.4 %. (Table 2) 46,XY,t(2;4;8)(q23;q27;p21) Patient The results for analysis of semen from the male with a chromosomal translocation are shown in Table 1. This patient was found to be borderline oligospermic. A total of 909 sperm were scored using fluorescence in situ hybridization for the chromosome 4 orange and chromosome 12 green probe combination. The frequency of chromosome 4 aneuploidy was 2.0% disomy and 1.3% nullisomy. A total of 940 sperm were scored for the chromosome 8 orange and chromosome 12 green probe combination. The frequency of chromosome 8 aneuploidy was 2.7% disomy and 2.1 % nullisomy. Chromosome 12 was not involved in the translocation, but was used as a control. The incidence of chromosome 12 aneuploidy was 0.2% for 1,849 sperm were scored. DISCUSSION The introduction of dual color fluorescence in situ hybridization to evaluate the chromosomal complement of cells in interphase has made it possible to accurately determine the frequency of disomy or nullisomy for each chromosome. Using this method, we evaluated 49,500 spermatozoa from fresh semen in an efficient, accurate, and relatively inexpensive manner for six different human chromosomes. Fluorescence in situ hybridization is easier than other methods to study chromosomes of spermatozoa. For example, using the human sperm/hamster egg fusion technique, Martin and Rademaker (10) established the karyotype of 6,821 sperm over an 8-year period. By comparison, it took approximately 40 minutes to score 500 sperm in this study. The human sperm-hamster egg fusion technique does permit the evaluation of the entire chromosomal constitution of a single sperm, but has the disadvantage of needing to be done on cells in metaphase. We used a dual probe strategy to study sperm, which permits assessment of the frequency of both disomy and nullisomy in spermatozoa. Previous studies on sperm used a single chromosome specific fluorescence probe (11-13, 15). Consequently those studies do not reliably account for nullisomy because the lack of a visible signal in a sperm nuclei could be a failure of probe hybridization. In our experience, occasional areas of any glass slide sperm preparation contain sperm that do not hybridize with the probes. These areas usually have air bubbles beneath the coverslip that prevent the probe mixture from interacting with the sperm. When using dual color fluorescence in situ hybridization, the problem of nullisomy versus technical error can be distinguished by using a control probe signal. Of course, even this approach would not distinguish between a sperm lacking both target chromosomes and technical artifact. However, this is most likely a rare problem that would not significantly interfere with attempts to establish the normal range for aneuploidy in sperm. We have determined that the median frequency of disomy and nullisomy for chromosomes 4, 7, 8, 12,18, and X and Yis 0.4%, 0.4%, 0.2%, 0.2%, 0.2%, and 0.2%, respectively. Thus, the frequency of me i otic nondisjunction for these chromosomes appears to be similar in males. It is difficult to estimate an overall frequency of aneuploidy (over all 23 chromosomes) because some sperm may be abnormal for several chromosomes. If the frequency of aneuploidy for all chromosomes was similar and there was no overlap, then an upper bound for overall frequency of aneuploidy would also be 1.4%. However, the estimated associations in this study suggest there is some overlap. In this study, the ratio of X- and Y-bearing sperm did not differ significantly from the expected 1:1 predicted from normal meiosis of the X and Y chromosome. This finding is consistent with previous fluorescence in situ hybridization investigations (12, 13, 16). The incidence of diploid spermatozoa was rare (7/49,500) in our study. Nevertheless, finding at least some diploid sperm provides direct evidence for their existence. Diploid sperm have been implicated in the possible origin of some hydatidiform moles. If a diploid sperm fertilizes a haploid or anuclear oocyte this could lead to the genesis of a partial (69,XXX or 69,XYY) or complete (46,XX, or 46,YY) mole, respectively (17). The evaluation of semen from a patient with a rare autosomal three way translocation, t(2;4;8) (q23;q27;p21), demonstrates one application of fluorescence in situ hybridization in clinical practice. In carriers of such translocations, meiosis is complicated by the complex pairing of the involved chromosomes. This results in atypical chromosome segregation that can produce sperm with multiple copies of the centromeric regions of chromosome 2, 4, and/or 8. In the sperm of this patient, the incidence of aneuploidy for chromosomes 4 and 8 was 3.3% and 4.8%, respectively. This is significantly higher than the normal range for both chromo- Lu et ai. Investigation of aneuploidy in sperm 397

5 somes 4 and 8 and is consistent with the meiotic errors that might be predicted for a complex translocation. We did not have a reliable probe for chromosome 2 to study this patient. We expect that the frequency of aneuploidy for chromosome 2 would be similar to chromosomes 4 and 8 in this patient. We established the incidence of aneuploidy for chromosome 12 in this patient as a control and found the frequency to be 0.2%. This is within the normal range for chromosome 12 and, as expected, appears to be undergoing normal meiosis independently of the chromosomes involved in the translocation. Our data indicate that the combined incidence of nondisjunction for chromosomes 4,7,8,12,18 and X and Y is 1.4% in normal males. Furthermore, no significant difference was apparent among the individual chromosomes we studied. These findings are consistent with several other cytogenetic studies (18). However, Martin et al. (19) suggest that in males, the nondisjunction of chromosomes 1, 21 and X and Y might occur more frequently than other chromosomes. Our data do not suggest any increased sex chromosome nondisjunction when compared with chromosomes 4,7,8, 12, or 18. The frequency of aneuploidy for different chromosomes varies between liveborns and spontaneous abortions. Because our results suggest the incidence of aneuploidy is similar for different chromosomes in males, it appears that this observation is then most likely due to differences in viability of different forms of aneuploidy and/or differences in maternal chromosome nondisjunction. The viability of conceptuses with different chromosomal aberrations is clearly not the same. This implies an important role for the differential viability of different trisomies in what is observed clinically. Trisomy 13, 18, 21 and the sex chromosomes are found in liveborns and trisomy 15, 16, and 22 are common in abortuses whereas other trisomies are rare (1, 3, 4). Autosomal monosomies are also rare in spontaneous abortuses or liveborns (20). Most likely, conceptuses with rare trisomies or monosomies are nonviable and either do not implant or abort so early that clinical identification is not possible. A significant number of per implantation embryo losses in humans have been shown by using sensitive {j-hcg assays (21). It has been estimated that at least one-third of all conceptions may terminate before midtrimester with one-half of these being unidentified pregnancy losses occurring before or shortly after implantation (22). Many of these pregnancy losses may be due to chromosomal aberrations that are not compatible with continued viability. This investigation confirms the usefulness of fluorescence in situ hybridization to study chromosome aberrations in sperm. This study also establishes the normal range for aneuploidy for six chromosomes and shows that the incidence of aneuploidy is similar among them. We suspect that nondisjunction of the 17 other chromosomes in the human karyotype is most likely similar to the 6 chromosomes we investigated. However, further studies are necessary to verify this. It is important to establish the normal range for aneuploidy for human gametes because this information provides a baseline for clinical studies. This information would be valuable for comparison in studies of men who [1] have been exposed to toxic agents, [2] carry chromosome abnormalities, or [3] have idiopathic fertility problems. Fluorescence in situ hybridization has also been used in attempts to study procedures that may separate X- and Y-bearing sperm (23, 24). 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