Hum. Reprod. Advance Access originally published online on September 22, 2006
Human Reproduction 2007 22(1):201-209; doi:10.1093/humrep/del357
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Morphology assessment and fluorescence in situ hybridization of the same spermatozoon using a computerized cell-scanning system
1 Infertility and IVF Unit Assaf Harofeh Medical Center, Tel-Aviv University, Zerifin and 2 BioView Ltd, Nes Ziona, Israel
3 To whom correspondence should be addressed at: Infertility and IVF Unit Assaf Harofeh Medical Center, Tel-Aviv University, Zerifin 70300, Israel. E-mail: dvoras{at}asaf.health.gov.il
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Abstract |
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BACKGROUND: Poor sperm morphology is statistically associated with an increase in the incidence of chromosome abnormalities. Our aim was to examine the possible correlation between chromosomal aberrations and sperm morphology in the same cell. METHODS: 12349 spermatozoa from 7 teratozoospermic and one globozoospermic patients, and from 3 fertile donors were analyzed using a system which scans for cell morphology and chromosomal ploidy in the same cell using digital technology. RESULTS: Chromosomal aberrations were detected in 5.3% of teratozoospermic cases and in 6.7% in the globozoospermic patient compared with 1.6% in donors (P < 0.0001). Chromosomal aberrations were more common in abnormally formed sperm compared with normal spermatozoa: 4.5% vs 1.3% in the teratozoospermic group and 2.0% vs 0.3% in the control group (NS), especially frequent among sperm with two heads or two tails (52.1–77.2%) or extreme head deformations (10.6–11.1%) irrespective of grouping, and in mild amorphous heads in the globozoospermic patients (20.2%). The frequency of chromosomal aberrations in morphologically normal sperm was comparable whether derived from teratozoospermic or normospermic patients. CONCLUSIONS: The computerized cell-scanning system demonstrated the relationship between chromosomal aberrations and sperm morphology in the same spermatozoon. The incidence of chromosomal aberrations was positively linked to abnormal sperm morphology, the more severe the abnormality, the higher the incidence of aneuploidy.
Key words: aneuploidy/fluorescence in situ hybridization/globozoospermia/morphological abnormalities/teratozoospermia
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Introduction |
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Recent studies have shown that infertile teratozoospermic men produce more cytogenetically abnormal spermatozoa than fertile men, despite a normal somatic karyotype (Egozcue et al., 1997
; Shi and Martin, 2001; Kahraman et al., 2004). Control of chromosome segregation during cell division could be dysregulated because of an altered intratesticular environment (Rives et al., 1999). Thus, severely teratozoospermic men may be at increased risk of producing aneuploid offspring (Lee et al., 1996; Bernardini et al., 1998; Obasaju et al., 1999; Pang et al., 1999; Pfeffer et al., 1999; Calogero et al., 2001, 2003; Templado et al., 2002). Patients with absolute teratozoospermia (0% normal forms) had a significantly higher incidence of aneuploidy in their sperm cells. This association was proved to be especially robust between sex chromosomes and spermatozoa with abnormal head morphology: enlarged heads (Yurov et al., 1996; Vicari et al., 2003), macrocephalic sperm head (Devillard et al., 2002; Lewis-Jones et al., 2003) or multiple tails (In’t Veld et al., 1997). The association of sperm morphology with chromosome aberrations has not been systematically examined in lesser degrees of teratozoospermia, and correlation between chromosomal content and morphology in individual spermatozoon has been fraught with technical difficulty. (Lee et al., 1996; Yurov et al., 1996; Bernardini et al., 1997; Almeida Santos et al., 2002; Martin et al., 2003). To ultimately improve sperm selection for successful ICSI, we endeavoured to examine the potential relationship between chromosomal aberrations and morphology in the same spermatozoon using a new computerized cell-scanning system.
