1 Department of Animal Science and Technology, Chung-Ang University, Gyungki-Do 456-756, 2 BET Research Institute, Chung-Ang University, Gyungki-Do 456-756, 3 Department of Obstetrics and Gynaecology, Ewha Woman's University MokDong Hospital, Seoul 158-051 and 4 Department of Obstetric and Gynaecology, Chung-Ang University Hospital, Seoul 100-272, Korea
5 To whom correspondence should be addressed. Email: mgpang{at}cau.ac.kr
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Abstract |
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Key words: aneuploidy/FISH/male infertility/meiosis/sperm
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Introduction |
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The successful implementation of ICSI has provided a unique means to allow couples suffering from severe male factor infertility to achieve their reproductive goals. If the infertile man has a germ cell in the ejaculate or if one can be retrieved from the reproductive tract, ICSI can be carried out with remarkable fertilization results. However, despite the great therapeutic advantages of the technique, ICSI provides solutions to clinicians often in the absence of an aetiological or pathophysiological diagnosis. Therefore, it is imperative that we continue our efforts toward the identification of the specific sperm defects involved as well as their origin (genetic, developmental, environmental or others).
To evaluate the cytogenetic make-up of sperm from oligoasthenoteratozoospermic (OAT) males, several centres have reported the incidence of aneuploidy and structural abnormalities in sperm from infertile men. Pang et al., (1994) performed a preliminary study on 15 OAT patients using fluorescence in situ hybridization (FISH) to determine aneuploidy for chromosomes 1, X and Y. There were significant increases in aneuploidy for all chromosomes analysed in OAT patients, compared with controls. Similar results were obtained and extended to other chromosomes (Moosani et al., 1995
; Aran et al., 1999
; Pang et al., 1999
; Pfeffer et al., 1999
; Ushijima et al., 2000
; Vegetti et al., 2000
). Recently, Schmid et al., (2004)
showed a significant increase in the average frequencies of sperm with duplications and deletions in oligozoospermic patients.
In order to assess the role that chromosome complement plays in normal and abnormal fertility, detailed molecular cytogenetic studies must be done on sperm samples from men with normal fertility, and also from men with abnormal fertility. Care should be taken to fully characterize each patient's clinical picture so that data on subjects with identical presentations can later be pooled. Correlations of meiotic cytology and FISH studies on sperm from the same individuals may also prove highly informative.
To understand more clearly the cytogenetic make-up of sperm from OAT patients, patients with low sperm count, poor motility and low levels of normal forms, FISH was used to determine numerical chromosome abnormalities. Frequencies of aneuploidy for chromosomes 13, 18, 21, X and Y, frequency of non-disjunction at meiosis I or II in sperm from normal men and OAT patients were examined. We also analysed the effect of semen parameters on the frequency of aneuploidy in OAT patients.
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Materials and methods |
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Sperm decondensation and sperm heading swelling
Semen samples were immediately washed after liquefaction and decondensed as previously described (Pang et al., 1999). Briefly, semen samples were washed once in phosphate-buffered saline (PBS). Following centrifugation, the pellet was suspended with 1 ml PBS containing 6 mmol/l EDTA and centrifuged. The pellet was resuspended with 1 ml PBS containing 2 mmol/l dithiothreitol, incubated for 45 min at 37 °C and centrifuged. Following centrifugation, the supernatant was discarded and the pellet was resuspended in fresh fixative (3 parts methanol:1 part glacial acetic acid). Slide preparation was by the smear method and short-term slide storage was at 4 °C.
