Detection of X- and Y -bearing human spermatozoa after motile sperm isolation by swim-up

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1 FERTILITY AND STERILITY Vol. 60, No.6, December 1993 Copyright 1993 The American Fertility Society Printed on acid-free paper in U s. A. Detection of X- and Y -bearing human spermatozoa after motile sperm isolation by swim-up Tie Lan Han, M.D.*t Sean P. Flaherty, Ph.D.*:j: Judith H. Ford, Ph.D.t Colin D. Matthews, M.D.* The University of Adelaide and The Queen Elizabeth Hospital, Woodville, South Australia, Australia Objective: To assess the ratio of X- to Y-bearing human spermatozoa in motile fractions isolated by the swim-up technique. Design: The proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in motile fractions isolated from the same samples by swim-up. X- and Y-bearing sperm were simultaneously identified using chromosome-specific DNA probes and double fluorescence in situ hybridization. Setting: Hospital-based university department. Participants: Ten healthy donors with normal semen characteristics. Main Outcome Measures: The distribution of haploid cells (X or Y), normal size cells with two sex chromosomes (XX, YY, or XY), and large cells containing two (XX, YY, or XY) or four (XXYY) sex chromosomes were measured in neat semen samples and in motile fractions prepared by swim-up. Results: Overall, 95% of sperm in the neat semen and swim-up fractions were labeled with the probes. The ratios of X- to Y-bearing sperm were 47.3:46.9 (neat semen) and 48.4:47.1 (swim-up fractions), which were not significantly different from a 1:1 ratio. The frequencies of sperm with normal size nuclei and two sex chromosomes (XX, YY, or XY) in the swim-up fractions were not significantly different from the controls, but there was a significant reduction in the proportion of cells with large nuclei and two (XX, YY, or XY) or four (XXYY) sex chromosomes in the swim-up fractions. Conclusions: The swim-up technique does not selectively enrich either X- or Y-bearing sperm. Because the isolation of motile spermatozoa is an important procedure for routine lui, IVF-ET, and GIFT, the results of this study are important reassurance that the sex ratio is not altered by this method of sperm preparation. Fertil SterilI993;60: Key Words: Human sperm, swim-up technique, fluorescence in situ hybridization, sex selection, X chromosome, Y chromosome Received May 12, 1993; revised and accepted August 20, Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital. t Genetics Department, The Queen Elizabeth Hospital. :j: Reprint requests: Sean Flaherty, Ph.D., Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia. The swim-up technique (1) is routinely used to obtain populations of highly motile human spermatozoa in many clinical lui, IVF -ET, and GIFT programs (2, 3). Previous studies have addressed the motility, morphology, fertilization rate, and nuclear maturity of human sperm prepared by swim-up and other methods (4-7). Although there have only been a few studies on the sex status and incidence of chromosomal abnormalities in motile sperm fractions prepared by swim-up, they suggest that the ratio of X-to Y -bearing sperm is not altered by this procedure (8-10). However, Check et al. (11) and Check and Katsoff (12) recently reported that infants 1046 Han et al. Identification of X and Y sperm Fertility and Sterility

2 derived from lui using sperm prepared by a modified swim-up procedure have a skewed male:female ratio. In this study, we have examined the effect of motile sperm isolation by swim-up on the ratio of X- to Y -bearing spermatozoa. A double fluorescence in situ hybridization technique (13) was used to simultaneously identify X- and V-bearing sperm. This method enables quick and efficient sexing of large numbers of sperm using chromosome-specific DNA probes and avoids the problems of sexing sperm using quinacrine staining or single probe fluorescence in situ hybridization methods. It also has advantages over quantitative polymerase chain reaction (PCR) methods (10) in that the sex status of each individual sperm is scored rather than the DNA from a population of cells. The ratio of X-to Y -bearing sperm was examined in neat semen and in motile fractions isolated from the same semen samples using the swim-up procedure. The results demonstrate that there is no change in the ratio of X- to V-bearing sperm after motile sperm isolation by the swim-up procedure. Semen Samples MATERIALS AND METHODS Single semen samples were obtained from 10 normal healthy donors who attended the Andrology Laboratory