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Materials and methods |
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Patients
Fifty infertile couples with normal peripheral blood karyotype were referred to our IVF centre during January–July 2003. Male patients provided semen samples, which were analysed for concentration and motility according to the World Health Organization (WHO, 1992) parameters. Morphology was performed according to Kruger’s strict criteria (Kruger, 1998). Semen volume of
1 ml, sperm concentration of 20 x 106/ml, 50% motile sperm and 14% of sperm with normal morphology were regarded as normal. Eight of the 50 patients whose sperm analysis had normal concentration and motility, but <7% normal forms, were recruited for the study. Seven of these had isolated teratozoospermia, with mean volume of 3.3 ± 0.5 ml, mean concentration of 83.8 ±(SD) 58.7 x 106/ml, total progressive motility 67.5 ± 23.4% and mean percentage fraction of sperm with normal forms 5.0 ± 2.0%. The eighth patient had globozoospermia, although concentration and motility were in normal ranges. Samples from three donors to our sperm bank were used as controls. They had normal semen analysis, with mean semen volume of 2.7 ± 1.2 ml, sperm concentration of 75.3 ± 18.3 x 106/ml, 65.0 ± 4.0% motile sperm and 12.0 ± 2.1% normal forms (Kruger’s criteria).
Assay characteristics
The method employed an automated cell-scanning system originally designed for diagnosis and follow-up of pathological cells in haematological diseases, which enables multisequential staining of cell preparations (DuetTM, BioView Ltd, Nes-Ziona, Israel). This method provides large-scale automated bright field and fluorescence scanning enabling simultaneous classification of morphology and fluorescence in situ hybridization (FISH) in the same cell, for many cells.
Bright field scanning procedure
Sperm cells from centrifuged native ejaculate of each individual were stained by eosin–nigrosin (Sigma, Israel) and then smeared on glass slides (Super Frost Plus, Meuzel-Glaser, Germany). Each slide was first analysed and scanned automatically by bright field microscopy using x20 or x40 objectives. Coordinates and images of cells confined to an area of a circle of 900-µm radius found on the slide were digitally recorded for future reference during the next phases of analysis.
One experienced biologist observed all cells for their viability and morphology and classified them according to Kruger’s criteria: a normal form was defined as a smooth, oval head measuring 3–5 µm in length and 2–3 µm in width with a well-defined acrosome comprising 40–70% of the sperm head, neck, midpiece and tail with no defects. ‘Round forms’ had various diameters: from 2 to 8 µm. ‘Amorphous head’ included sperm forms with mild head deformations. ‘Extremely amorphous head’ included macro or microcephalic head and cells including nuclear vacuoles. ‘Elongated head forms’ included those with long-post acrosome sections or tapering heads. ‘Midpiece defects’ were defined as cells with a rim of cytoplasm or a broken neck. Cells with ‘tail abnormalities’ had coiled tail defect or kinked tail. ‘Polyploid cells’ were those with two heads, two tails or both. Our laboratory’s standard for accuracy in morphology is compatible with international standards as examined in an international survey conducted by Comhaire et al. (1994), in which the correlation between participating laboratories was found to be statistically significant (r = 0.69, P < 00.1).
Destaining
The eosin–nigrosin staining was removed after immersion in ice-cold methanol/acetic acid 3:1 solution for 1 h and washed with phosphate-buffered saline (PBS). Slides were washed in 2x standard saline citrate (SSC) solution and incubated for 5 min in 1 mol/l Tris buffer, pH 9.5, containing 25-mmol/l dithiothreitol (DTT) (Martini et al., 1995). After decondensation, the slides were washed once in 2x SSC, once in 1x PBS and finally dehydrated through an ethanol series and air-dried. This decondensation treatment is well accepted, allows the maintenance of sperm structure and permits unequivocal differentiation between spermatozoa and other cells present in the ejaculate.
FISH
In situ hybridization using chromosome X centromeric sequence probe (Spectrum x Aqua; Vysis, Downer’s Grove, IL, USA), chromosome Y satellite III sequence probe (Spectrum Orange; Vysis) and chromosome 18 centromeric sequence probe (Spectrum x Green; Vysis) was performed on the same slide according to Harper et al. (1994). Sex chromosomes together with an 18 autosome were studied because trisomies 13,18,21 together with sex chromosome aneuploidy are the most common numerical chromosomal abnormalities in live human births (Nielsen and Wohlert, 1991; Vegetti et al., 2000). Slides and probes were co-denatured at 74°C for 5 min and hybridized for 2 h at 37°C using a HYBriteTM system (Vysis). Washings were performed for 5 min with 60% formamide/2x SSC and 5 min with 2x SSC at 42°C followed by two additional 5-min washes at room temperature with 0.001% NP-40/2x SSC. Finally, slides were mounted in Blue View Counter stain (Bio-BlueTM) (BioView Ltd).
