Journal of Andrology
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Journal of Andrology, Vol. 24, No. 1, January/February 2003
Copyright © American Society of Andrology

Analysis of Aneuploidy in Spermatozoa From Testicular Biopsies From Men With Nonobstructive Azoospermia

RENÉE H. MARTIN*,{dagger}, CALVIN GREENE{ddagger}, ALFRED W. RADEMAKER§, EVELYN KO{dagger} AND JUDY CHERNOS*,{dagger}

From the * Department of Medical Genetics and{ddagger} Department of Obstetrics and Gynecology, Faculty of Medicine, and {dagger} Department of Genetics, Alberta Children's Hospital, University of Calgary, Alberta, Canada; and § Cancer Center Biometry Section, Northwestern University, Chicago, Illinois.

Correspondence to: Dr Renée H. Martin, Medical Genetics Clinic, 1820 Richmond Road SW, Calgary, Alberta, Canada T2T 5C7 (e-mail: rhmartin{at}ucalgary.ca).
Received for publication April 2, 2002; accepted for publication August 22, 2002.

   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Testicular sperm biopsy combined with intracytoplasmic sperm injection (ICSI) allows men with azoospermia the possibility of fathering a child. However, little information exists on the risk of chromosome abnormalities in their sperm. Multicolor fluorescence in situ hybridization (FISH) analysis was used to determine the frequency of sperm diploidy and disomy for the sex chromosomes in six men with normal karyotypes and non-obstructive azoospermia. A new method using microwave decondensation and codenaturation of sperm nuclei yielded a much larger number of sperm nuclei for FISH analysis than our previous study of men with azoospermia. A total of 59 916 sperm were analyzed; more than 9000 sperm were scored for each man. The men with nonobstructive azoospermia had an increased frequency of sperm chromosomal disomy for YY, XY, total sex chromosomal disomy, and diploidy compared with 18 normal controls, but only YY disomy reached statistical significance. One infertile man had a frequency of 3.8% XY disomy and 4.3% diploidy, which was 13-fold and 7-fold higher than control donors, respectively. Our results suggest that some men with nonobstructive azoospermia have a significantly increased frequency of sex chromosomal abnormalities than normal men, but that the overall frequency of abnormalities is similar to that found in infertile men with abnormal semen parameters.

     Key words: Sperm chromosome analysis, male infertility, fluorescence in situ hybridization


Intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility to the extent that even men with azoospermia can benefit from this technique. Spermatozoa from the testes can be extracted by testicular biopsy and used in ICSI to produce a conception. However, it has been known for decades that male infertility is closely tied to chromosome abnormalities, and there is concern that the use of ICSI might increase the risk of a chromosomally abnormal child (Martin, 1996), especially in more severe types of infertility such as nonobstructive azoospermia. Chromosome analysis of fetuses and newborns from ICSI pregnancies has demonstrated an increased risk of approximately 3% (Van Steirteghem et al, 1999; Aboulghar et al, 2001). Van Opstal et al (1997) have shown that the majority of these abnormalities are paternal in origin. A number of reports have demonstrated that infertile men with abnormalities in various semen parameters have an increased frequency of chromosome abnormalities in their sperm, particularly involving the sex chromosomes (Moosani et al, 1995; Bernardini et al, 1997; Lahdetie et al, 1997; McInnes et al, 1998; Pang et al, 1999; Templado et al, 2002). However, few studies have examined the chromosomal normality of spermatozoa extracted from the testes of men with azoospermia. In a previous study we reported on 3324 spermatozoa from testicular biopsies in three men with nonobstructive azoospermia (Martin et al, 2000). We have developed a technique that allows maximum yield of fluorescence in situ hybridization (FISH) data in samples with very low sperm concentrations. In this study, we report on FISH analysis in a large sample of spermatozoa (59 916) from six men with nonobstructive azoospermia.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Infertile Patients