at The Queen Elizabeth Hospital. The samples were produced by masturbation, allowed to liquefy at room temperature for 15 to 30 minutes, and then were analyzed according to standard procedures (14). Sperm morphology was assessed using World Health Organization criteria on smears stained by the modified Papanicolaou technique. The semen values for the 10 donors are summarized in Table l. Each sample was divided into two aliquots. One aliquot of semen (control) was prepared for double fluorescence in situ hybridization, whereas a motile fraction was isolated from the other aliquot using the swim-up procedure and then prepared for fluorescence in situ hybridization. Swim-up Procedure Motile sperm were isolated by swim-up from a washed pellet (2). Briefly, 1 ml of liquefied semen was diluted with 2 ml of HEPES-buffered human tubal fluid medium (15) containing 7.5% human serum and centrifuged at 300 X g for 10 minutes. Table 1 Semen Characteristics of the 10 Semen Samples Progressive Donor Volume Concentration motility Morphology ml xicl'/ml % % normal ND* Mean * ND, not determined. The pellet was resuspended in 2 ml of medium and the centrifugation step was repeated. The final pellet was carefully overlaid with 0.5 ml of fresh medium, taking care not to disturb it, and the tube was then incubated upright at 37 C for 30 to 60 minutes. Up to 0.25 ml of the upper layer (motile fraction) was removed and analyzed as described above. The progressive motility of this fraction was routinely 80% to 95%. Pretreatment of Sperm A modification of the method described by West et al. (16) was used to pretreat sperm nuclei to allow access ofthe DNA probes. Neat semen or the motile fraction (10 to 20 X 10 6 /ml) was mixed with phosphate-buffered saline (PBS, ph 7.4) containing 6 mm ethylenediaminetetraacetic acid (EDT A) and then was centrifuged at 175 X g for 5 minutes. The pellet was resuspended to the same sperm concentration in PBS containing 2 mm dithiothreitol (DTT) and incubated at room temperature for 45 minutes with regular mixing. After centrifugation, the sperm were fixed with methanol:glacial acetic acid (3:1), dropped onto slides, and air dried. Mitotic Chromosome Spreads Mitotic chromosome spreads from human male peripheral blood lymphocytes were used as positive controls. They were prepared using the method of Buckle and Craig (17). DNA Probes The X chromosome-specific (TRX) and Y chromosome-specific (HRY) probes have been described Vol. 60, No.6, December 1993 Han et ai. Identification of X and Y sperm 1047

3 r in our previous publications (13, 18). Biotin-7- da TP was incorporated into the TRX probe using a nick translation kit (GIBCO-BRL, Glen Waverley, Victoria, Australia), while digoxigenin-dutp was incorporated into the HRY probe by random priming using a DIG DNA-labeling kit (Boehringer-Mannheim, Castle Hill, New South Wales, Australia). Fluorescence In Situ Hybridization The complete protocol for double fluorescence in situ hybridization has been described by Han et al. (13). Briefly, a mixture of the TRX (40 ng) and HRY (20 ng) probes were applied to each slide and simultaneously hybridized to the sperm DNA for 16 to 18 hours at 37 C. After thorough washing, the probes were visualized using a multi-step immunofluorescence procedure that used fluorescein isothiocyanate (FITC) for visualization of TRX and tetramethylrhodamine isothiocyanate for visualization ofhry. The following steps were used: Preincubation in 5% nonfat dry milk; FITC-avidin DCS (cell sorter grade; Vector Laboratories, Burlingame, CA); Biotinylated anti-avidin (Vector Laboratories) and mouse anti-digoxigenin (Boehringer-Mannheim); FITC-avidin DCS and tetramethylrhodamine isothiocyanate-rabbit anti-mouse immunoglobulin (Ig)G (Dako, Glostrup, Denmark); and tetramethylrhodamine isothiocyanate-swine anti-rabbit IgG (Dako). The slides were washed with Tris-buffered saline (ph 7.5) containing 0.05% Tween 20 and the appropriate serum (goat, mouse, rabbit, swine) between each incubation step. Finally, the slides were dehydrated, air dried, and mounted with a glycerol:pbs mixture containing 4,6-diamidino-2- phenylindole (Sigma Chemical Co., St. Louis, MO) as a nuclear counterstain and 2% l,4-diazo bicyclo 2,2,2] octane (Sigma Chemical Co.) as an antifade reagent. The slides were examined at X1,250 with a Leitz microscope equipped with epifluorescence and three different filter blocks (A, 12/3, and N-2). At least 1,000 sperm were scored from each preparation (range 1,000 to 1,030). Sperm with a single distinct green (FITC) or red (tetramethylrhodamine