Disomy frequency estimated by multicolour fluorescence in situ hybridization, degree of nuclear maturity and teratozoospermia in human spermatozoa

Transcription

1 Reproduction (2001) 121, Research Disomy frequency estimated by multicolour fluorescence in situ hybridization, degree of nuclear maturity and teratozoospermia in human spermatozoa F. Morel, C. Roux* and J. L. Bresson Service de Cytogénétique-Immunocytologie-Biologie du Développement et de la Reproduction, CECOS Besançon, Franche-Comté, Centre Hospitalier Universitaire Saint Jacques, EA 3185: Génétique et Reproduction, Faculté de Médecine et Pharmacie, Besançon, France The aim of this study was to determine whether sperm morphology and nuclear maturity are associated with an increase in the prevalence of disomy in human spermatozoa. Semen samples were obtained from 60 patients selected at random from a population of men undergoing semen analysis as part of consultation for infertility. Semen analysis and aniline blue staining were carried out on each ejaculate to assess nuclear maturity. Disomy frequencies were determined using dual colour and X-Y-8 multicolour fluorescence in situ hybridization on the four groups of samples with the five lowest and the five highest teratozoospermia values (groups 1 and 2), and the five lowest and the five highest percentages of aniline blue staining (groups 3 and 4). The prevalence of autosomal disomy was significantly higher in group 4 compared with group 3, but differences between groups 1 and 2 were not significant. No significant differences in the prevalence of gonosomal disomies or diploidies were observed among the groups. These results indicate a link between chromosomal meiotic segregation and the dynamic process of nucleoproteins during gametogenesis. The difference observed between the frequency of autosomal and gonosomal disomy using aniline blue staining was unexpected and may be due to the extended isolation of the gonosomes in the heterochromatic body. Introduction Many studies (Spriggs et al., 1995; Van Hummelen et al., 1996; Downie et al., 1997; Guttenbach et al., 1997a; Mercier et al., 1998; Rives et al., 1998; Rubes et al., 1998) have evaluated the frequency of disomy in human spermatozoa using fluorescence in situ hybridization (FISH). These studies have minimized the contribution to chromosomal abnormalities due to errors during meiosis in males. In most studies, the frequency of disomy in chromosomal pairs is estimated to be between 0.15 and 0.3%. From these values we calculated the incidence of aneuploidies for 23 chromosomal pairs to be between 6.9 and 13.8%, based on the hypothesis that the mean frequency of nondisjunction remains the same for each chromosomal pair, including an equal number of hypohaploidies and hyperhaploidies (Pellestor et al., 1996; Morel et al., 1997). Nevertheless, only a few studies have attempted to correlate the incidence of disomies with the main sperm characteristics (Miharu et al., 1994; Moosani et al., 1995; Finkelstein et al., 1998; McInnes et al., 1998; Pang et al., 1999). *Correspondence christophe.roux@ufc-chu.univ-fcomte.fr An investigation by Morel et al. (1998) on a population of men undergoing consultation for couple infertility showed that the frequency of disomy was correlated with the degree of nuclear immaturity as evaluated by the percentage of nuclei stained by aniline blue. There was no evidence of a relationship between the frequency of disomy and age, necrozoospermia, motility, sperm concentration, or the multiple abnormalities index, although a correlation with teratozoospermia came close to significance. The aim of this study was to continue to develop the work by Morel et al. (1998) by comparing the frequency of disomy and diploidy in spermatozoa from groups of men with spermatozoa of normal morphology with those from men that had severe teratozoospermia, and by comparing spermatozoa from men that exhibited normal nuclear maturity with those in which many spermatozoa showed nuclear immaturity. Sample collection Materials and Methods Ejaculates were obtained from 60 patients selected at random from a population of men undergoing semen analysis as part of consultation for couple infertility. For each patient, sperm analyses were completed on the day on which the samples were obtained. In addition, aniline blue 2001 Journals of Reproduction and Fertility /2001