Fluorescence scanning
Slides after FISH procedure were searched by Bio ViewTM automatically for target cells with fluorescence signals. One embryologist (D.S.) reconfirmed the results. In cases of unclear FISH signals, a geneticist (M.R.) confirmed the interpretation of the embryologist. Fluorescent illumination was achieved by a standard 100-W mercury lamp (HBO103/W2; Osram, Munchen, Germany), a triple colour filter (V6200; Chroma, Brattle-boro, USA) and an aqua colour filter (61008; Chroma, Cc/YFP/dsR).
The high-speed automated scanning (
5000 cells/h) was performed in full colour using x63 objective (0.16 m per pixel). A colour camera enabled the system to snap a single image per cell, which contained all its colour details, which was analysed without further digital processing. The system was programmed to search for cells presenting the specific chromosomal normality and abnormality associated with sperm ploidy. Cells were searched for and targeted. Slides with a hybridization rate >98% were analysed with few exceptions, including only intact spermatozoa bearing a similar degree of decondensation. Disrupted or overlapping spermatozoa were excluded from the analysis. Spermatozoa 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.
Only clear hybridization signals, similar in size, separated from each other by at least one signal domain and clearly positioned within the sperm head were considered. Because the system gave a combined picture of four separate images taken from four different planes with a difference of 0.6 µm between each of them, 2.4 µm was screened which covered all the possibilities for a signal.
Divided (split) signals were not scored as disomies. Spermatozoa were scored as nullisomic if they showed no signal for a given chromosome, whereas the signal of the other chromosomes tested is present. Finally, a spermatozoon was considered diploid if it manifested two signals for each tested chromosome. The lack of FISH signals in a spermatozoon head that was positive for Bio-BlueTM stain was considered a case of ‘no hybridization’.
Combined analysis of morphology and FISH
A pair of images was presented on a screen for each cell. Cells could be observed by the combination of morphology and FISH. For example, the system could search automatically for host XX or XY cells, and then the morphology of each host cell could be shown in parallel. The system could also search automatically or manually for specific cell types for their ploidy as demonstrated by figure 1.
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Statistical analysis
Statistical evaluation using one-way analysis of variance (ANOVA) was performed after ARCSIN
P (where P is the proportion) transformation to come closer to a normal distribution. Fisher’s 
2 exact test was used where appropriate. A P-value of 0.05 was considered statistically significant. |
Results |
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A total of 5500 individual sperm from seven teratozoospermic patients, 2500 from one globozoospermic patient and 7000 from the three fertile donors (controls) were automatically scanned. In 4511 (82.0%), 2138 (85.5%) and 5700 (81.4%) cells, respectively, morphology and ploidy of each cell were determined based on the corresponding bright field and fluorescent images. A mean (±SD) of 644.0 ± 365.0 sperm nuclei per slide was scored for teratozoospermic patients, 2139.3 ± 1480.3 sperm nuclei for normal patients and 2504 nuclei per slide for globozoospermic patient.
Of 4511 sperm from the teratozoospermic patients, 223 (4.9%) had normal appearance according to bright field. In the controls, 684 (11.9%) of 5700 sperm were found to have normal morphology. No spermatozoa with normal morphology were visualized among the globozoospermic cells as shown by figure 2. Table I summarizes mean and SD of sperm morphology distribution of the sperm cell population per patient.
