Globozoospermia is associated with chromatin structure abnormalities: Case report

Enzo Vicari, Anna Perdichizzi, Adele De Palma, Nunziatina Burrello, Rosario D'Agata and Aldo E. Calogero,1

Division of Endocrinology, Andrology and Internal Medicine, Department of Biomedical Sciences, University of Catania, Catania, Italy


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
A recent study has shown normal sperm chromatin structure in a patient with globozoospermia. However, the poor success rate following intracytoplasmic sperm injection (ICSI) with the use of this sperm suggests that sperm nuclear abnormalities may be present. We report here a study of the sperm DNA integrity and chromosome aneuploidy and diploidy rates of a patient with 100% round-headed sperm. The sperm chromatin packaging quality was assessed by using flow cytometry after staining the DNA with propidium iodide. DNA fragmentation, possibly indicative of apoptosis, was evaluated by flow cytometry using the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling (TUNEL) assay. The sperm chromosome aneuploidy and diploidy rates were evaluated by fluorescence in-situ hybridization using {alpha}-centromeric probes for chromosomes 8, 12, 18, X and Y. The patient with globozoospermia had a significantly higher number of sperm with chromatin decondensation (35 ± 1.1%) and positive for the TUNEL assay (37 ± 1.7%) compared with that found in four normal controls (4.7 ± 0.4 and 22.5 ± 1.2% respectively). In contrast, the total sperm aneuploidy (0.16%) and diploidy (0.05%) rates for the chromosomes studied were within the range found in 14 normozoospermic men. To the best of our knowledge, this is the first case report of round-headed sperm that has shown an elevated number of sperm with abnormal chromatin structure and DNA strand breaks.

Key words: aneuploidy/chromatin structure/diploidy/globozoospermia/sperm


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Globozoospermia was first described in 1971 (Schirren et al., 1971Go). It is an uncommon condition probably occurring in 0.1% of all andrology patients, and it is characterized by the complete absence of the acrosome vesicle and the disorganization of the mid-piece and tail (often exhibiting structural abnormalities and abnormal mitochondria). These abnormalities are found in all the sperm in the patient's ejaculate and lead to the inability to bind the human oocyte zona pellucida and to produce sperm–oocyte fusion. Other sperm abnormalities such as nuclear membrane and chromatin condensation disorders in the area of the nucleus have been described. The hereditary background to this condition has not yet been established (Carrell et al., 1999Go). A polygenic and polymorphic mode, a monogenic mode, a dominant inheritance and a homozygous autosomal gene defect have all been suggested (Trokoudes et al., 1995Go).

Two types of globozoospermia have been described (Singh, 1992Go). Type I is characterized by a complete lack of acrosome and acrosomal enzymes and by a spherical arrangement of the chromatin. These sperm are unable to penetrate the zona pellucida, causing primary infertility. Type II has some acrosomal covering with a conical nucleus, which may be surrounded by large cytoplasmic droplets indicating secondary degenerative changes. It is assumed that infertility in this type of globozoospermia is caused by subsequent poor motility (Singh, 1992Go).

Sperm DNA status in those patients with globozoospermia is not well known. Carrell and collaborators have found an elevated sperm aneuploidy rate, studied by fluorescence in-situ hybridization (FISH), for chromosomes 13, 21, X and Y (Carrell et al., 1999Go) and 15 and 18 (Carrell et al., 2001Go) in one of two infertile siblings with globozoospermia, whereas a normal aneuploidy rate has been reported by other authors (Viville et al., 2000Go). A recent case report has shown that only 13% of sperm demonstrated DNA denaturation following the application of sperm chromatin structure assay (SCSA). A similar percentage has been obtained by using the COMET assay (Larson et al., 2001Go) These results were similar to those obtained in normal controls, suggesting that globozoospermia is not associated with abnormal chromatin structure or DNA strand breaks. However, globozoospermia is a severe form of teratozoospermia and as such it is possible that round-headed sperm may have an abnormal chromatin structure and/or DNA strand breaks both of which have been reported to cause early pregnancy loss (Ibrahim et al., 1988Go) or to increase the risk of malformations, cancer and genetic diseases in the offspring (Potts et al., 1999Go). Therefore, the evaluation of the sperm DNA status in patients with globozoospermia is fundamental.

