Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound5

Q.Van Dyk1, S. Lanzendorf2, P. Kolm3, G.D. Hodgen2 and M.C. Mahony2,4

1 Reproductive Medicine and Infertility Associates, P.A., 360 Sherman Street, St Paul, MN, 2 The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA, and 3 Biostatistics, Eastern Virginia Medical School, Norfolk, VA, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Spermatozoa–zona pellucida binding selects for human spermatozoa with progressive motility, normal morphology and functional competency. We postulated that this gamete interaction would also act to select against spermatozoa with chromosomal numerical aberrations. Spermatozoa from 41 men participating in the intracytoplasmic sperm injection (ICSI) programme were evaluated for the incidence of aneuploidy of chromosomes 18, X and Y. The hemizona assay was utilized to determine whether zona-bound spermatozoa from these patients have a reduced incidence of aneuploidy compared with those selected by motility only in a standard swim-up procedure. Using multicolour fluorescence in-situ hybridization (FISH) with DNA probes specific for chromosomes 18, X and Y, the disomy rates for chromosomes 18, X, Y and XY were found to be 0.31, 0.27, 0.29 and 0.14% respectively in the swim-up motile fraction, and 0.31, 0.33, 0.32 and 0.19% respectively in the pellet fraction. Analysing the zona-bound spermatozoa, the disomy rates for chromosome 18, X, Y and XY were found to be 0.02, 0.15, 0.12 and 0.07% respectively. The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up motile fraction or the pellet fraction (P < 0.0001). The incidence of chromosome 18 aneuploidy, including both chromosome 18 disomy and nullisomy, in the swim-up motile fractions was significantly increased in patients with an abnormal or borderline hemizona index compared with those with a normal hemizona index (P < 0.05). We also found that a high incidence of sperm aneuploidy was associated to a certain extent with low fertilization rate, and with failure to achieve pregnancy through ICSI. This study suggests that the human zona pellucida has the capacity to select against aneuploid spermatozoa by an as yet undetermined mechanism.

Key words: chromosomal aneuploidy/FISH/hemizona assay/human spermatozoa/ICSI


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intracytoplasmic sperm injection (ICSI) is the most invasive, yet the most successful, assisted reproduction technique currently offered to patients with male factor infertility (Palermo et al., 1992Go). Concerns over the potential genetic risks of this procedure surfaced soon after the first clinical application of ICSI (Cummins and Jequier, 1995Go; Meschede et al., 1995Go; Persson et al., 1996Go). Depending upon the study, the percentages of abnormal karyotypes among ICSI pregnancies range from 1% to a strikingly high incidence of 40% (Govaerts et al., 1995Go; In't Veld et al., 1995; Liebaers et al., 1995Go). More recently, a follow-up study of 1082 ICSI pregnancies and children revealed a rate of 0.83% each for sex-chromosome aberrations and autosomal aberrations, which is significantly higher than the incidence of sex-chromosome aberrations in the general population (Bonduelle et al., 1998Go). These sex chromosome anomalies among ICSI pregnancies are likely to be of paternal origin (Van Opstal et al., 1997Go).

Patients participating in ICSI programmes have on average more severe fertility problems than patients in regular IVF programmes. There is an increased incidence of chromosomal structural aberrations among subfertile and especially severely subfertile men. Men with low sperm counts have been reported with a higher incidence of reciprocally balanced translocations (Baschat et al., 1996Go). Chromosomal abnormalities can be present in ~15% of the azoospermic men and in ~6–7% of men with a low sperm count with or without other abnormal semen characteristics (Bourrouillou et al., 1992Go). The situation is further complicated by the fact that even chromosomally normal individuals can produce aneuploid spermatozoa (Rudak et al., 1978Go; Martin et al., 1987Go; Han et al., 1993Go; Holmes and Martin, 1993Go). A significantly higher incidence of aneuploidy was noted in semen from oligoasthenoteratozoospermic men (infertility due to semen with low sperm concentration, poor sperm motility and abnormal sperm morphology) compared with those from normal men (Pang et al., 1995Go; Lahdetie et al., 1997Go). Thus, the higher incidence of chromosomal abnormality in the patient group, coupled with the probability of increased chromosomal abnormalities in their spermatozoa, complicate the risk factor analysis of ICSI.

No significant deviation in the incidence of newborns with severe disorders born after IVF (1.5%) from the general population (~1–2%) has been observed (American Fertility Society, 1993Go). While IVF does not appear to result in increased genetic defects, ICSI circumvents several steps necessary in the natural fertilization process, such as spermatozoa–zona pellucida binding and penetration, and fusion of the spermatozoon–oocyte cell membrane. The possible role of these steps to function as a barrier against defective spermatozoa still awaits investigation.

The zona pellucida is an extremely stringent tool to select live and functional spermatozoa. Zona-bound spermatozoa can be generally characterized as having good motility, normal morphology, ability to bind tightly to the zona pellucida, hyperactivate and to acrosome react (Coddington et al., 1990Go; Menkveld et al., 1991Go; Oehninger et al., 1997Go). Sperm performance in the hemizona assay, a zona pellucida binding assay, was found to be a valuable parameter of predicting the outcome of in-vitro fertilization (Coddington et al., 1994Go). Thus, the zona pellucida has proven to be an important barrier against physiologically and functionally abnormal spermatozoa.