The six infertile patients all had nonobstructive azoospermia, normal 46,XY somatic karyotypes, and normal FSH concentrations. The men were 26-40 years of age with a mean of 33.8 years. Sperm samples were retrieved by testicular biopsy and were frozen in the same cryoprotective medium as control donors (Martin et al, 1991). The testicular pathologies of the patients were as follows: patients A and B had incomplete maturation arrest, patient C had hypospermatogenesis, patient D had complete maturation arrest, a histology report was not available for patient E, and patient F had essentially normal spermatogenesis with dilatation of the tubules. Patients A, B, D, and F had successful pregnancies after sperm retrieval and ICSI with the birth of a normal girl, female twins (one with a heart problem), normal female twins, and a normal girl and boy, respectively. The patients were recruited from the University of Calgary Infertility Clinic. The study was approved by the institutional ethics committee and all donors gave informed consent.

Normal Control Donors

Normal healthy men with no history of childhood diseases, chronic disorders, environmental exposure, substance abuse, radiation exposure, or prescription drug use made up the control group. The 18 men were between the ages of 23-58 years with a mean of 35.6 years. All donors had normal semen profiles (World Health Organization, 1992); 11 were of proven fertility and 7 were of unproven fertility (Kinakin et al, 1997). Sperm samples were produced by masturbation and were cryopreserved until FISH analysis. We have previously demonstrated that cryopreservation has no effect on the frequency or type of chromosomal abnormalities in human spermatozoa (Martin et al, 1991).

Sperm Preparation

Frozen testicular biopsy samples were washed twice with Tris-NaCl and centrifuged at 600 x g for 6 minutes. The supernatant was removed, the pellet was resuspended in less than 100 µL Tris-NaCl, and two 5-µL aliquots of suspension were applied over the same 2 cm2 area on a microscope slide, and were air-dried after each application. Slides were dehydrated in 80% methanol at -20°C for 20 minutes, then dried overnight at room temperature. The sperm DNA on the slides was microwave-decondensed with 200 µL of 10 mM dithiothreitol (DTT; Sigma, Oakville, ON) in 0.1 M Tris (550 watts, 15 seconds), followed by 200 µL of 10 mM lithium diiodosalicylate (LIS; Sigma)/1 mM DTT in 0.1 M Tris (550 watts, 1.5 minutes). Slides were rinsed with two 200 µL aliquots of 2x SSC/0.1% NP-40 (Non-idet P-40, Sigma), air-dried at room temperature, then dehydrated in 80% methanol at -20°C for 20 minutes, air-dried, and used immediately.

Microwave Codenaturation of Sperm and Probes and Hybridization

Slides were warmed to 37°C, XY1 probe solution was applied to the sperm area, and a coverslip was sealed in place. Sperm and probe were microwave codenatured at 1100 watts for 80 seconds, followed by hybridization in a humidified chamber at 37°C for 18-24 hours. Preparation of chromosome probes and probe solution is described elsewhere (Kinakin et al, 1997).

One at a time, slides were laid flat on a preheated slide warmer and posthybridized with 200 µL of 2x SSC at 70°C for 2 minutes. After rinsing twice with 200 µL of 2x SSC/0.1% NP-40, hybridized areas were treated for 10-20 seconds with 0.05 µg/µL propidium iodide (Sigma), air-dried in the dark at room temperature for about 5 minutes, coverslipped in antifade solution (0.25 mg/mL p-phenylenediamine, Sigma), and stored in the dark. Complete details of the microwave decondensation/codenaturation technique are described elsewhere (Ko et al, 2001).

Scoring of Sperm Nuclei

Slides were counted using a Zeiss Axiophot microscope fitted with four filter sets: fluorescein isothiocyanate (FITC), rhodamine/FITC, DAPI, and rhodamine/FITC/DAPI. Two same-colored signals were counted as individual signals if they were separated by at least one signal diameter (1/2 signal diameter for the overlarge Y signal) and were of similar size, shape, and intensity. The chromosome 1 signal was used as an internal autosomal control to distinguish between disomy and diploidy. One person was involved in the assessment of slides. The time required to analyze the slides was approximately three to five times that of normal men because of the dearth of spermatozoa on the slides.