2 784 F. Morel et al. staining and FISH were performed on fixed slides. All tests (spermocytogram, aniline blue staining, FISH) were performed on the same ejaculate from each patient. The following distinct samples were selected: (i) the five sperm samples with the lowest teratozoospermia values: group 1; (ii) the five sperm samples with the highest teratozoospermia values: group 2; (iii) the five sperm samples with the lowest percentage of aniline blue staining: group 3; and (iv) the five sperm samples with the highest percentage of aniline blue staining: group 4. These samples were studied in dual FISH (chromosomes 15 and 18) and in triple FISH (chromosomes X, Y and 8). The men selected had no history of chemotherapy, radiotherapy, chronic illness or exposure to potential mutagens. All patients were informed about the investigations and gave their consent before the study. The study was also approved by the Ethics Committee of the Besançon University Hospital. Semen analysis Semen from all patients was analysed according to World Health Organization Guidelines (WHO, 1992), except for sperm morphology, which was evaluated on 100 spermatozoa according to the method used by David et al. (1975) which differentiates head, mid-piece and tail defects. The degree of chromatin condensation was examined on smears that were fixed in 3% (v/v) glutaraldehyde in 0.2 mol phosphate buffer l 1 for 30 min and stained with 5% (w/v) aniline blue at ph 3.5 for 5 min (Terquem and Dadoune, 1983). Sperm nuclei that stained blue were considered to be immature. The percentage of blue sperm heads was calculated from observation of 200 spermatozoa per preparation. Aneuploidy analysis Sperm preparation. Each semen sample was washed twice in PBS and 100 µl sperm suspension was placed on to the slide. The slides were air dried, fixed in methanol:acetic acid (3:1) and stored at room temperature. Sperm nuclei were partially decondensed for 2 15 min with a decondensation solution of 0.2 mol Tris HCl l 1 (ph 8.6) containing 1.25% (w/v) papain, 0.16% (v/v) dithioerythritol and 0.5% (v/v) dimethylsulfoxide under a phase-contrast microscope. Different durations of decondensation were used due to different sperm sensitivity to the decondensation solution. Molecular DNA probes. Four DNA probes for the centromeric regions of chromosome X (DXZ1; M. Morris, Geneva), chromosome 15 (D15Z; Oncor, Illkirch), chromosome 8 (pjm128; American Type Culture Collection, Rockville, MD) and chromosome 18 (L1.84; American Type Culture Collection) were used, as well as one DNA probe for the heterochromatin region of chromosome Y (phy2.1; J. H. Cooke, Edinburgh) (Cooke et al., 1982). The probes were labelled by nick translation with biotin- 7-deoxyadenosine triphosphate (Gibco BRL; Cergy Pontoise) (DXZ1 and D15Z probes), digoxigenin-11-deoxyuridine triphosphate (Boehringer Mannheim, Meylan) (pjm128 and L1.84 probes) or fluorescein-12-deoxyuridine triphosphate (Boehringer Mannheim) (phy2.1 probe) according to kit instructions (Boehringer Mannheim). The specificity and sensitivity of each probe were tested to metaphase spreads as described in a study by Mercier and Bresson (1995) using FISH on uncultured amniotic cells. Hybridization procedure. Two-colour FISH was used with a mixture of denatured D15Z and L1.84 probes to determine the frequency of autosomal disomy. Dual FISH with two autosomal probes enabled diploid cells (somatic or spermatogenetic lines) to be distinguished from disomic spermatozoa. Three-colour FISH was performed to determine the frequency of sex chromosomal disomy with a mixture of denatured phy2.1, DXZ1 and pjm128 probes. Triple FISH (X and Y probes and one autosomal probe) enabled the diploid cells to be distinguished from XY spermatozoa. In dual FISH, digoxigenin-labelled hybrids were detected using tetra-rhodamine isothiocyanate conjugate (TRITC)- labelled antidigoxigenin (Boehringer Mannheim). Biotinylatedlabelled hybrids were detected using fluorescein isothiocyanate (FITC)-labelled avidin (Vector Laboratories, Biosys, Compiègne) and then biotinylated goat anti-avidin (Vector Laboratories) and FITC-labelled avidin. In triple FISH, biotinylated-labelled hybrids were detected using aminomethylcoumarin acetic acid (AMCA)-labelled avidin (Vector Laboratories) and then biotinylated goat antiavidin (Vector Laboratories) and AMCA-labelled avidin. Finally, the slides were counterstained with a medium containing glycerol, an anti-fading reagent (Vector Laboratories), and propidium iodide (Sigma Aldrich, Saint- Quentin). Data collection and analysis. The slides were analysed using a Zeiss Axiophot epifluorescence microscope equipped with a CCD camera for image acquisition and enhancement (Cytovision, Applied Imaging). Slides were included for scoring when the efficiency of hybridization was > 95%. A minimum of sperm nuclei were counted for each sample (10 4 sperm nuclei in 15/18 dual FISH and 10 4 sperm nuclei in X/Y/8 multi-fish) using strict selection criteria: spermatozoa with clumped or overlapping nuclei, disrupted nuclei with indistinct margins, heads but no tails, or nuclei that were not swollen or were swollen to more than three times their original size due to excessive decondensation were excluded from scoring (Mercier et al., 1996; Morel et al., 2000). Statistical analysis Mann Withney U tests were performed to compare semen parameters and the frequency of disomy and

3 Nuclear immaturity and disomy in human spermatozoa 785 Table 1. Characteristics of the 60 human sperm samples used in the study Variable Mean Median Quartile 1 Quartile 3 Minimum Maximum Volume (ml) Sperm concentration (10 6 ml 1 ) Sperm motility (%) Necrozoospermia (%) Teratozoospermia (%) Multiple abnormalities index Aniline blue staining (%) diploidies between spermatozoa in groups 1 and 2 and between those in groups 3 and 4. Results Semen characteristics of the 60 sperm samples The mean ejaculate volume, sperm concentration, motility, necrozoospermia and multiple abnormalities index are shown (Table 1). The frequency of teratozoospermia and the percentage of nuclei stained by aniline blue in the 60 semen samples tested varied significantly. Teratozoospermia ranged from 25 to 99% with a mean of 62.9%. The percentage of nuclei stained by aniline blue ranged from 4 to 67% with an average of 30.2% (Table 1). Definition of the groups From determinations of semen characteristics, four groups of five sperm samples were selected (Tables 2 and 3): (i) group 1, samples with the five lowest values for teratozoospermia (range 25 40%); (ii) group 2, samples with the five highest values for teratozoospermia (range 90 99%); (iii) group 3, samples with the lowest values for the percentage of aniline blue staining (range 4 10%); and (iv) group 4, samples with the highest values for the percentage of aniline blue staining (range 53 67%). Two patients were included both in groups 1 and 3, and two patients were included both in groups 2 and 4. Comparisons of the mean values of chromosome 8, 15, 18, X, Y diploidy and disomy frequencies in groups 1 and 2 No significant differences in the frequency of autosomal and gonosomal disomies or diploidies were observed between the group with the lowest values of abnormal spermatozoa and the group with the highest teratozoospermia values (Table 2). Comparisons of the mean values of chromosome 8, 15, 18, X, Y diploidy and disomy frequencies in groups 3 and 4 Significant differences were found in the frequency of autosomal disomy for chromosomes 8, 15 and 18 (P < 0.05) between the group considered to be normal in terms of nuclear maturity and the group with a high degree of nuclear immaturity. However, no significant differences were observed in the frequency of gonosomal disomy or diploidies (Table 3). Discussion During the past few years, many studies (Miharu et al., 1994; Moosani et al., 1995; Pang et al., 1995; Bernardini et al., 1997; McInnes et al., 1998; Pang et al., 1999) have compared the frequency of disomy in spermatozoa from fertile and infertile men (infertility due to oligo-, oligoastheno-, oligoterato-, oligoasthenoteratozoospermia and unexplained infertility) and have reached different conclusions. Some