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Sperm morphology, performed according to computerized scanning, correlated with the morphology determined previously by the biologist at our male infertility unit in all groups. Sperm with mild amorphous head abnormalities were significantly less frequent in the teratozoospermic patients compared with the controls (45.3 ± 7.3 versus 61.7 ± 6.9). Prominent deformations were seen in 49.9% of the sperm in the teratozoospermic group. Among them were extreme amorphous head, elongated shape, midpiece defect combination, tail defect, two heads, two tails or both. Such prominent deformities were only observed in 28.4% of the sperm in the normozospermic group (P < 0.001). Spermatozoa with extreme amorphous heads were significantly more common among the teratozoospermic patients compared with the controls (6.9 ± 4.05 versus 1.9 ± 2.09%, P = 0.03). Sperm with round heads were equally frequent in the two groups but most commonly seen in the globozoospermic cases. Two heads, two tails or both were very uncommon in all instances.
Elongated head shape and midpiece deformities appeared with a similar frequency in the teratozoospermic and normal patients, whereas tail deformities were significantly more frequent in the first and not in the latter group (18.0 ± 10.1 versus 5.0 ± 3.1). No elongated shapes or tail defects were seen in the globozoospermic patient, although 6% of sperm had midpiece defects.
Pooled analysis of chromosomal aberrations disclosed 198 (4.4%) sperm cells with chromosomal disturbances of 4511 of the sperm in the teratozoospermic cases, 121 (2.0%) sperm of 5700 in the normal patients and 144 (6.7%) chromosomally defective sperm of 2138 in the globozoospermic patient. Table II summarizes the incidence of the different chromosomal aberrations including all kinds of nulisomy, disomy and diploidy when analysed per patient. There were significantly more disomic sperm among the teratozoospermic patients (P < 0.02) compared with normal patients figure 3 shows different diploid sperm from a teratozoospermic patient. In the teratozoospermic patients as well as in the globozoospermic patient, disomy rate was multiplied between 2- and 4-fold compared with nulisomy and diploidy. In total, the incidence of chromosomal aberrations was significantly higher (P < 0.04) among the sperm of the teratozoospermic patients as compared with sperm from normal donors.
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Table III lists sex and autosomal chromosomal aberrations, as they appear for each of the seven teratozoospermic patients. These abnormalities include sex chromosome disomy (24,XX: 24,YY and 24,XY), autosomal chromosome disomy (24,Y, 1818 and 24,X, 1818), sex chromosome nullisomy, 18 chromosome nullisomy and diploidy (46,XY, 1818: 46,XX, 1818 and 46YY, 1818). All appeared at random within patient groups. Ranges and SDs were relatively extreme and expressed large differences among the teratozoospermic individuals.
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Table IV summarizes the distribution of the chromosomal aberrations among the different sperm forms when analysed per patient. The incidence of chromosomal aberrations was somewhat higher in sperm with abnormal shapes as compared with that found in morphologically normal sperm (4.5 ± 4.17 versus 1.3 ± 2.15% and 2.0 ± 1.28 versus 0.3 ± 0.58%) in the terato and normozoospermic groups, respectively (not significant). When analysed by pooling all spermatozoa in each group, the differences were found to be statistically significant [P < 0.05 (Fisher’s
2 exact test)]: comparing the morphologically abnormal shapes [4.5% (195/4288)] with the normal ones [1.4% (3/223)] in the teratozoospermic group. A significant difference of P < 0.0001 was noted in aneuploidy in the normozoospermic group in the abnormal-shaped sperm [2.3% (117/5016)] as opposed to normal-shaped cells [0.6% (4/684)] (P < 0.0001). The frequency of chromosomal aberrations in morphologically normal sperm was comparable in the teratozoospermic and normospermic patients. This, however, was not the case when pooling all spermatozoa with abnormal shapes. Here, higher incidence of chromosomal aberrations was found in the teratozoospermic group compared with the controls: 4.5% (195/4288) versus 2.3% (117/5016), P < 0.0001: the highest percentage of sperm with chromosomal aberrations was found among those with two heads or two tails, no matter to what group they belonged to. Sperm with extreme head deformations had also a relatively high incidence of chromosomal abnormality (11.1 and 10.6% respectively). All other morphological pathologies were associated with a low incidence of chromosomal aberrations. The incidence of chromosomal aberrations among sperm cells with elongated head, midpiece defects and tail deformations was relatively low, with no significant differences among the different groups. In the globozoospermic patient, significantly higher percentages of chromosomal aberrations were found in all sperm shapes compared with the fertile and teratozoospermic population, with an exception for the group with two tails or two heads, where the percentages were similar to that found in teratozoospermic patients.