This study reports the sperm DNA status of a patient with globozoospermia. The sperm chromatin packaging quality was assessed by applying propidium iodide (PI) staining, whereas DNA fragmentation was studied by using the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling (TUNEL) procedure. The sperm aneuploidy rate for chromosome 8, 12, 18, X and Y was also evaluated by means of FISH.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patient and normal control selection
The patient was a 39-year-old man who consulted the Andrology and Reproductive Endocrinology Unit (AREU), University of Catania Medical School, for primary infertility of 6 years duration. He had no previous history of cryptorchidism or varicocele and was not exposed to environmental and/or workplace gonadotoxins (i.e. heat, heavy metal, radiation, pesticides, etc.). Repeated semen analysis showed asthenozoospermia and monomorphic teratozoospermia with 100% round-headed sperm (WHO, 1999Go) (Table IGo). Physical examination showed a normal body mass index, no gynecomastia, a mean testicular volume of 14.5 ml and no laboratory, clinical or ultrasound signs suggestive of male accessory gland infection. Serum LH, FSH, testosterone, 17ß-estradiol and prolactin levels were normal. Four healthy men with normal sperm parameters were selected as controls for sperm chromatin packaging quality and DNA fragmentation. Our laboratory reference values for sperm aneuploidy rates were obtained from 14 healthy normozoospermic men.


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Table I. Conventional sperm parameters of the patient with globozoospermia
 
PI staining
Semen samples were diluted, gently resuspended in calcium- and magnesium-free phosphate-buffered saline (PBS) containing albumin 0.1% and centrifuged at 500 g for 10 min at room temperature. The seminal fluid was removed and the sperm were stained with PI as previously reported (Nicoletti et al., 1991Go) with slight modifications. Briefly, 1x106 cell aliquots were gently incubated in 0.5 ml PBS containing 50 µg/ml of PI (Sigma Chemical, Milan, Italy), 0.1% sodium citrate, and 0.1% Nonidet P40 (Sigma Chemical), 100 KU of RNAse type A (Sigma Chemical) in the dark, at room temperature. After 30 min, flow cytometry (FCM) analysis was performed using the flow cytometer EPICS XL (Coulter Electronics, Krefel, Germany) equipped with a 488 nm argon laser as light source. In this study, only one of the three fluorescent detectors available was used to measure the fluorescence corresponding to the red colour of PI (FL-3 detector 620 nm wavelength band). Ten thousand events were measured for each sample at a flow rate of 200–300 events/s. The debris was gated-out by drawing a region on forward versus side scatter dot plot enclosing the population of sperm that were of interest. To ascertain whether the gate thus drawn included only sperm, we evaluated the ploidy of this cell population (haploid). For this purpose, human peripheral blood mononuclear cells (diploid), collected from healthy donors, were simultaneously analysed during each experiment, as an internal standard. In order to gate-out, and thus exclude from the analysis doublets and cell aggregates, a `doublet discrimination module' was used. The peak width was estimated using the coefficient of variation (CV) of the signals within each peak.

TUNEL assay
Sperm were labelled using the Apoptosis Mebstain kit (Beckman Coulter), purchased from Instrumentation Laboratory (Milan, Italy). To obtain a negative control, TdT was omitted from the reaction mixture. The positive control was obtained by pre-treating the sperm with 1 µg/ml deoxyribonuclease I, RNAse-free (Sigma Chemical) at 37°C for 60 min before labelling. DNA fragmentation was evaluated by FCM. Ten thousand events were measured for each sample at a flow rate of 500 events/s. The debris was eliminated following the same procedure. Light-scattering and fluorescence data were obtained at a fixed gain setting in logarithmic mode. The percentage of FITC-labelled sperm was determined in the FL-1 channel of the FCM.

FISH analysis
Sperm FISH analysis was performed as previously reported (Calogero et al., 2001Go). Briefly, sperm were washed three times in phosphate buffered saline (PBS), pH 7.2, centrifuged at 650xg for 10 min and fixed in methanol/acetic acid (3:1). They were spread on slides and the slides were washed in 2x standard saline citrate solution (SSC) and incubated for 5 min in 1 mol/l Tris buffer, pH 9.5, containing 25 mmol/l dithiothreitol.

Double- and a triple-colour FISH were carried out, using {alpha}-centromeric probes for chromosomes 8, 12, 18, X and Y, purchased from the University of Bari (Bari, Italy). The probe mixture for triple FISH consisted of a repetitive DNA sequence of centromeric probes: chromosome X (pDMX1), labelled with FITC; for chromosome Y (pLAY5.5), labelled with Cy3; and for chromosome 12 (pBR12), labelled with FITC and Cy3. The probe mixture for double-colour FISH also consisted of a repetitive DNA sequence of centromeric probes for chromosome 8 (pZ8.4) and for chromosome 18 (2Xba), labelled FITC and Cy3, respectively.