studies found an increased frequency of chromosomal abnormalities in infertile men (Moosani et al., 1995; Bernardini et al., 1997; Lähdetie et al., 1997; McInnes et al., 1998; Pang et al., 1999), indicating that infertility may be a risk factor for chromosomal aneuploidy in spermatozoa. However, other studies (Miharu et al., 1994; Guttenbach et al., 1997b) found no such differences between fertile and infertile men, indicating that generalized aneuploidy in spermatozoa is not a major reason for unexplained infertility. Finkelstein et al. (1998) used parameters of sperm volume, concentration, vitality, motility and morphology to study the relationship between chromosomal nondisjunction and semen quality in two groups of men that showed considerable differences in semen quality. The results from this study indicated an increased rate of aneuploidies in sex cells derived from low quality semen. The divergent opinions from these studies may result from different methodological approaches or recruitment of patients. However, the disparity of the tested samples makes it difficult to compare the results (Morel et al., 1997, 1999). Few studies have compared the frequency of chromosomal abnormalities with an analysis of the morphological characteristics of spermatozoa (Martin and Rademaker, 1988; Rosenbusch et al., 1992; Lee et al., 1996; Yurov et al., 1996; In t Veld et al., 1997; Bernardini et al., 1998; Estop et al., 1998; Rives et al., 1999). After fusion of male gametes with zona-free hamster eggs, Rosenbusch et al. (1992) confirmed the results of a study by Martin and Rademaker (1988) in which no relation was observed between the morphological and chromosomal abnormalities. This finding

4 786 F. Morel et al. Table 2. Comparison of semen parameters and mean values of chromosome 8, 15, 18, X and Y diploidy and disomy frequencies between the groups of spermatozoa with the lowest (group 1, n = 5) and highest (group 2, n = 5) teratozoospermia values Group 1 Group 2 Variable Mean Minimum Median Maximum Mean Minimum Median Maximum P values Teratozoospermia (%) < 0.01 Head defects Large head Small head Mid-piece defects only Tail defects only Semen volume (ml) NS Sperm concentration (10 6 ml 1 ) < 0.01 Sperm motility (%) < 0.05 Necrozoospermia (%) NS Multiple abnormalities index < 0.01 Aniline blue staining (%) NS Disomies X NS Y NS XY NS NS NS NS Diploidies 15/ NS X/Y/ NS NS: not significant. indicated that an assessment of sperm morphology cannot be used as an indicator of chromosomal damage in human spermatozoa. Lee et al. (1996) analysed the chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes. These authors concluded that the incidence of structural chromosome aberrations was significantly higher in spermatozoa with amorphous, round and elongated heads than in those with morphologically normal heads, whereas the incidence of aneuploidy was not significantly different between the two groups and no increase of chromosomal aberrations was found in spermatozoa with either large heads or small heads. Yurov et al. (1996) found that most spermatozoa with large heads contained a diploid chromosome number. Bernardini et al. (1998) found that certain types of morphologically abnormal spermatozoa (spermatozoa with enlarged heads and those with two tails and one head) and immature germ cells present in ejaculated semen carry high baseline rates of disomy and diploidy. According to Estop et al. (1998), who studied the meiotic segregation of 24 spermatozoa obtained from a 47,XXY male, the percentage of spermatozoa with an abnormal number of sex chromosomes increased from 17% in spermatozoa with normal morphology to 61% in spermatozoa with abnormal morphology. Rives et al. (1999) found no relationship between teratozoospermia and the frequency of aneuploidy in sperm nuclei of infertile men. In a study on 57 different semen samples, Morel et al. (1998) demonstrated, using a linear regression model, that the correlation between teratozoospermia and disomy frequencies bordered on statistical significance. In the present study, there was no significant difference in the frequency of autosomal and gonosomal disomy or diploidies between the group