We postulate that bypassing the zona pellucida in the fertilization process poses an increased risk of the oocyte being fertilized by a spermatozoon with a genetic defect. We therefore focused this study on determining whether zona pellucida-bound spermatozoa exhibited lower aneuploidy rates than spermatozoa selected by motility alone. At the Jones Institute for Reproductive Medicine, ICSI is offered to patients with male factor infertility and to patients who have previously failed to achieve pregnancy through conventional IVF. The hemizona assay is used for the identification and diagnosis of male infertility, and to guide the selection of therapy. In this study, we determined the aneuploidy incidences for chromosomes X, Y and 18 in spermatozoa from patients participating in the ICSI programme. The incidences of aneuploidy for the three chromosomes in the patients' spermatozoa isolated in swim-up supernatants and the pellet were compared with that in hemizona-bound spermatozoa. In addition, the relationships among different sperm characteristics [including concentration, motility, morphology, hemizona assay (HZA) index, hypo-osmotic swelling test index], fertilization index, pregnancy status and the incidence of chromosome aneuploidy in spermatozoa from ICSI patients were also analysed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study was conducted with the approval of the Institutional Review Board of the Eastern Virginia Medical School. Forty-one patients (age range 30 to 54 years) participating in the ICSI programme were included in this study during the period from November 1996 to June 1997.

Semen preparation and evaluation
Semen samples were obtained by masturbation after 2–4 days of sexual abstinence, and analysed after complete liquefaction within 1 h of collection. Sperm concentration and motility were evaluated using the HTM-IVOS Motility Analyser (Hamilton-Thorne Research Inc., Danvers, MA, USA). A 3 µl fraction of the sample was loaded onto a 4-chamber microcell slide (Fertility Tech, Rockaway, NJ, USA) and then transferred to the HTM-IVOS. Data were collected on randomly selected fields along the length of the microcell chamber until at least 100 motile spermatozoa were analysed. The analysis was performed at 37°C. Sperm morphology was analysed using the strict criteria method. Morphology slides were prepared by making a thin smear of semen on clean slides, and stained by the Diff-Quick (Baxter Health Corp., McGaw Park, IL, USA) staining technique (Kruger et al., 1986Go). From each specimen, the morphology of at least 200 spermatozoa was determined at x1000 magnification.

The motile sperm fractions were separated by swim-up. A 0.5 ml fraction of semen was diluted with 1.5 ml of Ham's F-10 medium (GIBCO Lab., Grand Island, NY, USA) supplemented with 0.3% human serum albumin (HSA) (Irvine Sci., Santa Anna, CA, USA), and centrifuged at 300 g for 7 min. The sperm pellet was suspended in 0.5 ml medium and re-centrifuged for 5 min. A 0.5 ml aliquot of the medium was then layered gently over the pellet, and the specimen was incubated at 37°C and 5% CO2 in water-saturated air. At the end of the 1 h incubation, the supernatant was gently harvested without disturbing the pellet. The sperm concentration and motility in the swim-up supernatant was also evaluated using the HTM-IVOS Motility Analyser.

Hemizona assay (HZA)
The HZA was performed using a protocol described previously (Oehninger et al., 1991Go). Briefly, salt-stored human oocytes were microbisected into matching hemizonae using Narishige micromanipulators (Narishige, Tokyo, Japan) mounted on a phase-contrast inverted microscope (Nikon Diaphot, Garden City, NY, USA). Hemizonae were transferred into Ham's F-10 medium with 0.3% HSA droplets one day before the initiation of HZA, and stored at 4°C. Droplets of each patient's sperm swim-up suspension (500 000 motile sperm/100 µl) were placed in a Petri dish, along with control (from known fertile donors) sperm droplets of the same motile sperm concentration. One hemizona of a pair was placed in the droplet of the patient's spermatozoa, while the other matching hemizona was placed in the control sperm droplet. The droplets were covered with mineral oil and incubated for 4 h (37°C, 5% CO2 in water-saturated air). At the end of the incubation, each hemizona was removed and rinsed through multiple droplets of fresh medium to dislodge loosely attached spermatozoa. The number of spermatozoa tightly attached to the outer surface of each hemizona was counted. For each hemizona pair, the HZA index was calculated as follows: (number of patient spermatozoa bound to the hemizona/number of control spermatozoa bound to the hemizona)x100. The final HZA index was the average of the three individual HZA indices. A final HZA index >=35 was considered normal, an index between 14 and 35 was considered borderline, and an index <14 was considered abnormal.