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Discussion |
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Numerous studies have analysed sperm aneuploidy in patients with severe male infertility, in an effort to prevent transmitting genetic abnormalities to a resulting conceptus (Engel et al., 1996
; Bonduelle et al., 1999; Lamb, 1999; Schlegel, 1999). This risk seems to be amplified using ICSI in the teratozoospermic population, where higher rates of sperm non-disjunction are found (Lee et al., 1996; In’t Veld et al., 1997; Bernardini et al., 1998; Obasaju et al., 1999; Pfeffer et al., 1999; Harkonen et al., 2001). Obtaining information concerning the correlation between sperm morphology and its genetic integrity to enable such differentiation could be extremely informative.
In the current study, a novel computerized cell-scanning system was utilized to study the potential relationship between chromosomal aberrations and sperm morphology in the same cell. Higher rates of chromosomal aberrations were found in teratoazoospermic patients including one globozoospermic patient compared with patients with normal sperm (4.4 and 6.7 versus 2.0%). Carrell et al. (2004) studied aneuploidy for X, Y, 18 and 21 chromosomes in three groups of patients. His results revealed 15.9% of sperm with aberrations for men with rare ultrastructure defects (Round-only syndrome or severe tail agenesis), 4.0% for patients with severe teratoasthenooligozoospermia syndrome and 1.2% for fertile controls.
In our study, the disomy rate was 2- to 4-fold greater than nullisomy or polyploidy in all three groups of patients. These results confirm previous data showing significantly higher rates of diploidy and disomy in sperm or sperm fractions containing high numbers of morphologically abnormal shapes (Ryu et al., 2001). This increase of disomy is logical because disomies include diverse numerical disturbances in the chromosomal complement, with either an excess or absence of the chromosomes of each pair resulting from abnormal chromosome segregation (Devillard et al., 2002).
Our aim was to elucidate which sperm population correlated with an increased rate of non-disjunction. At first, a distinction between normal and abnormal sperm shape population was carried out. When analysed by pooling all spermatozoa in each group, it was clearly demonstrated that the incidence of chromosomal aberration was higher among sperm with abnormal shapes as compared with normal ones. This was true in both the teratozoospermic and the fertile population. When analysed per patient, the difference did not reach a statistical significance because of the diversion in the teratozoospermic population and the small number of patients in the control group. Ryu et al. (2001) analysed small numbers of sperm (100–150) from infertile patients (<4% normal morphology by Kruger’s criteria) using standard haematoxylin staining, whereas the location of normal appearing sperm was recorded using an electronic microstage locator. FISH analysis of these ‘normal’ sperm showed aneuploidy in a range from 1.8 to 5.5% in the infertile group as compared with 0–2.6% in fertile controls. The mean frequency of aneuploidy among morphologically abnormal sperm was 29% in infertile cases versus 13% aneuploid sperm among the controls. The authors concluded that normal morphology is not an absolute indicator for the selection of genetically normal sperm. Our data show that the frequency of aneuploidy is greater among morphologically abnormal sperm from teratozoospermic and globozoospermic patients compared with that of sperm of a fertile population (4.5 and 6.7 versus 2.0%, respectively). However, the incidence of chromosomal non-disjunction among normally shaped cells did not show such a marked difference between the teratozoospermic and the fertile groups (1.3 versus 0.3%), respectively.
The automated cell-scanning system we used provides simultaneous evaluation of morphology and FISH in the same cell in a large cell population. This technique also enables us to investigate the distribution of genetically abnormal sperm among the subpopulations. A large group of sperm was amorphous, with mild morphological deformations; their similarity to the normal cell population might be the cause for their relatively high presence in the fertile patients group (62.7%) compared with the teratozoospermic group (47.2%). This might be also the explanation for their rare presence in the globozoospermic patient in whom even a single normal spermatozoon could not be found. The amorphous sperm population repeated the pattern of increased chromosomal aberrations in the teratozoospermic patients compared with the fertile ones.