FISH
To investigate both the autosomes and the sex chromosomes, simultaneous three-probe three-colour FISH was performed using probe sets for chromosomes 13, 18 and 21 (Set I) and X, Y and 18 (Set II). Probes used for FISH were from loci 13q22 contig (RB1), D18Z1, 21q22.1 contig (D21S259-D21S341-D21S342), DXZ1 and DYZ3. All probes were directly labelled with fluorescent dyes, chromosome 13 with Spectrum Green, chromosome 21 and the X chromosome with Spectrum Orange, the Y chromosome with Spectrum Aqua, and chromosome 18 with Spectrum Aqua (Set I) or Spectrum Green (Set II). Alpha satellite and contig DNA probes were obtained from Vysis, Inc. (USA). All hybridizations for patients' and control sperm were performed at the same time. Approximately 3000 sperm per subject were scored with each autosome probe set and 2000 sperm per subject were scored using the X, Y, 18 probe set. Three-probe FISH was used to differentiate disomy due to non-disjunction from diploidy due to non-reduction. Simultaneous scoring of three chromosomes also provided an internal control to differentiate nullisomy from lack of hybridization. The hybridization mixture contained 1 µl of three probes each, 6 µl of formamide, and 1 µl of 20 x standard saline citrate (SSC) (total volume =10 µl). Hybridization mixes were added to pre-warmed slides (42 °C) and covered with 22 x 22 mm coverslips which were sealed with rubber cement. Slides were denatured at 80 °C for 5 min. All slides were hybridized in a moist chamber for 6 h at 42 °C. The slides were then washed three times (5 min each) in 50% formamide, 2 x SSC, pH 7.0, followed by 5 min 2 x SSC, 0.1% NP-40, pH 7.0, all at 37 °C. Transition to antifade was accomplished by a 5 min PBS wash at room temperature. Coverslips were added over 13 µl antifade with or without counterstain. Microscopy was performed using a Nikon E600 epifluorescent microscope. Multiple fluorescent signal detection was accomplished using an automatic filter wheel with four different excitation filters. Images were captured using ChIPS Genetic Workstations and Image Software (GenDix, Inc., Korea).
Scoring criteria
Nuclei were scored only if they were not over-decondensed, did not overlap and were intact with clearly defined borders. A sperm was scored as disomic for a particular chromosome if it showed two signals for that chromosome and one each for the simultaneously probed chromosomes. For disomy to be counted, the distance between the two signals had to equal or be greater than the diameter of one fluorescent domain. Two spots separated by less than the diameter on one domain were scored as a single signal. The absence of signal for a single chromosome was scored as nullisomy for that chromosome. Sperm showing signal for none of the chromosomes of a probe set were scored as such but were not included in the calculation of nullisomy frequencies as this outcome may be an artefact resulting from unsuccessful hybridization due to inadequate nuclear decondensation. A cell was scored as diploid if there were two signals for each probed autosome and two sex chromosomes present.
Statistical analyses
Using 2-analyses, we compared the frequencies of aneuploidy for individual chromosomes both within patients and between patients. Due to the heterogeneity of aneuploidy frequencies both within and between patients, these data were not pooled. However, data from controls were homogeneous and were therefore pooled. Linear regression analysis was used to correlate total aneuploidy frequencies and sperm parameters. All data analyses were done by SAS system (version 8.0).
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Results |
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Discussion |
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Our data showed that individual patients had significantly higher proportions of disomic, nullisomic and diploid sperm nuclei compared to normal control group. Also between-patient heterogeneity exists for all OAT patients. The factor(s) responsible for the heterogeneity present both within and between samples from OAT patients is obscure. Cytogenetic heterogeneity in the OAT population might account for some of the cytogenetic variability seen between patients but would be implausible to generate the observed heterogeneity seen between chromosomes within patients. It is possible that mutations within a gene or genes associated with meiosis may predispose a particular chromosome to non-disjoin at a higher frequency than other chromosomes within the same gamete. The results of our data provide suggestive evidence for the existence of chromosome-specific patterns of non-disjunction in OAT patients.
Our data suggest that the frequency of aneuploid sperm seems to be influenced by sperm count. Frequencies of sperm chromosome abnormalities increased with decreased sperm count in OAT patients. Our findings in OAT patients are in agreement with other previous reports which show that the frequency of meiotic non-disjunction increases when the sperm count decreases (Rives et al., 1998; Vegetti et al., 2000
; Martin et al., 2003
). Also, our results are in accordance with a study done in somatic cells from infertile males. Data suggest that the frequency of chromosome abnormalities in somatic cells in males selected for infertility is inversely proportional to the sperm concentration (Vendrell et al., 1999
). Therefore our studies confirm the direct correlation between the sperm count and the frequency of aneuploidy, i.e. meiotic errors.
Vegetti et al. (2000) reported that the risk of a chromosomal aneuploidy in sperm seems to be inversely correlated with sperm count and total progressive motility in patients with abnormal semen parameters. Moreover, Calogero et al. (2001) reported that sperm aneuploidy rate was negatively correlated with sperm concentration and particularly with the percentage of normal forms. Interestingly, we did not find a significant correlation between aneuploidy rates and abnormal motility and abnormal morphology of sperm.