Hypo-osmotic swelling test (HOST)
HOST was carried out on spermatozoa in both the swim-up motile fractions and the pellet fractions. The test was performed according to a previously published method (Jeyendran et al., 1984Go), but with some modifications. Briefly, an aliquot of 10 µl sample (either swim-up supernatant or resuspended swim-up pellet) was added to a droplet containing 100 µl hypo-osmotic solution (7.35 g sodium citrate and 13.5 g fructose in 1 l distilled water). The droplet was partially covered with mineral oil and incubated (37°C, 5% CO2 in water-saturated air) for 30 min. Spermatozoa with thickening or curling tails were scored as positive, while spermatozoa with straight tails were scored as negative. A total of 100 spermatozoa were counted, and the HOST index expressed as the percentage of positive spermatozoa in the sample following the incubation.

Fixation and decondensation of the sperm nuclei
Spermatozoa in the swim-up supernatant fraction, pellet fraction, and hemizona–spermatozoa complexes were washed separately in three changes of phosphate-buffered saline (PBS; 0.15 mol/l NaCl, 10 mmol/l sodium phosphate, pH 7.2), and fixed on a clean glass slide with freshly prepared fixative (methanol:acetic acid; 3:1). The slides were air-dried and stored at –20°C until evaluation. For FISH analysis, fixed spermatozoa were washed in 2x SSC (0.3 mol/l NaCl, 30 mmol/l sodium citrate, pH 7.5) to remove any residual fixative and incubated in 0.1 mol/l Tris–HCl buffer, pH 8.0, containing 50 mmol/l dithiothreitol. After sperm nuclei decondensation, the slides were washed once in 2x SSC, dehydrated through an ethanol series (70–90–100%), and air-dried (Jones et al., 1987Go).

Fluorescence in-situ hybridization (FISH) analysis
The DNA probes (Vysis, Downers Grove, IL, USA) used in this study recognize the alpha satellite DNA of the centromeric region of human chromosomes X (Xp11.1-q11.1), Y (Yp11.1-q11.1) and 18 (18p11.1-q11.1). The probes detecting chromosome X, Y and 18 were labelled with fluorescent haptens CEP (chromosome enumeration probe) SpectrumGreen, CEP SpectrumOrange and CEP SpectrumAqua respectively (Figure 1Go). The process of FISH was carried out according to the manufacturer's recommendation (Vysis). Briefly, slides were denatured at 75°C for 5 min in 70% formamide/2x SSC, followed by dehydration through an ethanol series (70–85–100%). Slides were air-dried, and warmed to 45–50°C before the denatured probe mixture was applied. The area with the probe mixture was covered with a coverslip, sealed with rubber cement, and the slides were placed in a humidified box in a 37°C incubator. Post-hybridization washes were carried out after overnight hybridization incubation. Slides were first immersed in three changes of 50% formamide/2x SSC for 10 min each, and then washed with 2x SSC for another 10 min. Slides were finally washed for 5 min in 2x SSC/0.1% NP-40. All the washing buffers were incubated at 45°C. Slides were then air-dried in the dark, and the counterstain, 1,4-diamidino-2-phenylindole (DAPI II) was added just before viewing.



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Figure 1. Hemizona-bound sperm nuclei hybridized with alpha-satellite centromeric probes (x600 magnification) specific for chromosomes X (green), Y (orange) and 18 (aqua) and were counterstained with DAPI II.

 
Microscopy
FISH signals were analysed with a Nikon epifluorescence microscope equipped with an appropriate triple bandpass filter set for SpectrumOrangeTM, SpectrumGreenTM, SpectrumAquaTM and DAPI (Vysis). A sperm nucleus was scored only if it was intact and not overlapped. An X or Y chromosome in a sperm nucleus was recognized by a green or an orange fluorescent spot respectively. Chromosome 18 was recognized by the presence of an aqua fluorescent spot in the sperm nucleus. Sperm nuclei were scored as disomic for sex chromosomes when an extra X or Y signal and a single aqua fluorescent spot were clearly visible within the nucleus, were comparable in brightness and size, and were at least one domain apart. Sperm nuclei were scored as nullisomic for sex chromosome when only a single chromosome 18 signal was visible. Sperm nuclei were considered diploid when an extra X or Y chromosome signal and two chromosome 18 signals were present. At least 500 spermatozoa were evaluated in each swim-up and pellet sample, and all hemizona-bound spermatozoa were evaluated.

Statistical analysis
Sperm parameters such as concentration, motility, morphology and HOST index were analysed as continuous variables, and Spearman's rank order correlation was used to interpret the relationship between those parameters and the incidence of sperm aneuploidy. HZA index, ICSI indications and pregnancy status were analysed as categorical data, and the Wilcoxon rank-sum test was used to interpret the relationship between these categories and the incidence of sperm aneuploidy. The significance level chosen was P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Semen specimens from 41 patients who participated in the ICSI programme were included in this study. A summary of semen parameters including sperm concentration, percent motility, percentage of spermatozoa with normal morphology according to strict criteria, hypo-osmotic swelling index and hemizona index, as well as fertilization and pregnancy rates are presented in Table IGo. In total, 21 839 sperm nuclei from the motile fractions, 21 936 sperm nuclei from the pellet fractions, and 4081 sperm nuclei from the hemizona-bound fractions were analysed. The mean number of sperm nuclei per patient evaluated was 533 ± 13 for the motile fractions, 535 ± 14 for the pellet fractions, and 117 ± 14 for the hemizona-bound fractions. Hybridization efficiency was 97%. No difference in hybridization efficiency among the three sperm groups was observed.