isothiocyanate) spot were classified as haploid X or Y -bearing sperm, respectively. Cells that contained two or more spots were scored separately and were divided into groups with normal-size or large nuclei (two to four times the normal size) (19). Statistical Analysis The x 2 test for homogeneity was used to compare the overall proportions of X-and Y -bearing sperm for the 10 samples. A x 2 test was used to compare the proportions of haploid X, haploid Y, XY cells with normal-size nuclei and XY cells with large nuclei in the control and swim-up groups. A Fisher's Exact test was used to compare the proportions of XX, YY, and XXYY cells with large nuclei in the control and swim-up groups. A P value < 0.05 was considered significant. RESULTS Mitotic chromosome spreads and interphase cells from male lymphocyte preparations exhibited clear double-labeling signals. In chromosome spreads there was a distinct green (FITC) spot over the centromere of the X chromosome and a red (tetramethylrhodamine isothiocyanate) spot over the long arm of the Y chromosome. A total of 10,046 sperm (controls) and 10,059 sperm (swim-up) were scored. In the neat semen, 95% of the sperm exhibited a positive hybridization signal and this varied from 90.1 % to 98.5% for individual samples. Similarly, 95.9% of sperm in the swim-up fractions were labeled, with individual samples giving hybridization efficiencies of 91.6% to 99.0% (Table 2). The overall ratio of X- to V-bearing sperm in the neat semen samples was 47.3:46.9, which was not significantly different from a 1:1 ratio. The ratios for individual samples were also not significantly different from 1:1 (Table 2). The ratio of X- to Y bearing sperm in the swim-up fractions was Table 2 X and Y Labeling Results for Individual Semen Samples Neat semen* Swim-up* Donor Labeled X Y Labeled X Y * Values are percentages Han et al. Identification of X and Y sperm Fertility and Sterility

4 Table 3 Frequencies of Haploid and Aneuploid/Polyploid Cells Before and After Swim-up* Neat semen Swim-up Haploid X t Haploid Y t Normal-size nuclei XX t yy t XY t Large nuclei XX yy XY XXYY * Values are percentages. t No significant difference between groups. + Swim-up group is significantly different from neat semen group, P < Swim-up group is significantly different from neat semen group, P < :47.1, which was not significantly different from the ratio in the neat semen or from a 1:1 ratio. There were no significant differences between the control and swim-up fractions in the frequency of haploid X or Y sperm or sperm with normal-size nuclei and two sex chromosomes (XX, YY, or XY). However, the swim-up fractions contained a significantly lower proportion of cells with large nuclei and two (XX, YY, or XY) or four (XXYY) sex chromosomes (Table 3). DISCUSSION In this study, a double fluorescence in situ hybridization technique (13) was used to simultaneously identify X- and Y -bearing human sperm. This technique has several advantages over other methods of sexing sperm. First, it uses probes which are specific for the X and Y chromosomes, rather than a stain such as quinacrine, which brightly stains several regions of the genome in addition to the Y chromosome. Second, a representative sample of the entire population of sperm is studied rather than only a subgroup that is capable of fertilizing zona-free hamster eggs (20, 21). Third, double labeling with X - and Y -specific probes identifies X-and Y -bearing sperm simultaneously and therefore the sex status of the sperm is determined with certainty. In contrast, fluorescence in situ hybridization using a single sex chromosome probe (18, 22, 23) only identifies either the X- or Y -bearing sperm and it is assumed that the nonlabeled cells carry the other sex chromosome. This can produce very misleading results if the hybridization efficiency is low, as unlabeled sperm will be mistaken for sperm that contain the other sex chromosome. Finally, the high labeling efficiency (95%) allows confidence that the X:Y ratio is not unduly biased by inefficient labeling. Recent studies using double fluorescence in situ hybridization (13), sperm karyotyping after penetration of hamster eggs (20, 21), and quantitative PCR (10) have shown that the ratio of X- to Y bearing sperm in human semen is 1:1. The results of this study confirm those findings. It has been suggested that X- and Y-bearing sperm can be separated on the basis of a differential motility of the two populations of sperm (24). Because the swim-up technique selects motile sperm, we were interested to determine whether the isolation