However, we can assume that there is a significant correlation between the higher frequencies of aneuploidy or diploidy and the lower sperm number. This assumption is well supported by the findings in our patients. However, it is likely that some of the sperm with defective meiosis gradually reduced during the stages of maturation and fertilization. Which suggests meiotic quality control system is relatively strong but not perfect in man. Vegetti et al. (2000) suggested that this higher meiotic error in sperm, which is inversely correlated with sperm count and motility, should not be ignored, since data from Pang et al. (1999)
indicate that if each chromosome shows this slight increase in aneuploidy rate, it is possible to speculate that the total rate of aneuploidy is
3374% in sperm from OAT patients.
All the chromosomes studies had an increased frequency of aneuploidy in the OAT patients. Interestingly, sperm count significantly and inversely correlated with the frequencies of diploidy, and aneuploidies for chromosomes 13 and 21 in our OAT patients. Martin et al. (2003) reported an inverse correlation between the frequency of sperm chromosome abnormalities and the sperm count for sex chromosome disomy and diploidy. Warburton and Kinney (1996)
hypothesized that chromosome differential susceptibility to aneuploidy is related to chromosomal differences in structures and behaviours. However, further studies are required.
The much higher incidences of chromosomal abnormalities after ICSI reported by In't Veld et al. (1995) and Van Opstal et al. (1997)
are consistent with our data. Previously, Martin (1996)
suggested that there may be a risk of transmitting chromosomal abnormalities to offspring following ICSI. Rosenbusch and Sterzik (1996)
discussed the possibility of irregular chromosome segregation following ICSI. The elevated frequency of autosomal non-disjunction, which we observed, may not markedly impact the frequencies of autosomal abnormalities in newborns because all resulting autosomal monosomies and most trisomies are lethal and autosomal aneuploidy in oocytes is common. It is unclear what the contribution of aneuploid sperm is to the frequency of implantation and the incidence of early spontaneous abortions containing aneuploid cells of paternal origin. However, the elevated frequencies of aneuploidy observed in the sperm from this population of OAT males suggest that they may be at increased risk for transmitting genetic abnormalities to their offspring. This low pregnancy rate and high implantation failure may be associated with aneuploidy in the male gametes. Aneuploidy in sperm may be associated with lack of implantation and/or the ability to carry a fetus to term. Further studies are required.
Fortunately, sperm cells from these OAT patients are unlikely to fertilize an ovum successfully by themselves. Also, there is an earlier and a stronger selection against autosomal trisomic and monosomic embryos in the human body. The only full numerical autosomal anomalies surviving to birth are trisomy 13 (probability of survival to birth of 2.8%), trisomy 18 (probability of 5.4%) and trisomy 21 (probability of 22.1%), but only trisomy 21 allows survival into puberty and adulthood (Jacobs and Hassold, 1995). Most numerical autosomal anomalies originate during maternal meiosis I (trisomies 8, 13, 15, 16, 18 and 21) although some cases (up to 10%) are paternal in origin (Nicolaidis and Peterson, 1998
). However, most embryos exhibiting sex chromosome abnormalities cross the pregnancybirth barrier and have a probability of survival to birth of 55.3% for XXY, 70% for XXX, 100% for XYY and 3% for XO. Triploids survive to birth only exceptionally (Jacobs and Hassold, 1995
). As a result, most infertile males with meiotic disorders are sterile or have a history of low reproductive efficiencies. Our data with in accordance with those of Aran et al. (1999)
, which showed a lower implantation rate (16%) and a higher abortion rate (33%) in infertile males.
In conclusion, we confirm that OAT patients had significantly higher frequencies of disomic, nullisomic and diploid sperm nuclei compared to a normal control group. Also between-patient heterogeneity was observed in all OAT patients. Sperm count inversely correlated with the frequencies of diploidy and aneuploidies for all chromosomes tested in OAT patients. Twenty-two series of ICSI and 18 embryo transfers were performed in 20 couples. Only three cases achieved successful pregnancies.
IVF protocols incorporating ICSI are being performed and this technique is now the treatment of choice for OAT males. Therefore, the high frequency of aneuploidy raises concerns that there may be an increased incidence of aneuploid babies, especially sex chromosome abnormalities. The risk of chromosome abnormalities in the offspring of patients undergoing ICSI must be carefully evaluated.