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Table I. Sperm parameters of 41 ICSI patients evaluated for the incidence of aneuploidy of chromosomes 18, X, and Y
 
In addition to examining aneuploidy rates in the patient population, testing was completed for donors whose specimens were utilized as controls in the hemizona assay. The rates of sex-chromosome aneuploidy were 0.38% in the swim-up motile fractions, 0.17% in the pellet fraction, and 0.22% in the spermatozoa–hemizona complexes. The rate of chromosome 18 aneuploidy was 0.14% in both the swim-up motile fractions and the pellet fraction, and 0.02% in the spermatozoa–hemizona complexes. Rates of total diploidy, a summary of 46,XX, 46,YY and 46,XY, were 0.17% in the swim-up motile fractions, 0.14% in the pellet fraction, and 0.04% in the spermatozoa–hemizona complexes.

Overall assessment of aneuploidy incidence in spermatozoa from ICSI patients
The incidence of sperm sex-chromosome nullisomy and disomy in the swim-up motile fractions, in the pellet fractions and in the spermatozoa–hemizona (HZ-bound) complexes is presented in Table IIGo. The pellet samples had a greater percentage of sex-chromosome aneuploidy (1.69%) than the swim-up samples (1.41%), but this difference was not statistically significant. Both the swim-up and the pellet sperm samples had a significantly greater percentage of sex-chromosome aneuploidy than the HZ-bound sperm samples (0.61%, P < 0.0001 for both comparisons). The incidence of chromosome 18 nullisomy and disomy in the swim-up motile fractions, in the pellet fractions, and in the HZ-bound complexes are presented in Table IIIGo. No difference in incidence of chromosome 18 aneuploidy between the swim-up samples (0.60%) and the pellet samples (0.72%) was observed, but both were significantly greater than the incidence in HZ-bound spermatozoa (0.10%, P < 0.0001 for both comparisons). We also observed that the incidence of sex-chromosome aneuploidy was found to be significantly greater than the incidence of chromosome 18 aneuploidy in the swim-up motile fractions, the pellet fractions and the HZ-bound fractions (P < 0.0001 for all three comparisons) (Table IVGo).


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Table II. The incidence of sperm sex-chromosome nullisomy and disomy in the swim-up motile fractions (SU), the pellet fractions (Pellet), and the spermatozoa–hemizona complexes (S-HZ) in 41 ICSI patients
 

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Table III. The incidence of sperm chromosome 18 nullisomy and disomy in the swim-up motile fractions (SU), the pellet fractions (Pellet), and the spermatozoa–hemizona complexes (S-HZ) in 41 ICSI patients
 

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Table IV. The incidence of sex-chromosome aneuploidy and the incidence of chromosome 18 aneuploidy in the swim-up motile fractions (SU), in the pellet fractions (Pellet), and in the spermatozoa–hemizona complexes (S-HZ) in 41 ICSI patients
 
The incidence of diploid spermatozoa in the swim-up motile and pellet fractions and in the HZ-bound complexes is presented in Table VGo. The incidence of diploid spermatozoa in the pellet samples (0.35%) was significantly greater than that in the HZ-bound spermatozoa (0.07%, P < 0.0001). This incidence in the swim-up samples (0.26%) was not significantly different from either the pellet samples or the HZ-bound spermatozoa.


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Table V. The incidence of diploid spermatozoa in the swim-up motile fractions (SU), in the pellet fractions (Pellet), and in the spermatozoa–hemizona complexes (S-HZ) in 41 ICSI patients
 
Correlation between the incidence of aneuploidy in spermatozoa and semen parameters
Semen concentration
There was a moderate but significant correlation between the incidence of sex chromosome nullisomy in the swim-up samples and the semen concentration (r = 0.33, P = 0.0332). Similarly, the correlation between the incidence of sex-chromosome aneuploidy in the swim-up samples and the semen concentration was moderate, but significant (r = 0.31, P =0.0481).

Semen motility
There was a moderate, but significant, correlation between the incidence of chromosome X disomy in the swim-up motile fractions and the semen motility (r = 0.31, P = 0.0451), between the incidence of sex chromosome nullisomy in the swim-up motile fractions and the semen motility (r = 0.31, P = 0.0494), and between the incidence of chromosome 18 disomy in the swim-up motile fractions and the semen motility (r = 0.38, P = 0.0140). Both the incidence of sex-chromosome aneuploidy and the incidence of chromosome 18 aneuploidy in the swim-up motile fractions correlated with the semen motility (r = 0.33, P = 0.0345 and r = 0.032, P = 0.0381 respectively). The incidence of disomy in the swim-up motile fractions was significantly correlated with the semen motility (r = 0.37, P = 0.0171), and the incidence of nullisomy in the pellet fractions was significantly correlated with semen motility (r = 0.32, P = 0.0404).