of motile sperm by this routine procedure alters the ratio of X- to Y -bearing sperm. The results of this study clearly show that the swim-up procedure isolates motile sperm fractions with a 1:1 ratio of X- to Y -bearing sperm. This agrees with previous studies on swim-up fractions using the hamster egg system (8, 9) and with the recent quantitative PCR study of Lobel et al. (10). Check et al. (11) and Check and Katsoff (12), however, reported a high percentage of male births after insemination with sperm isolated by a modified swim-up procedure and, furthermore, they found that the proportion of sperm that possessed a Y body after quinacrine staining was increased by the modified swim-up procedure. Thus our results contrast with their findings. This may be due to the lack of specificity of quinacrine staining (25) and the fact that they did not verify the ratio of X-to Y -bearing sperm using specific DNA probes or technical differences in the swim-up procedure, because the method we used (swim-up from a washed pellet) is but one of several ways to perform a swim-up. Nevertheless, clinical results from our IVF-ET and GIFT programs also suggest that there is no change in the ratio of X- to Y -bearing sperm after swim-up. Between 1982 and 1987, a total of 350 live births from 262 IVF -ET /G 1FT pregnancies were derived from sperm isolated by swim-up. The data consists of 186 singleton, 64 twin, 11 triplet, and 1 quadruplet pregnancy, and the proportion of male to female births was 92 to 94 (singletons) and 78 to 86 (multiple pregnancies). Overall, 170 male and 180 female infants were born, giving a male to female ratio of 48.6:51.4, which is not significantly different from a 1:1 ratio. In this study we also differentiated between haploid sperm and those with XX, YY, or XY complements Vol. 60, No.6, December 1993 Han et al. Identification of X and Y sperm 1049

5 r to estimate the frequencies of aneuploidy and diploidy in the control and swim-up fractions. Joseph et al. (19) used head size to discriminate between disomic haploid sperm and diploid sperm, the rationale being that diploid sperm would have a large nucleus because of the diploid number of chromosomes whereas disomic haploid sperm would have a normal-size nucleus. If we apply this classification system to the data in Table 3, sperm with two sex chromosomes and a normal-size nucleus would be putative disomic cells whereas those with a large nucleus would be putative diploid or tetraploid (XXYY) cells. The frequencies of putative disomic (XX, YY) and haploid XY sperm were similar in the neat semen and swim-up samples, which suggests that the level of aneuploidy in the isolated motile sperm fraction was comparable to the total sperm population. The frequencies of putative disomic X (0.25% to 0.26%), disomic Y (0.23% to 0.27%), and haploid XY (0.15% to 0.16%) sperm in the control and swim-up samples were comparable to our earlier fluorescence in situ hybridization studies on human semen (13, 18) and to other studies that used fluorescence in situ hybridization and a single Y chromosome-specific DNA probe (22, 23). Our results also concur with those of Benet et al. (8) and Brandriff et al. (9) who found that there were similar levels of chromosomal abnormalities in the motile sperm population and the total sperm population. There was a significant reduction in the proportion of putative diploid and tetraploid cells in the swim-up fractions, with the most pronounced decrease occurring in the putative diploid XY group. The exact identity of this small population of cells is uncertain, but they may comprise a mixed population of nonmotile sperm, immature germ cells, and leukocytes, which mostly remain in the pellet after swim-up. This would explain the current finding that the frequency of these cells decreased in the swim-up fractions. However, classifying sperm as disomic or diploid on the basis of head size alone is at best only an approximation as the differences in head size might also be due to other undisclosed factors, such as the heterogeneous response of human sperm to nuclear decondensation with DTT. A more accurate method of distinguishing disomic and diploid sperm would be to use an autosomal probe in conjunction with the X and Y probes, which would conclusively establish the ploidy status of each sperm without relying on morphological criteria. Thus, diploid sperm should have two sex chromosomes and two autosomes, whereas disomic haploid cells would have two sex chromosomes but only one autosome. In conclusion, these double fluorescence in situ hybridization