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Acknowledgements |
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References |
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Calogero AE, De Palma A, Grazioso C, Barone N, Romeo R, Rappazzo G and D'Agata R (2001) Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters. Hum Reprod 16, 11721179.
In't Veld P, Brandenburg H, Verhoeff A, Dhont M and Los F (1995) Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet 346, 773.[CrossRef][ISI][Medline]
Jacobs PA and Hassold TJ (1995) The origin of numerical chromosome abnormalities. Adv Genet 33, 101133.[ISI][Medline]
Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, Van Zyl JA and Smith K (1986) Sperm morphology features as a prognostic factor in in vitro fertilization. Fertil Steril 46, 11181123.[ISI][Medline]
Martin RH (1996) The risk of chromosomal abnormalities following ICSI. Hum Reprod 11, 924925.[ISI][Medline]
Martin RH, Rademaker AW, Greene C, Ko E, Hoang T, Barclay L and Chernos J (2003) A comparison of the frequency of sperm chromosome abnormalities in men with mild, moderate, and severe oligozoospermia. Biol Reprod 69, 535539.
Moosani N, Pattinson H, Carter M, Cox D, Rademaker A and Martin R (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil Steril 64, 811817.[ISI][Medline]
Nicolaidis P and Peterson MB (1998) Origin and mechanisms of nondisjunction in human autosomal trisomies. Hum Reprod 13, 311319.
Pang MG, Zackowski JL, Ryu BY, Moon SY, Acosta AA and Kearns WG (1994) Aneuploidy detection for chromosomes 1, X, and Y by fluorescence in situ hybridization in human sperm from oligoasthenoteratozoospermic patients. Am J Hum Genet 55, 644.
Pang MG, Hoegerman SF, Cuticchia AJ, Moon SY, Doncel GF, Acosta AA and Kearns WG (1999) Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y fluorescence in situ hybridization (FISH) in sperm from nine oligoasthenoteratozoospermic (OAT) patients undergoing ICSI. Hum Reprod 14, 12661273.
Pfeffer J, Pang MG, Hoegerman SF, Osgood CJ, Stacey MW, Mayer J, Oehninger S and Kearns WG (1999) Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection. Fertil Steril 72, 472478.[CrossRef][ISI][Medline]
Rives N, Mazurier S, Bellet D, Joly G and Mace B (1998) Assessment of autosome and gonosome disomy in human sperm nuclei by chromosome painting. Hum Genet 102, 616623.[CrossRef][ISI][Medline]
Rosenbusch B and Sterzik K (1996) Irregular chromosome segregation following ICSI? Hum Reprod 11, 23372338.
Schmid TE, Brinkworth MH, Hill F, Sloter E, Kamischke A, Marchetti F, Nieschlag E and Wyrobek AJ (2004) Detection of structural and numerical chromosomal abnormalities by ACM-FISH analysis in sperm of oligozoospermic infertility patients. Hum Reprod 19, 13951400.
Skakkebaek NE, Bryant JI and Philip J (1973) Studies on meiotic chromosomes in infertile men and controls with normal karyotypes. J Reprod Fertil 35, 2336.[ISI][Medline]
Ushijima C, Kumasako Y, Kihaile PE, Hirotsuru K and Utsunomiya T (2000) Analysis of chromosomal abnormalities in human spermatozoa using multi-colour fluorescence in situ hybridization. Hum Reprod 15, 11071111.
Van Opstal D, Los FJ, Ramlakhan S, Van Hemel JO, Van Den Ouweland AMW, Brandenburg H, Pieters MHEC, Verhoeff A, Vermeer MCS, Dhont M and In't Veld PA (1997) Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection. Hum Reprod 12, 682686.[Abstract]
Vegetti W, Van Assche E, Frias A, Verheyen G, Bianchi MM, Bonduelle M, Liebaers I and Van Steirteghem A (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in situ hybridization in infertile men. Hum Reprod 15, 351365.
Vendrell JM, Garcia F, Veiga A, Calderon G, Egozcue S, Egozcue J and Barri PN (1999) Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia. Hum Reprod 14, 375378.
Warburton D and Kinney A (1996) Chromosomal differences in susceptibility to meiotic aneuploidy. Environ Mol Mutagen 28, 237247.[CrossRef][ISI][Medline]
Submitted on October 27, 2004; resubmitted on January 30, 2005; accepted on January 31, 2005.
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