Semen morphology
There was moderate, significant correlation between the incidence of sex-chromosome nullisomy in the swim-up motile fractions and semen morphology (r = 0.38, P = 0.0150). The incidence of 46,XY diploidy was not significantly correlated with semen morphology (r = –0.30, P = 0.536).

HOST index
The incidence of chromosome 18 nullisomy in the swim-up fractions was negatively and significantly correlated with the HOST index of the pellet fractions (r = –0.38, P = 0.0429). The incidence of sex-chromosome disomy XY was also negatively and significantly correlated with the HOST index of the pellet fractions (r = –0.41, P = 0.0280). Interestingly, the higher the incidence of chromosome 18 aneuploidy in the motile fraction, the lower was the percentage of live spermatozoa found in the pellet fraction, according to HOST.

HZA index
The incidence of chromosome 18 disomy in the swim-up motile fractions was significantly greater in patients with abnormal HZA index (<14) than in patients with borderline HZA index (14 to <35) or normal HZA index (>=35) (P = 0.0247). The incidence of chromosome 18 aneuploidy (including both chromosome 18 disomy and nullisomy) in the swim-up motile fractions was also significantly greater in patients with abnormal HZA index than in patients with borderline HZA index or normal HZA index (P = 0.0356). In other words, samples with higher chromosome 18 aneuploidy rate in the motile fraction had a poorer binding capacity for human zona pellucida.

Fertilization
There were significant negative correlations between the incidence of sex-chromosome disomy 24,XY in the swim-up samples and the rate of fertilization (r = –0.45, P = 0.0033), between the incidence of diploid 46,XY in the swim-up samples and the rate of fertilization (r = –0.41, P = 0.0074), and between the incidence of diploid 46,XY in the pellet samples and the rate of fertilization (r = –0.46, P = 0.0022). Thus, we found that the presence of diploid 46,XY spermatozoa or sex-chromosome disomy 24,XY spermatozoa in the semen sample indicated a lower fertilization rate using ICSI.

Pregnancy
The incidence of diploid 46,YY in swim-up motile fractions was significantly greater in non-pregnant patients (0.71%) than in pregnant patients (0.56%, P = 0.0174). The total rate of diploidy (including diploidy 46,XX, 46,YY and 46XY) in swim-up motile fractions in non-pregnant patients (1.81%) was also significantly greater than that in pregnant patients (0.50%, P = 0.0107). Thus, the increased presence of diploid spermatozoa in the semen sample was related to a reduced pregnancy rate.

Age of the patients
There were no significant correlations existing between the age of the patients and the aneuploidy incidences in spermatozoa from the swim-up motile fractions (P > 0.07) or the pellet fractions (P > 0.14).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The introduction of ICSI technology has brought revolutionary change to modern assisted reproductive medicine, and has contributed greatly to the overall success of infertility treatment. However, since spermatozoa used in ICSI frequently are obtained from semen of extremely poor quality, there is concern over the achieved genetic normalcy rate of ICSI pregnancy. In this study, we investigated whether the zona pellucida can function as a barrier against spermatozoa with numerical chromosome error.

Data presented were collected from 41 ICSI patients. The analysis of aneuploidy was performed on swim-up motile fractions, pellet fractions and hemizona-bound spermatozoa. The emphasis of our study was to examine the capacity of human zona pellucida to select against aneuploid spermatozoa by examining differences in rates among the three groups. Since our objective was not to assess the relationship between peripheral and sperm aneuploidy, we did not complete either karyotypes or FISH analysis of peripheral blood specimens. As a result, we are presently unable to determine whether the aneuploidy rates observed are due to inherited or to de-novo chromosomal aberrations. The original semen specimens were not available as the clinical procedures were prioritized. FISH analysis of sperm chromosome aneuploidy was carried out independently of other assays or procedures; hence, the results of sperm aneuploidy incidence were not biased by the information from other tests.

The incidence for sex-chromosome aneuploidy and chromosome 18 aneuploidy in hemizona-bound spermatozoa (0.61 and 0.10% respectively) was significantly lower than that in both the swim-up motile fractions (1.41 and 0.60% respectively) and in the pellet fractions (1.69 and 0.72% respectively). These data are in agreement with previous reports on sex-chromosome aneuploidy frequency in ICSI patients (Storeng et al., 1998Go). The frequency of sex-chromosome disomy in the swim-up motile and pellet fractions was much higher than that reported for donors or randomly selected men of couples seeking infertility treatment (Martin et al., 1996Go; Martinez-Pasarell et al., 1997aGo; Morel et al., 1997Go). Conversely, the sex chromosome disomy incidence was much lower than that reported for severe oligoasthenoteratozoospermic patients (Pang et al., 1995Go). This was not unexpected, as patients included in this study had a wider range of semen parameters.

