Chromosome aneuploidy in the spermatozoa of two men with globozoospermia

Abstract

The objective of this study was to determine the aneuploidy level in spermatozoa in two men with globozoospermia. Sperm nuclei were analysed by fluorescence in-situ hybridization (FISH) in two infertile males with globozoospermia. Dual FISH for chromosomes 7 and 9, 13 and 21, and triple FISH for chromosomes X, Y, and 18 was performed. The main outcome measured was meiotic segregation differences between both globozoospermic men and controls. A statistically significant difference in disomies 13 and 21 was found between patients 1 and 2. The diploidy rate of spermatozoa of patient 1 (0.876%) was significantly increased compared with that of patient 2 (0.304%) and control men (0.293%). In conclusion there seems to be a slightly increased frequency of aneuploidy in round-headed spermatozoa. However, it is unlikely that these aneuploid spermatozoa would be used in assisted reproduction techniques.

Introduction

First described by Schirren et al. (1971), globozoospermia is a rarely described morphologic disorder of spermatozoa presenting with round-heads observed in less than 1% of infertile men. The lack of the acrosome is the main characteristic of this type of teratozoospermia associated with severe infertility. The acrosomeless spermatozoon is unable to go through the zona pellucida and fuse with the oocyte. Singh (1992) described two types of globozoospermia: type I with round-headed spermatozoa lacking the acrosome and acrosomal enzymes and type II with round-headed spermatozoa having remnants of the acrosome. The physiopathology of this anomaly is still unclear. Studies of sperm DNA status demonstrated abnormal chromatin structures and DNA strand breaks (Vicari et al., 2002).

The genetic defect is still unknown and different modes of inheritance (polygenic, autosomal dominant, autosomal recessive, etc.) have been proposed (Trokoudes et al., 1995; Stone et al., 2000). Kang-Decker et al. (2001) reported Hrb protein deficient mice with round-headed acrosomeless and infertility.

ICSI is the sole treatment for globozoospermic patients, with various fertilization successes (Lundin et al., 1994; Stone et al., 2000; Zeyneloglu et al., 2002). The first case of fertilization and pregnancy by ICSI, with round-headed spermatozoa, was described 9 years ago (Lundin et al., 1994). Few studies have analysed sperm aneuploidy in spermatozoa from globozoospermic patients (Viville et al., 2000; Carrell et al., 2001; Vicari et al., 2002; Martin et al., 2003).

In this study, we evaluated the aneuploidy rates of chromosomes 7, 9, 13, 18, 21, X, Y and diploidy rates in sperm of two unrelated globozoospermic patients, using dual and triple fluorescence in-situ hybridization (FISH), in order to determine whether there is an increased risk of numerical anomalies in the spermatozoa. These chromosomes were chosen because chromosomes 13, 18, 21, X and Y are associated with viable aneuploidy, while chromosomes 7 and 9, belonging to a different group, are very rarely observed in amniotic cells and/or spontaneous abortions.

Materials and methods

Patients

A couple presented with a 3-year history of infertility. The 32-year-old man (patient 1) had infertility due to a globozoospermia diagnosed one year earlier by electron microscopy. He had a negative familial history. His karyotype on peripheral lymphocytes was normal. Although the semen sample used for FISH studies was normospermic, this patient had a long history of oligoasthenoteratospermia (Table I).

The second couple presented with a 2-year history of infertility. The 39-year-old man (patient 2), with a negative familial history of infertility, had a complete globozoospermia and a normal karyotype. According to Singh's classification, both patients belonged to type I globozoospermia (Singh, 1992).

Four men included in an ISCI programme because of female infertility served as controls. Their clinical history and physical examination revealed no abnormality. They had normal sperm characteristics according to the criteria of the World Health Organization (1999) and normal 46,XY karyotype.

Prior to this study, the patients and controls were informed of the investigations and gave their consent. Human ejaculates were obtained from all six men. The characteristics of the sperm from the patients and controls are described in Table II.

Analyses of aneuploidy

The sperm samples were washed in PBS and 20 μl of sperm were dropped and fixed on a slide with Carnoy's solution (methanol/acetic acid: 3v/1v). The sperm nuclei were partially decondensed for 2 min with a solution of NaOH (1 mol/l), then washed in 2×SSC for 10 min.

The sperm samples were analysed in dual FISH 7–9 with specific alphoid probes of chromosome 7 (probe D7Z1, spectrum green, Abbott^®, Rungis, France) and chromosome 9 (spectrum orange, Abbott^®), in dual FISH 13–21 with a specific cocktail probe of 13q14 and 21q22 (LSI 13q14 and LSI 21q22, dual colour, PNAT2113, QBIOgene^®, Illkrich, France), and triple FISH X-Y-18 with specific alphoid probes of chromosome X (probe DXZ1, spectrum green, Abbott^®), chromosome Y (probe DYZ3, spectrum orange, Abbott^®) and chromosome 18 (D18Z1, spectrum aqua, Abbott^®).