Statistical Analysis

Statistical analysis was performed by a two-tailed Z statistic (Rosner, 1995).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
A total of 59 916 sperm was analyzed from six men with nonobstructive azoospermia with more than 9000 sperm scored for each man. The frequencies of XY, XX, and YY disomy as well as diploidy and proportion of X- and Y-bearing sperm in these infertile men were compared with results obtained from 363 157 spermatozoa from 18 normal control donors (Kinakin et al, 1997). The individual results of FISH analysis in the infertile men are presented in Table 1. The men with nonobstructive azoospermia had a greater frequency of sex chromosomal disomy for XY, YY, and total sex chromosomal disomy and diploidy compared with control donors, but only YY disomy reached statistical significance (P = .02, two-tailed Z statistic). One infertile man (patient F) had a frequency of 3.8% XY disomy and 4.3% diploidy, 13-fold and 7-fold higher than control donors, respectively.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
It is extremely difficult to obtain data on the chromosome constitution of testicular sperm because the number of spermatozoa is expected to be very small. Previously, we studied three men with nonobstructive azoospermia and we were able to analyze 1779 spermatozoa for sex chromosomal abnormalities (Martin et al, 2000). In this small sample, we did not find a significantly increased frequency of sperm aneuploidy compared with control donors. These three men (patients A, B, and C) were reanalyzed in this study along with three new patients using a new method of microwave decondensation and denaturation. This method proved effective in preserving the spermatozoa on slides. Conventional FISH decondensation, denaturation, and posthybridization techniques result in a significant loss of spermatozoa from slides and this is exacerbated in cases of testicular tissue, wherein the other cells present seem to cause an even greater loss of sperm. In this study, the same cryopreserved testicular sample was used for donors A, B, and C as the previous study, but the total number of spermatozoa available for analysis increased dramatically from 214 to 10013 in donor A, 1236 to 10025 in donor B, and 329 to 10053 in donor C. Sample sizes in the three new patients were also approximately 10 000 sperm analyzed per male. This new larger sample demonstrated an increased frequency of YY and XY disomy and diploidy in infertile men compared with control donors with only YY disomy reaching statistical significance. However, it is interesting that the mean increased frequency of disomy for any category is only twofold to threefold higher than that observed in control donors. Thus the frequency of sperm chromosome abnormalities in these men with nonobstructive azoospermia is similar to that in infertile men with sperm in the ejaculate displaying abnormalities in various sperm parameters (Moosani et al, 1995; Bernardini et al, 1997; Lahdetie et al, 1997; Templado et al, 2002). However, one man in our study (patient F) did have a much higher frequency of XY disomy and diploidy with values 13-fold and 7-fold higher than control donors, respectively. This demonstrates that there is variation among patients with nonobstructive azoospermia and that some men have near-normal frequencies of sperm chromosome abnormalities (eg, patient B), whereas others have frequencies that are definitely increased (eg, patient F).Go


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  		Chromosomal abnormalities in testicular spermatozoa from men with nonobstructive azoospermia
 

To our knowledge, only four other laboratories have reported sperm chromosomal abnormalities in patients with nonobstructive azoospermia. Bernardini et al (2000) reported three men with nonobstructive azoospermia and were able to analyze approximately 200 sperm cells per patient. They found a highly significant increase in the frequency of autosomal disomy for chromosomes 1 (2.4%) and 17 (4.9%), as well as sex chromosomes (11.7% for XY, 5.8% for XX, and 6.2% for YY cells) compared with control donors. Levron et al (2001a,b) studied nine men with nonobstructive azoospermia. They analyzed a total of 153 sperm in 9 infertile patients and found a significantly increased frequency of total aneuploidy in patients compared with 6 control donors. The frequencies of XY, XX, and YY disomy were 3.3%, 2.6%, and 1.3% respectively. Palermo et al (2002) studied 490 sperm from 5 men with nonobstructive azoospermia. They found an increased frequency of autosomal disomy (2.0%) and sex chromosomal disomy (4.3%) in azoospermic patients, with a significantly higher overall aneuploidy frequency in azoospermic patients (11.8%) compared with ejaculated sperm from normal men (1.5%). Burello et al (2002) studied 2723 testicular sperm from 6 men and found a significantly increased frequency of sperm disomy for chromosomes 8 (1.03%), 18 (1.23%), X (1.23%), and Y (0.77%). Thus all these studies have suggested a significantly increased frequency of chromosomal abnormalities in testicular sperm samples. The sample sizes to date have been unavoidably small: before this study approximately 4000 testicular sperm had been analyzed from 23 men. With our new microwave decondensation and codenaturation technique we have been able to substantially increase the sample size in our study to 59 916 sperm from 6 men. Even with this much larger sample we have not shown a great increase in frequency of sperm aneuploidy, however, it is clear that there is considerable variation in the risk of aneuploidy in testicular sperm, as shown by our donor F.