Compared with samples selected based on motility only, there was a reduced frequency of spermatozoa with chromosome aneuploidy and diploidy bound to hemizona. Clearly, zona selection did not completely block the fertilization by spermatozoa with numerical chromosomal aberration. However, the selectivity of the zona pellucida might be the single significant mechanism at the gamete level of blocking fertilization by an abnormal spermatozoon. Using zona-free hamster oocytes, it was demonstrated (Martinez-Pasarell et al., 1997bGo) that sperm capacity to fuse with the oocyte membrane and subsequently form pronuclei did not reduce the incidence of either aneuploidy or diploidy.

As expected, no significant difference was observed between the rates of nullisomy and disomy. Sex-chromosome aneuploidy was significantly greater than the incidence of chromosome 18 aneuploidy. Subfertile men have been reported to exhibit decreased metaphase II/metaphase I ratios in their testicular biopsy specimens (Lamont et al., 1981Go). Since a decreased number of cells reaching metaphase II correlates with an increased percentage of cells with unpaired sex-chromosomes (Chandley et al., 1976Go), our results are consistent with those of microscopic studies of testicular specimens. Similarly, a study of fertile donors showed sex-chromosome aneuploidy frequencies to be greater than those of autosomal aneuploidy (Martin et al., 1996Go).

We noted a negative correlation between chromosome 18 aneuploidy, but not sex-chromosome aneuploidy, and the HZA index. Although it is premature to speculate that aneuploid spermatozoa are defective in spermatozoa–zona binding capacity, there is obviously an inferred association between the high frequency of aneuploidy and reduced zona-binding capacity in human spermatozoa. Interestingly, earlier studies reported a significant association between HZA index and fertilization rate in vitro (Coddington et al., 1994Go). The human hemizona has been demonstrated previously to select not only for motile spermatozoa, but also for functionally normal spermatozoa (Oehninger et al., 1997Go). Although the incidence of chromosome 18 aneuploidy is less than that of sex-chromosome aneuploidy in human spermatozoa, it is apparently associated to some degree with impaired sperm function, namely zona-binding.

This study and those of others have strongly proven that the incidence of sperm aneuploidy among ICSI patients is significantly higher. For example, sex-chromosome aneuploidy 1.41% (swim-up) and 1.69% (pellet) in ICSI patients versus 0.38% reported here compared well with 0.42% for normal fertile donors reported previously (Martin et al., 1996Go). Similarly, chromosome 18 aneuploidy at 0.60% (swim-up) and 0.72% (pellet) in ICSI patients was significantly greater than 0.14% reported here, and compared with 0.11% for normal fertile donors (Spriggs et al., 1995Go). Cytogenetic analysis of the largest number of ICSI pregnancies also appears to support our finding, since the incidences of de-novo and inherited autosomal aberrations are higher than those reported for the general population (Nielsen and Wohlert, 1991Go; Bonduelle et al., 1998Go). It has been well established that the incidence of chromosomal aberrations among children born through conventional IVF is not significantly different from that of the general population. Selection at the fertilization level is supported by the fact that pregnancies with sperm-derived aneuploidy are less common than expected from analysis of sperm chromosomes in men with and without constitutional chromosome rearrangements (Brandiff et al., 1986Go; Martin et al., 1987Go; Martin, 1988aGo,bGo). Thus, the potentially higher incidence of aneuploidy in pregnancies through ICSI could be the result of two factors: (i) increased incidence of aneuploidy in spermatozoa from participating patients; and (ii) the bypassing of zona pellucida selection against aneuploid spermatozoa.

It is demonstrated here that the zona pellucida selects against spermatozoa with chromosome numerical disorder. However, this selectivity of the zona pellucida cannot yet be generalized, as its mechanism is unclear and it is also unknown whether this selectivity extends to spermatozoa with structural chromosomal disorders. Also, it is understood that the zona pellucida does not select against spermatozoa carrying single gene defects, otherwise the Mendelian laws of inheritance would not be true.

The relationships between the incidence of aneuploidy and other semen parameters were analysed in an attempt to explain whether aneuploid spermatozoa tended to be morphologically abnormal or were motility-impaired; and hence whether zona selection could be mediated by these two mechanisms. This study did not establish such a relationship. We found an association between sperm motility and the frequency of sex-chromosome and chromosome 18 aneuploidy. We observed an association between the incidence of sex-chromosome nullisomy in the swim-up motile fraction and the morphology index of the original semen. We found it difficult to draw a firm conclusion, since spermatozoa in the motile fraction make up a very small portion of the total spermatozoa in the semen sample. Our findings are consistent with the report that all nine de-novo sex-chromosome aberration pregnancies were obtained using spermatozoa from severe oligoasthenoteratozoospermic patients, while no association could be found to link this defect with any specific semen parameters (Bonduelle et al., 1998Go). In another study, no correlation between sperm morphology and chromosomal aberrations could be reported (Martin and Rademaker, 1988Go). With spermatozoa exhibiting a low strict morphology index, a link between chromosome disomy and specific head deformation of the sperm head was reported (Estop et al., 1997Go). In analysing sperm pronuclei, it was found (Lee et al., 1996Go) that head defects were associated with chromosomal structural aberrations, but not numerical aberrations. We did find the incidence of chromosome 18 disomy in spermatozoa to be negatively associated with the hypo-osmotic swelling test index, suggesting that an increase in chromosome 18 disomy is associated with increased sperm membrane defects.