with the lowest values of morphologically abnormal spermatozoa and the group with the highest teratozoospermia values. In these two groups, the distribution of sub-classes of morphological abnormalities was similar, but few changes in the frequency of disomy between these groups cannot rule out a biological relationship between teratozoospermia and disomy. In the present study, the percentage of nuclei stained by aniline blue showed that a high frequency of autosomal disomy is correlated with a lack of nuclear maturity. Moreover, there was a significant difference in sperm concentration and teratozoospermia between groups 3 and

5 Nuclear immaturity and disomy in human spermatozoa 787 Table 3. Comparison of semen parameters and mean values of chromosome 8, 15, 18, X and Y diploidy and disomy frequencies between the groups of spermatozoa with the lowest (group 3, n = 5) and highest (group 4, n = 5) percentage of aniline blue staining Group 3 Group 4 Variable Mean Minimum Median Maximum Mean Minimum Median Maximum P values Teratozoospermia (%) < 0.05 Head defects Large head Small head Mid-piece defects only Tail defects only Semen volume (ml) NS Sperm concentration (10 6 ml 1 ) < 0.05 Sperm motility (%) NS Necrozoospermia (%) NS Multiple abnormalities index NS Aniline blue staining (%) < 0.01 Disomies X NS Y NS XY NS < < < 0.05 Diploidies 15/ NS X/Y/ NS NS: not significant. 4; however, this difference is not important because these parameters were not correlated with disomy frequencies (Morel et al., 1998). To date, only one study (Morel et al., 1998) has attempted to correlate the frequency of meiotic errors with the degree of nuclear immaturity as revealed by aniline blue staining. Aniline blue stains lysine-rich nucleoprotein (Terquem and Dadoune, 1983; Roux and Dadoune, 1989). During human spermatogenesis there is a marked remodelling of chromatin, which turns progressively from a diffuse state in somatic cells and early spermatids into a highly condensed state in late spermatids and mature spermatozoa. In the testis, the restructuring of the chromosomal material during spermatogenesis is accompanied by a gradual replacement of part of the somatic histones (H1, H2A, H2B, H3, H4) by testis-specific subtypes (TH2A, TH2B, TH3, H1t). Most histone variants appear during the meiotic prophase (Grimes, 1986). During spermiogenesis, there is a double nuclear basic protein transition: somatictype histones rich in lysine or their testis-specific variants are partially or totally replaced by intermediate basic proteins (transition proteins TP1 and TP2 or pro-protamines HPI1 and HPI2). These intermediate basic proteins, HPI1 and HPI2, are synthesized in large amounts in human spermatids during the elongation phase, but can only be detected in small amounts in mature spermatids when the depositing of protamine from the P1 (HP1) and P2 (HP2, HP3, HP4) families is complete (Gusse et al., 1986; Balhorn, 1989; Pringent et al., 1998). However, histones (essentially TH2B) are partially retained in mature spermatids and sperm nuclei. In normal human spermatozoa, histones represent approximately 20% of all nuclear basic proteins and they are highly acetylated (Gusse et al., 1986; Gatewood et al., 1987). In conclusion, at least three hypotheses can be put forward to explain significant increases in the prevalence of autosomal disomy between the samples with the five lowest and five highest percentage values of aniline blue staining: (i) a chromosomal segregation abnormality during meiosis could disturb the replacement of nucleoproteins during the double transition in spermiogenesis and allow the persistence of lysine-rich nucleoproteins in spermatozoa; (ii) a pre-meiotic or meiotic chromatin abnormality associated with disturbance in somatic and/or testicular variant histone pattern could disturb both the meiotic process and the construction of definitive nucleoprotein