Sperm with two heads, two tails or both (also called polyploid sperm) had the highest incidence of chromosomal abnormalities in all three groups. There are some case reports, which describe patients who suffer from absolute teratozoospermia, usually with a homogeneous microscopic pattern with a systemic sperm defect present in most spermatozoa. To this variety belong double-headed sperm, with failure of complete separation of the intracellular bridges during the division of secondary spermatocytes (Dym et al., 1971; Baccetti et al., 2001), multiple tails that fail to separate during spermatogenesis or dysfunction of centriolar microtubules (Escalier, 1983; Zamboni, 1987; Baccetti et al., 2004) and large-headed sperm (macrocephalic heads) which are thought to result from cytoplasm retention (immature forms) (Chemes et al., 1999; Viville et al., 2000; Morel et al., 2001; Devillard et al., 2002; Vicari et al., 2003). It has been suggested that a recessive gene may predispose to non-disjunction during cell division (Spriggs et al., 1996). Genetic analysis showed 90–100% correlation between these kinds of cells and their genetic disintegrity (Yurov et al., 1996; Baccetti et al., 2001; Devillard et al., 2002; Lewis-Jones et al., 2003).
In our study, the incidence of sperm with two heads, two tails or both was very low and similar in all three groups. These results might point to a coincidental disturbance rather than a genetic one. Sperm with extreme head deformations had also relatively high percentage of chromosomal aberrations. They were found in higher incidence in the teratozoospermic and globozoospermic population. Lee et al. (1996) found no increase in chromosome aberrations among spermatozoa with a small or large head, but the incidence of structural chromosomal aberrations was approximately four times higher in spermatozoa with an amorphous head than in sperm with a morphologically normal head.
The most frequently found sperm morphopathology comprises non-specific and variable anomalies in different sperm components, which have no common defect, and results from random alterations in sperm organelles and have no genetic impact (Chemes and Rawe, 2003). These anomalies include elongated head shapes, midpiece and tail defects. The incidence of chromosomal aberrations in these pathological forms among the different study groups was not significantly different because of their low prevalence. On the contrary, the incidence of chromosomal aberrations among the three study groups of sperm with mild pathology was significantly different because of their high prevalence.
The globozoospermic patient belongs to the group of systemic sperm defects, in the sense that there is a common sperm phenotype that predominates, which is associated with an extremely low potential for establishing a normal pregnancy (Zeyneloglu et al., 2002). Several studies found an increased frequency of aneuploidy involving sex chromosomes (Martin et al., 2003), aneupoidy for chromosome 15 (Carrel et al., 2001) chromatin structure anomalies (Vicari et al., 2002) and Y chromosome microdeletion. Others demonstrated that globozoospermia was not associated with sperm karyotype abnormalities (Rybouchikin et al., 1997; Vicari et al., 2002). In our study, overall sperm aneuploidy in the globozoospermic patient was rather high (6.7%); however, within the subgroup of round-headed sperm, aneuploidy was 4.7%, making it the lowest average for other abnormal shapes. The implication of these results is that microinjection of round-headed cells is preferable to that of other abnormally formed cells because these have a lower incidence of chromosomal aberrations.
In conclusion, the use of a novel computerized cell-scanning system enabled us, for the first time, to evaluate the potential relationship between chromosomal aberrations and sperm morphology in the same spermatozoon in a large sample of sperm cells. Specific morphologic abnormalities of sperm (two heads, two tails, extreme amorphous) were associated with higher chromosomal aberrations. These cells, which can easily be identified with x400 magnification, as used in an ICSI procedure, should not be used for oocyte injection. Normal shapes are not an absolute indication for a normal genetic complement; however, they show the lowest incidence of chromosomal aberrations and thus should always be preferred. Other abnormal sperm forms might be chromosomally defected especially in a teratomorphic population; however, these occur relatively rarely. Therefore, they can be used for ICSI in cases where normal sperm are not present. As for the globozoospermic patient, it seems that round-headed sperm have less chromosomal aberrations when compared with other abnormal sperm forms.
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Submitted on September 1, 2005; resubmitted on June 12, 2006; resubmitted on August 5, 2006; accepted on August 11, 2006.
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