This study demonstrated that the zona pellucida did select against spermatozoa with chromosome aberrations, and in particular against aneuploidy. We are still unclear about the underlying mechanism of this selection. Gathering facts from this study and those of others, there is apparently evidence to support the association between sperm chromosomal aneuploidy and severe oligoasthenoteratozoospermia, between sperm chromosomal aberration and sperm head defects, between sperm chromosomal aneuploidy and defects in sperm membrane integrity, between sperm chromosomal aneuploidy and impaired spermatozoa–zona binding, between sperm chromosomal aneuploidy and impaired sperm pronuclei formation, and finally, between sperm chromosomal aneuploidy and failure to achieve pregnancy with ICSI.

We demonstrated here the importance of zona selectivity of normal spermatozoa. At present, it may be premature to apply these zona-selected spermatozoa in clinical practice. However, improving the genetic normalcy of spermatozoa in an era of the widespread use of ICSI has significant importance, and should certainly be considered.


    Notes
 
4 To whom correspondence should be addressed at: The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia 23507, USA. E-mail: mahonymc{at}evms.edu Back

* Presented at the 23rd Annual Meeting of the American Society of Andrology, Long Beach, California, USA, March 1998 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
American Fertility Society (1993) Assisted reproductive technology in the United States and Canada: 1991 results from the Society for Assisted Reproductive Technology generated from the American Fertility Society Registry. Fertil. Steril., 59, 956–962.[ISI][Medline]

Baschat, A.A., Kupker W., al Hasani S. et al. (1996) Results of cytogenetic analysis in men with severe subfertility prior to intracytoplasmic sperm injection. Hum. Reprod., 11, 330–333.[Abstract]

Bonduelle, M., Aytoz, A., Van Assche, E. et al. (1998) Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum. Reprod., 13, 781–782.[Free Full Text]

Bourrouillou, G., Bujan, L. and Calvas, P. (1992) Role and contribution of karyotyping in male infertility. Prog. Urol., 2, 189–195.[Medline]

Brandiff, B., Gordon, L., Ashworth, L.K. et al. (1986) Cytogenetics of human meiotic segregation in two translocation carriers. Am. J. Hum. Genet., 38, 197–208.[ISI][Medline]

Chandley, A.C., Maclean, N. and Edmond P. (1976) Cytogenetics and infertility in men II: testicular histology and meiosis. Ann. Hum. Genet. 40, 165–76.[ISI][Medline]

Coddington, C.C., Fulgham D.L., Alexander N.J., et al. (1990) Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T-6 antibody. Fertil Steril., 54, 504–508.[ISI][Medline]

Coddington, C.C., Oehninger, S.C., Olive, D.L. et al. (1994) Hemizona index (HZI) demonstrates excellent predictability when evaluate sperm fertilizing capacity in in vitro fertilization patients. J. Androl., 15, 250–254.[Abstract/Free Full Text]

Cummins, J.M. and Jequier, A.M. (1995) Concerns and recommendations for intracytoplasmic sperm injection (ICSI) treatment. Hum. Reprod., 10 (Suppl. 1), 138–143.[ISI][Medline]

Estop, A.M., Vandermark, K.K., Munne, S. et al. (1997) Sperm morphology and chromosome aneuploidy in men with infertility as determined by fluorescence in situ hybridization (FISH). Fertil. Steril., 68 (1S), 208S.

Govaerts, I., Englert, Y., Vamos, E. et al. (1995) Letters to the editor: Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet, 346, 1095–1096.

Han, T.L., Flaherty, S.P., Ford, J.H. et al. (1993) Detection of X- and Y-bearing human spermatozoa after motile sperm isolation by swim up. Fertil. Steril., 60, 1046–1051.[ISI][Medline]

Holmes, J.M. and Martin, R.H. (1993) Aneuploidy detection in human sperm nuclei using fluorescence in situ hybridization. Hum. Genet., 91, 20–24.[ISI][Medline]

In't Veld, P., Brandenburg, H., Verhoeff, A. et al. (1995) Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet, 346, 773.[ISI][Medline]

Jeyendran, R.S., Van der Ven, H.H., Perez-Pelaez, M. et al. (1984) Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil., 70, 219–228.[Abstract]

Jones, K.W., Singh, L. and Edwards, R.G. (1987) The use of probes for the Y chromosome in pre-implantation embryo cells. Hum. Reprod., 2, 439–445.[Abstract]

Kruger, T.F., Menkveld, R., Stander, F.S.H. et al. (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil. Steril., 46, 1118–1123.[ISI][Medline]

Lahdetie, J., Saari, N., Ajosenpaa-Saari, M. et al. (1997) Incidence of aneuploid spermatozoa among infertile men studied by multicolor fluorescence in situ hybridization. Am. J. Med. Genet., 71, 115–121.[ISI][Medline]

Lamont, M.A., Faed, M.J. and Baxby, K. (1981) Comparative studies of spermatogenesis in fertile and subfertile men. J. Clin. Pathol., 34, 145–150.[Abstract]

Lee, J.D., Kamiguchi, Y. and Yanagimachi, R. (1996) Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes. Hum. Reprod., 11, 1942–1946.[Abstract]

Liebaers, I., Bonduelle, M., Van Assche, E. et al. (1995) Letters to the editor: Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet, 346, 1095.