The slides were denatured in a PCR machine with a solution of 70% formamide/2xSSC (pH 7.5) at 80°C for 150 s, immersed in a 70, 90 and 100% ethanol series for 3 min each and dried by air. The probes, precipitated and denatured at 80°C for 8 min, were applied directly to the slides which were then covered with a coverslip and sealed with rubber cement. Hybridization occurred overnight in a dark humidified container at 37°C, after which one coverslip was removed and the slides were immersed in a post-hybridization wash of 50% formamide/2xSSC for three times at 37°C for 5 min, 2xSSC for three times at 42°C for 5 min and 2xSSC/0.1% Tween 20 at room temperature for 5 min. The slides were then mounted in 4',6-diamidino-2-phenylindole (DAPI) counterstain and antifade. The slides were observed using an Axiophot fluorescent microscope (C.Zeiss, Oberkochen, Germany) with an appropriate set of filters, namely single band DAPI, FITC and Cy3. Only intact sperm bearing a similar degree of decondensation and clear hybridization signals were scored; disrupted or overlapping sperm were excluded from analysis. Sperm were regarded as abnormal if they presented two (or more) distinct hybridization signals for the same chromosome, each equal in intensity and size to the single signal found in normal monosomic nuclei. We considered only clear hybridization signals, similar in size, separated from each other by at least one signal domain and clearly positioned within the sperm head. Divided (split) signals were not scored as disomies. Sperm were scored as nullisomic if they showed no signal for a given chromosome, when the signal of the other chromosome tested was present. Finally, a spermatozoon was considered diploid in cases where it manifested two signals for each tested chromosome and when the tail, as well as the normal oval shape of a sperm head, were evident. The absence of FISH signals in a spermatozoon head showing DAPI stain was considered to be a case of no hybridization.

Statistical analysis
Data are shown as mean ± SEM. Statistical analysis was carried out using unpaired Student's t-test. P-value < 0.05 was considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
The conventional sperm parameters of the patient with globozoospermia are reported in Table IGo. He was astheno-teratozoospermic.

PI staining
Two different cell populations were detected in the semen sample of the patient with globozoospermia and in normal controls (Figure 1Go). The first fluorescence peak corresponded to sperm with mature chromatin, whereas the second peak corresponded to haploid cells with an increased DNA accessibility to PI (sperm with immature chromatin). A third peak including diploid cells was also observed. The percentage of sperm with immature chromatin was significantly higher (P < 0.001) in the patient with globozoospermia (Table IIGo).



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Figure 1. Frequency distribution histograms of sperm from a normozoospermic man (left panel) and the patient with globozoospermia (right panel) stained with propidium iodide. A, normal sperm; B, immature sperm; C, diploid cells.

 

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Table II. Mean (± SEM) percentage and coefficients of variation of sperm with mature and immature chromatin in a patient with globozoospermia and four normal controls as measured by flow cytometry following staining with propidium iodide
 
TUNEL assay
Representative frequency histograms following TUNEL assay of sperm obtained from a normozoospermic man, including negative and positive controls, and of the patient with globozoospermia are shown in Figure 2Go. The patient with globozoospermia had a significantly higher percentage of sperm with DNA fragmentation (37.0 ± 1.7%, three different ejaculates) compared to that found in four normozoospermic men (22.5 ± 1.2%) (P < 0.005).



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Figure 2. Frequency distribution histograms of sperm obtained from a normozoospermic man (panel A) and the patient with globozoospermia (panel B) stained with isothiocyanate-labelled dUTP. Panels C and D show the frequency distribution of sperm stained with isothiocyanate-labelled dUTP without using TdT (negative control) or with DNase (positive control), respectively. The horizontal lines represent sperm positive for fluorescein isothiocyanate-labelled dUTP.

 
Sperm aneuploidy rate
A total of 3885 sperm was analysed for the patient with globozoospermia. A mean of 3879 sperm (range: 3512–4018) was scored for each of the 14 normozoospermic men who were enrolled to establish the laboratory reference values. The disomy rates of the chromosome analysed are reported in Table IIIGo. The patient with globozoospermia had a total sperm aneuploidy rate of 0.16% (normal range 0.3–1.55%) and a diploidy rate of 0.05% (normal range 0–0.20%). X- and Y-bearing sperm had the expected 1:1 ratio.


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Table III. Sperm disomy rates analysed by triple (X, Y and 12)- and double (8 and 18)-colour FISH in a patient with globozoospermia
 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
The degree of nuclear chromatin condensation can be assessed by means of various techniques such as Aniline blue (Foresta et al., 1992Go), CMA3 (Bianchi et al., 1993Go), or the uptake of an intercalating compound (Acridine orange, PI, ethidium bromide); subsequent analysis can be performed by means of either fluorescence microscopy or flow cytometry (Spanò et al., 1984Go). This latter technique is probably the most objective and recommended (Gledhill et al., 1990Go). Moreover, it has been reported that the sperm which bind most fluorochrome contain endogenous DNA nicks, which may indicate anomalies in the chromatin packaging process occurring during spermiogenesis in some patients (Bianchi et al., 1993Go). Other authors have, however, suggested that DNA nicks could be related to apoptosis since they are characteristically present in apoptotic somatic cells and in germ cells which are being eliminated (Gorczyca et al., 1993).