6 788 F. Morel et al. patterns; and (iii) an initial cytological disorder in gametogenesis could appear during meiosis and subsequently influence the composition of the definitive nucleoproteins in spermatozoa. The differences observed in the frequency of gonosomal and autosomal disomy in terms of the aniline blue staining results were unexpected. This disparity may be due to a difference in the behaviour of the autosomal and gonosomal chromosomes during meiosis, and is indicated by the extended isolation of the gonosomes in the heterochromatic body. The authors wish to thank the Centre d Etude et de Conservation des Oeufs et du Sperme humain, Besançon - Franche Comté. The authors are also grateful to J. H. Cooke of the Western General Hospital of Edinburgh (United Kingdom) and to M. Morris of the General Hospital of Geneva (Switzerland) for providing phy 2.1 and DXZ1, respectively. They also thank P. Albert for her help with the proofreading of the manuscript and T. Elmrini and E. Monnet (Public Health Department of the Besançon Medical School) for their statistical expertise. This work was supported by the Fondation pour la Recherche Médicale (FRM) and the Association Régionale pour le Développement des Etudes Biologiques en Génétique et Reproduction Humaines, Besançon, France. References Balhorn R (1989) Mammalian protamines: structure and molecular interactions. In Molecular Biology of Chromosome Function pp Ed. KW Adolph. Springer-Verlag, New York Bernardini L, Martini E, Geraedts JPM, Hopman AHN, Lanteri S, Conte N and Capitanio GL (1997) Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in situ hybridization Molecular Human Reproduction Bernardini L, Borini A, Preti S, Conte N, Flamigni C, Capitanio GL and Venturini PL (1998) Study of aneuploidy in normal and abnormal germ cells from semen of fertile and infertile men Human Reproduction Cooke JH, Schmidtke J and Gosden JR (1982) Characterisation of a human Y chromosome repeated sequence and related sequences in higher primates Chromosoma David G, Bisson JP, Czyglik F, Jouannet P and Gernigon C (1975) Anomalies morphologiques des spermatozoïdes humains Journal de Gynécololgie Obstétrique et Biologie de la Reproduction Downie SE, Flaherty SP, Swann NJ and Matthews CD (1997) Estimation of aneuploidy for chromosomes 3, 7, 16, X and Y in spermatozoa from 10 normospermic men using fluorescence in situ hybridization Molecular Human Reproduction Estop AM, Cieply KM, Wakim A and Feingold E (1998) Meiotic products of a Klinefelter 47,XXY male as determined by sperm fluorescence in situ hybridization analysis Human Reproduction Finkelstein S, Mukamel E, Yavetz H, Paz G and Avivi L (1998) Increased rate of nondisjunction in sex cells derived from low-quality semen Human Genetics Gatewood JM, Cook GR, Balhorn R, Bradbury EM and Schmid CW (1987) Sequence-specific packaging of DNA in human sperm chromatin Science Grimes SR (1986) Nuclear proteins in spermatogenesis Comparative Biochemistry and Physiology Gusse M, Sautiere P, Belaiche D, Martinage A, Roux C, Dadoune JP and Chevaillier P (1986) Purification and characterization of nuclear basic proteins of human sperm Biochemica Biophysica Acta Guttenbach M, Engel W and Schmid M (1997a) Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review Human Genetics Guttenbach M, Martinez-Exposito JM, Michelmann HW, Engel W and Schmid M (1997b) Incidence of diploid and disomic sperm nuclei in 45 infertile men Human Reproduction In t Veld PA, Broekmans FJM, De France HF, Pearson PL, Pieters MHEC and Van Kooij RJ (1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa Human Reproduction Lähdetie J, Saari N, Ajosenpaa-Saari M and Mykkanen J (1997) Incidence of aneuploid spermatozoa among infertile men studied by multicolor fluorescence in situ hybridization American Journal of Medicine and Genetics Lee JD, Kamiguchi Y and Yanagimachi R (1996) Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes Human Reproduction McInnes B, Rademaker A, Greene CA, Ko E, Barclay L and Martin RH (1998) Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men Human Reproduction Martin RH and Rademaker A (1988) The relationship between sperm chromosomal abnormalities and sperm morphology in humans Mutation Research Mercier S and Bresson JL (1995) Prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization on uncultured amniotic cells: experience with 630 samples Annales de Génétique Mercier S, Morel F, Roux C, Clavequin MC and Bresson JL (1996) Analysis