Before hybridization, the sperm DNA slides were immersed in a jar of 2×SSC/0.4%NP40 solution for 30 min at 37°C and then passed through an ethanol series of growing concentration and allowed to air dry (Morel et al., 2003).

The denaturation was performed simultaneously on sperm nuclei and probes, for 1 min at 75°C in dual FISH 7–9 and triple FISH X-Y-18 and for 2 min at 80°C in dual FISH 13–21. The slides were incubated overnight at 42°C for specific alphoid probes and at 37°C for specific cocktail dual probe 13–21. Post-hybridization washes included 45 s in 0.4×SSC/0.3%NP40 at 72°C followed by 20 s in 2×SSC/0.1%NP40 at room temperature.

The slides were counterstained with 4′,6-diaminidino-2-phenylindol (DAPI) and analysed using a Zeiss Axioplan microscope (Zeiss, Le Pecq, France). Subsequent image acquisition was performed using a CCD camera with Isis (In situ imaging system) (MetaSystems, Altlussheim, Germany). A total of 5000 nuclei per sample were analysed whenever possible (Morel et al., 2003).

Statistical analyses

An independent χ² test or Yates test was used to compare the results obtained for each patient with globozoospermia with control men. Moreover, the same statistical analyses were used to compare both globozoospermic patients. The significance level was set at P<0.05.

Results

Table III shows the aneuploidy and diploidy rates in spermatozoa in both globozoospermic patients and the controls. No statistically significant difference was observed in the frequency of disomy of chromosomes 7, 9, 13, 18, and 21 between patients and controls. However, the statistical analyses revealed a significant difference in the disomies of acrocentric chromosomes 13 and 21 between patients 1 and 2 (P=0.038 and 0.017, respectively).

The diploidy rate of spermatozoa of patient 1 (0.876%) was significantly increased compared with that of patient 2 (0.304%) and control men (0.26%) (P<0.001). This increase was observed in double FISH 7–9, double FISH 13–21 and triple FISH X-Y-18. Furthermore, a high incidence of 46,XY nuclei (0.723%) compared with 46,XX (0.106%) and 46,YY (0.127%) was found in patient 1, but not in patient 2.

Discussion

Globozoospermia is a morphological abnormality characterized by acrosomeless spermatozoa. Production of the acrosome is a post-meiotic event in spermatogenesis.

In this study, we observed no statistically significant difference in the aneuploidy rates between globozoospermic patients and controls. We noted a significant increase of disomies 13 and 21 in patient 1 compared to patient 2. A significantly higher diploidy rate was also found in one globozoospermic man than in normal males. Diploidy is a non-segregation abnormality occurring during spermatogenesis due to a lack of cytodieresis or an incomplete meiosis. The abnormal segregation of chromosomes during meiosis may be related to oligozoospermia rather than morphologic abnormalities in patient 1. Indeed, many authors reported a link between sperm aneuploidy and oligoasthenozoospermia (Pang et al., 1999; Pfeffer et al., 1999; Calogero et al., 2001). In studies on teratozoospermia, Templado et al. (2002) and Vegetti et al. (2000) did not find a relationship between aneuploidy and morphologic abnormalities, as for patient 2.

Few studies reported molecular cytogenetic analyses in round-headed spermatozoa (Table IV). They yielded controversial results. Carrell et al. (2001) analysed the spermatozoa of a globozoospermic man whose wife had a spontaneous abortion of a fetus with trisomy 15. They found 4% of the spermatozoa to be disomic 15. However, they did not observe an increased rate of disomy 15 in one of his siblings with globozoospermia, but in another brother (1%) without sperm morphologic abnormality (Carrell et al. 2001). Therefore, the increased frequency of disomy 15 may not be related to globozoospermia.

No significant increase in numerical chromosomal abnormalities was found in two other patients with globozoospermia (Viville et al., 2000; Vicari et al., 2002). However, Viville et al., (2000) only studied sex chromosomes using chromosome 1 as an internal control. A fifth globozoospermic and asthenospermic man was studied by Martin et al. (2003). They found a similar rate of aneuploidy for chromosomes 1, 15, X, and Y, but a significant increase in disomy XY, in the patient than in controls (Martin et al., 2003).

In conclusion, it seems to have a slightly increased frequency of aneuploidy in round-headed spermatozoa. However, it is unlikely that these aneuploid spermatozoa would be used in assisted reproduction techniques. The association between this severe teratozoospermia and numerical chromosomal abnormalities is unclear. Although it could be merely fortuitous, the increase observed in some patients could reflect disturbances in spermatogenesis, as observed in other types of infertility (Shi and Martin, 2001).

References

Author notes

1Laboratoire d'Histologie, Embryologie et Cytogénétique, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, 2Service de Cytogénétique, Cytologie et Biologie de la Reproduction, CHU Morvan, Brest, 3Service de Génétique Clinique, CHRU, Lille, 4Centre de Génétique Chromosomique, Hôpital St Vincent de Paul, Lille and 5Service de Biologie de la Reproduction, CHRU, Lille, France