experiments clearly demonstrate that the swim-up procedure used in this study isolated a motile sperm fraction containing a 1:1 ratio of X- to V-bearing sperm. Because the swim-up method is an important procedure for routine lui, IVF-ET, and GIFT, the results are important reassurance that the sex ratio of sperm in the motile fraction is not altered. Acknowledgments. We thank the staff of the Andrology Laboratory at The Queen Elizabeth Hospital for assistance with donor recruitment and semen processing and Barbara Godfrey, M.A., of the Reproductive Medicine Unit at The Queen Elizabeth Hospital for collating clinical data. REFERENCES 1. Drevius LO. The 'sperm rise' test. J Reprod Fertil 1972; 24: Kirby CA, Flaherty SP, Godfrey BM, Warnes GM, Matthews CD. A prospective trial of intrauterine insemination of motile spermatozoa versus timed intercourse. Fertil Steril 1991;56: Avery SM, Elder KT. Routine gamete handling: semen assessment and preparation. In: Brinsden PR, Rainsbury P A, editors. A textbook of in vitro fertilization and assisted reproduction. Carnforth, UK: Parthenon Publishing Group, 1992: Russell LD, Rogers BJ. Improvement in the quality and fertilization potential of a human sperm population using the rise technique. J Androl 1987;8: Englert Y, Van den Bergh M, Rodesch C, Bertrand E, Biramane J, Legreve A. Comparative auto-controlled study between swim-up and Percoll preparation of fresh semen sampies for in-vitro fertilization. Hum Reprod 1992;7: Ng FLH, Liu DY, Baker HWG. Comparison of Percoll, mini-percoll and swim-up methods for sperm preparation from abnormal semen samples. Hum Reprod 1992;7: Le Lannou D, Blanchard Y. Nuclear maturity and morphology of human spermatozoa selected by Percoll density gradient centrifugation or swim-up procedure. J Reprod Fertil 1988;84: Benet J, Genesca A, Navarro J, Templado C. Cytogenetic studies in motile sperm from normal men. Hum Genet 1992;89: Brandriff BF, Gordon LA, Haendel S, Ashworth LK, Carrano AV. The chromosomal constitution of human sperm selected for motility. Fertil Steril 1986; 46: Lobel SM, Pomponio RJ, Mutter GL. The sex ratio of normal and manipulated human sperm quantitated by the polymerase chain reaction. Fertil SteriI1993;59: Check JH, Shan is BS, Cooper SO, Bollendorf A. Male sex preselection: swim-up technique and insemination of women after ovulation induction. Arch Androl 1989;23: Check JH, Katsoff D. A prospective study to evaluate the 1050 Han et al. Identification of X and Y sperm Fertility and Sterility

6 efficacy of modified swim-up preparation for male sex selection. Hum Reprod 1993;8: Han TL, Ford JH, Webb GC, Flaherty SP, Correll A, Matthews CD. Simultaneous detection of X- and V-bearing human sperm by double fluorescence in situ hybridization. Mol Reprod Dev 1993;34: World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge, U.K.: The Press Syndicate of the University of Cambridge, 1987: Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985; 44: West JD, West KM, Aitken RJ. Detection of V-bearing spermatozoa by DNA-DNA in situ hybridisation. Mol Reprod Dev 1989; 1: Buckle VJ, Craig IW. In situ hybridization. In: Davies KE, editor. Human genetic diseases. Oxford, U.K.:IRL Press, 1986: Han TL, Webb GC, Flaherty SP, Correll A, Matthews CD, Ford JH. Detection of chromosome 17- and X-bearing human spermatozoa using fluorescence in situ hybridization. Mol Reprod Dev 1992;33: Joseph AM, Gosden JR, Chadley AC. Estimation of aneuploidy levels in human spermatozoa using chromosome specific probes and in situ hybridization. Hum Genet 1984; 66: Brandriff B, Gordon L, Ashworth L, Watchmaker G, Moore D, Wyrobeck AJ, et al. Chromosomes of human sperm: variability among normal individuals. Hum Genet 1985;70: Martin RH, Rademaker A W, Hildebrand K, Long-Simpson L, Peterson D, Yamamoto J. Variation in the frequency and type of sperm chromosomal abnormalities among normal men. Hum Genet 1987;77: Guttenbach M, Schmid M. Determination of Y chromosome aneuploidy in human sperm nuclei by nonradioactive in situ hybridization. Am J Hum Genet 1990;46: Wyrobeck AJ, Alhborn T, Balhorn R, Stanker L, Pinkel D. Fluorescence in situ hybridization to Y chromosomes in decondensed human sperm nuclei. Mol Reprod Dev 1990;27: Ericsson RJ, Langevin CN, Nishino M. Isolation of fractions rich in human Y sperm. Nature 1973;246: van Kooij RJ, van Oost BA. Determination of sex ratio of spermatozoa with a deoxyribonucleic acid-probe and quinacrine staining: a comparison. Fertil SteriI1992;58: Vol. 60, No.6, December 1993 Han et a1. Identification of X and Y sperm 1051