   Acknowledgments
 
Many thanks to the donors who participated in this study, and to Linda Macaulay who prepared the manuscript.


   Footnotes
 
Supported by grant MA-7961 from the Canadian Institutes of Health Research. R.H. Martin holds a Canada Research Chair.


   References
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Aboulghar H, Aboulghar M, Manour R, Serour G, Amin Y, Al-Inany H. A prospective controlled study of karyotyping for 430 consecutive babies conceived through intracytoplasmic sperm injection. Fertil Steril. 2001;76:249 -254.[Medline]

Bernardini L, Gianaroli L, Fortini D, et al. Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients. Hum Reprod.2000; 15:2165 -2172.[Abstract/Free Full Text]

Bernardini L, Martini E, Geraedts J, Hopman A, Lanteri S, Conte N, Capitanio G. Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridization. Mol Hum Reprod.1997; 3:431 -438.[Abstract/Free Full Text]

Burello N, Calogero AE, De Palma A, et al. Chromosome analysis of epididymal and testicular spermatozoa in patients with azoospermia. Eur J Hum Genet.2002; 10:362 -366.[Medline]

Kinakin B, Rademaker A, Martin R. Paternal age effect of YY aneuploidy in human sperm, as assessed by fluorescence in situ hybridization. Cytogenet Cell Genet.1997; 78:116 -119.[Medline]

Ko E, Rademaker A, Martin R. Microwave decondensation and codenaturation: a new methodology to maximize FISH data from donors with very low concentrations of sperm. Cytogenet Cell Genet.2001; 95:143 -145.[Medline]

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Levron J, Aviain-Goldring A, Madgar I, Raviv G, Barkai G, Dor J. Studies on sperm chromosomes in patients with severe male factor infertility undergoing assisted reproductive technology treatment. Mol Cell Endocrinol. 2001b;183:523 -528.

Martin R. The risk of chromosomal abnormalities following ICSI. Hum Reprod.1996; 11:924 -925.[Free Full Text]

Martin R, Chernos J, Rademaker A. Effect of cryopreservation on the frequency of chromosomal abnormalities and sex ratio in human sperm. Mol Reprod Dev.1991; 30:159 -163.[Medline]

Martin R, Greene C, Rademaker A, Barclay L, Ko E, Chernos J. Chromosome analysis of spermatozoa extracted from testes of men with non-obstructive azoospermia. Hum Reprod.2000; 15:1121 -1124.[Abstract/Free Full Text]

McInnes B, Rademaker A, Greene C, Ko E, Barclay L, Martin R. Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men. Hum Reprod.1998; 13:2787 -2790.[Abstract/Free Full Text]

Moosani N, Pattinson H, Carter M, Cox D, Rademaker A, Martin R. Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil Steril. 1995;64:811 -817.[Medline]

Palermo G, Colombero L, Hariprashad J, Schlegel P, Rosenwaks Z. Chromosome analysis of epididymal and testicular sperm in azoospermic patients undergoing ICSI. Hum Reprod.2002; 17:570 -575.[Abstract/Free Full Text]

Pang M, Hoegerman S, Cuticchia A, Moon S, Doncel G, Acosta A, Kearns W. Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum Reprod.1999; 14:1266 -1273.[Abstract/Free Full Text]

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Templado C, Hoang T, Greene C, Rademaker A, Chernos J, Martin R. Aneuploid spermatozoa in infertile men: teratozoospermia. Mol Reprod Dev. 2002;61:200 -204.[Medline]

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