by two colour fluorescence in situ hybridization of the gonosomal equipment in spermatozoa of a 47,XYY male Molecular Human Reproduction Mercier S, Morel F, Fellmann F, Roux C and Bresson JL (1998) Molecular analysis of the chromosomal equipment in spermatozoa of a 46,XY,t(7;8)(q11.21;cen) carrier using fluorescence in situ hybridization Human Genetics Miharu N, Best RG and Young SR (1994) Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in situ hybridization Human Genetics Moosani N, Pattinson HA, Carter MD, Cox MD, Rademaker AW and Martin RH (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization Fertility and Sterility Morel F, Mercier S, Roux C, Clavequin MC and Bresson JL (1997) Estimation of aneuploidy levels for 8, 15, 18, X and Y chromosomes in 97 human sperm samples using fluorescence in situ hybridization Fertility and Sterility Morel F, Mercier S, Roux C, Elmrini T, Clavequin MC and Bresson JL (1998) Interindividual variations in the disomy frequencies of human spermatozoa and their correlation with nuclear maturity as evaluated by aniline blue staining Fertility and Sterility Morel F, Roux C and Bresson JL (1999) Aneuploidy of sex chromosome in sperm of 47,XYY men Archives of Andrology Morel F, Roux C and Bresson JL (2000) Segregation of sex chromosomes in spermatozoa of 46,XY/47,XXY men by multicolor fluorescence in situ hybridization (FISH) Molecular Human Reproduction Pang MG, Zackowski JL, Hoegerman SF, Friedman E, Moon SY, Cuticchia AJ, Acosta AA and Kearns WG (1995) Detection by fluorescence in situ hybridization of chromosome 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y aneuploidy in sperm from oligo-astheno-terato-zoospermic patients of an in vitro fertilization program American Journal of Human Genetics A 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 by fluorescence in situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection Human Reproduction Pellestor F, Quenesson I, Coignet L, Girardet A, Andreo B and Charlieu JP (1996) Direct detection of disomy in human sperm by the PRINS technique Human Genetics

7 Nuclear immaturity and disomy in human spermatozoa 789 Prigent Y, Troalen F and Dadoune JP (1998) Immunoelectron microscopic visualization of intermediate basic proteins HPI1 and HPI2 in human spermatids and spermatozoa Reproduction Nutrition Développement 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 Human Genetics Rives N, Saint Clair A, Mazurier S, Sibert L, Simeon N, Joly G and Mace B (1999) Relationship between clinical phenotype, semen parameters and aneuploidy frequency in sperm nuclei of 50 infertile males Human Genetics Rosenbusch B, Strehler E and Sterzik K (1992) Cytogenetics of human spermatozoa: correlations with sperm morphology and age of fertile men Fertility and Sterility Roux C and Dadoune JP (1989) Use of the acridine orange staining on smears of human spermatozoa after heat-treatment: evaluation of the chromatin condensation Andrologia Rubes J, Lowe X, Moore D, Perreault S, Slott V, Evenson D, Selevan SG and Wyrobek AJ (1998) Smoking cigarettes is associated with increased sperm disomy in teenage men Fertility and Sterility Spriggs EL, Rademaker AW and Martin RH (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 Cytogenetics and Cell Genetics Terquem A and Dadoune JP (1983) Aniline blue staining of human spermatozoa chromatin. Evaluation of nuclear maturation. In The Sperm Cell pp Ed. J André. Martinus Nijhoff, The Hague Van Hummelen P, Lowe XR and Wyrobek AJ (1996) Simultaneous detection of structural and numerical chromosome abnormalities in sperm of healthy men by multicolor fluorescence in situ hybridization Human Genetics World Health Organization (1992) Laboratory Manual for the Examination of Human Semen and Semen Cervical Mucus Interaction Cambridge University Press, Cambridge, UK Yurov YB, Saias MJ, Vorsanova SG, Erny R, Soloviev IV, Sharonin VO, Guichaoua MR and Luciani JM (1996) Rapid chromosomal analysis of germ-line cells by FISH: an investigation of an infertile male with largeheaded spermatozoa Molecular Human Reproduction Received 13 July First decision 8 September Revised manuscript received 21 December Accepted 15 January 2001.