Martin, R.H. (1988a) Cytogenetic analysis of sperm from a male heterozygous for a 13; 14 Robertsonian translocation. Hum. Genet., 80, 357–361.[ISI][Medline]

Martin, R.H. (1988b) Meiotic segregation of human chromosomes in translocation heterozygotes: report of a t(9;10)(q34;q11) and a review of the literature. Cytogenet. Cell Genet., 47, 48–51.[ISI][Medline]

Martin, R.H. and Rademaker, A. (1988) The relationship between sperm chromosomal abnormalities and sperm morphology in humans. Mutat. Res., 207, 159–164.[ISI][Medline]

Martin, R.H., Rademaker, A.W., Hildebrand, K., et al. (1987) Variation in the frequency of sperm chromosomal abnormalities among normal men. Hum. Genet., 77, 108–114.[ISI][Medline]

Martin, R.H., Spriggs, E. and Rademaker, A.W. (1996) Multicolor fluorescence in situ hybridization analysis of aneuploidy and diploidy frequencies in 225,846 sperm from 10 normal men. Biol. Reprod., 54, 394–398.[Abstract]

Martinez-Pasarell, O., Marquez, C., Coll, M.D. et al. (1997a) Analysis of human sperm-derived pronuclei by three-color fluorescent in-situ hybridization. Hum. Reprod., 12, 641–645.[Abstract]

Martinez-Pasarell, O., Vidal, F., Colls, P. et al. (1997b) Sex chromosome aneuploidy in sperm-derived pronuclei, motile sperm and unselected sperm, scored by three-color FISH. Cytogenet. Cell Genet., 78, 27–30.[ISI][Medline]

Menkveld, R., Franken, D.R., Kruger, T.F. et al. (1991) Sperm selection capacity of the human zona pellucida. Mol. Reprod. Dev., 30, 346–352.[ISI][Medline]

Meschede, D., De Geyter, C., Nieschlag, E. et al. (1995) Genetic risk in micromanipulative assisted reproduction. Hum. Reprod., 10, 2880–2886.[Abstract]

Morel, F., Mercier, S., Roux, C. et al. (1997) Estimation of aneuploidy levels for 8, 15, 18, X and Y chromosomes in 97 human sperm samples using fluorescence in situ hybridization. Fertil. Steril., 67, 1134–1139.[ISI][Medline]

Nielsen, J. and Wohlert, M. (1991) Chromosome abnormalities found among 34,910 newborn children: results from a 13 year incidence study in Arrhus, Denmark. Hum. Genet., 87, 81–83.[ISI][Medline]

Oehninger, S., Acosta, A.A., Veeck, L. et al. (1991) Recurrent failure of in vitro fertilization: role of the hemizona assay in the sequential diagnosis of specific sperm-oocyte defects. Am. J. Obstet. Gynecol., 164, 1210–1215.[ISI][Medline]

Oehninger, S., Mahony, M., Ozgur, K. et al. (1997) Clinical significance of human sperm-zona pellucida binding. Fertil. Steril., 67, 1121–1127.[ISI][Medline]

Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 17–18.[ISI][Medline]

Pang, M.G., Zackowski, J.L., Hoegerman, S.F. et al. (1995) Detection by fluorescence in situ hybridization of chromosome 7, 11, 12, 18, X and Y abnormalities in sperm from oligoasthenospermic patients of an in vitro fertilization program. J. Assist. Reprod. Genet., 12, 53S.

Persson, J.W., Peters, G.B. and Saunders, D.M. (1996) Genetic consequences of ICSI. Is ICSI associated with risks of genetic disease? Implications for counseling, practice and research. Hum. Reprod., 11, 921–932.[ISI][Medline]

Rudak, E., Jacobs, P.A. and Yanagimachi, R. (1978) Direct analysis of the chromosome constitution of human spermatozoa. Nature, 274, 911–913.[ISI][Medline]

Spriggs, E.L., Rademaker, A.W. and Martin, R.H. (1995) Aneuploidy in human sperm: results of two-and three-color fluorescence in situ hybridization using centromeric probes for chromosomes 1, 12, 15, 18, X, and Y. Cytogenet. Cell Genet., 71, 47–53.[ISI][Medline]

Storeng, R.T., Plachot, M., Theophile, D. et al. (1998) Incidence of sex chromosome abnormalities in spermatozoa from patients entering an IVF or ICSI protocol. Acta Obstet. Gynecol. Scand., 77, 191–197.[ISI][Medline]

Van Opstal, D., Los, F.J., Ramlakhan, S. et al. (1997) Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection. Hum. Reprod., 12, 682–686.[Abstract]

Submitted on July 30, 1999; accepted on March 28, 2000.