Our patient with globozoospermia had an elevated number of sperm with decondensed chromatin. The assessment of DNA fragmentation showed a percentage of TUNEL-positive sperm greater than that found in normal controls, suggesting an increased rate of apoptosis. However, it is noteworthy that cells showing a high degree of DNA fragmentation are not necessarily apoptotic (Evenson et al., 2002Go). These results indicated that, like other forms of teratozoospermia (Gandini et al., 2000Go), globozoospermia is probably associated with abnormal chromatin structures and DNA strand breaks. An increased rate of DNA fragmentation has also been reported by Baccetti and co-workers in patients with round-headed sperm (Baccetti et al., 1996Go). Our data are, however, at variance with a recent case report showing a normal chromatin packaging in a patient with globozoospermia (Larson et al., 2001Go). Although we do not have a definitive explanation for this, it may relate to the different methods employed to evaluate chromatin integrity (SCSA and single cell gel electrophoresis assay versus TUNEL). Alternatively, it may relate to the fact that only 69% of the sperm were found to have round heads in the patient reported in the latter study whereas all the sperm in our patient were globozoospermic. In addition, it should be highlighted that our patient, unlike the patient in a previous study (Larson et al., 2001Go), also had asthenozoospermia and a direct relationship between poor motility and the degree of DNA fragmentation has recently been suggested (Muratori et al., 2000Go).

Syms and co-workers (1984) have demonstrated that globozoospermic sperm could undergo nuclear decondensation when incubated with crushed hamster oocytes (Syms et al., 1984Go). Decondensation and pronuclear formation have also been detected after direct injection into hamster oocytes (Lanzendorf et al., 1988Go). Thus, if the inability to penetrate the oocytes is bypassed, fertilization could take place. This was made possible in humans with the introduction of the intracytoplasmic sperm injection (ICSI) technique (Palermo et al., 1992Go). Following the first pregnancies obtained from patients with globozoospermia by means of ICSI (Liu et al., 1995Go; Trokoudes et al., 1995Go), a number of globozoospermic patients have undergone this type of treatment. However, a low fertilization rate has been reported, unless oocytes have been activated with calcium chloride or ionophore (Battaglia et al., 1997Go; Rybouchkin et al., 1997Go; Stone et al., 2000Go) which implies a defective capability of the globozoospermic spermatozoon to activate the oocyte. We hypothesise that the poor status of chromatin condensation and DNA fragmentation may also contribute to the low fertilization rate reported in these patients. Accordingly, a poor chromatin packaging and/or damaged DNA have been implicated in the failure of sperm decondensation after ICSI, resulting in fertilization failure (Sakkas et al., 1996Go). Moreover, its has been shown that patients with male factor infertility display higher levels of loosely packaged DNA (Evenson et al., 1980Go; Foresta et al., 1992Go; Sailer et al., 1995Go; Golan et al., 1997Go).

Sperm aneuploidy is another abnormality which may impair ICSI outcome (Calogero et al., 2001Go). The normal sperm aneuploidy rate found in the globozoospermic patient reported in this study ruled out this hypothesis in this case. Accordingly, other studies have reported that the incidence of chromosomal abnormalities in round-headed sperm is similar to that in sperm from a fertile donor (Kullander and Rausing, 1975Go; Lalonde et al., 1988Go; Viville et al., 2000Go). However, a contribution of sperm aneuploidy to the low ICSI outcome success rate cannot be completely excluded in patients with globozoospermia since an elevated sperm aneuploidy rate has also been reported in one of two infertile siblings with globozoospermia indicating multigenic defects and/or variable expression of the syndrome (Carrell et al., 1999Go, 2001Go). Recently, these authors have reported the first case of sperm chromosome-15 aneuploidy in a patient with variable familial expression of round-headed morphology who had a child with trisomia 15 following ICSI (Carrell et al., 2001Go).

In conclusion, we have found an association between globozoospermia and abnormal sperm chromatin packaging and DNA strand breaks. The reported lower success rate in ICSI programmes for patients with globozoospermia (Battaglia et al., 1997Go; Rybouchkin et al., 1997Go; Stone et al., 2000Go) may be explained by this finding and this should be kept in mind when patients with this type of teratozoospermia are enrolled in ICSI programmes.


    Acknowledgements
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank David Farrugia, Lecturer, Faculty of Economics, University of Catania, for the English text editing.


    Notes
 
1 To whom correspondence should be addressed at: Cattedra di Endocrinologia, Ospedale Garibaldi, Piazza S. Maria di Gesù, 95123 Catania, Italy. E-mail: acaloger{at}unict.it Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 2, 2002; resubmitted on March 19, 2002; accepted on May 10, 2002.