Sperm selection for ICSI: shape properties do notpredict the absence or presence of numericalchromosomal aberrations

Ciler Celik-Ozenci1,2, Attila Jakab1, Tamas Kovacs1, Jillian Catalanotti1, Ramazan Demir2, Patricia Bray-Ward3, David Ward3 and Gabor Huszar1,4

1 The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology and 3 Department of Genetics, Yale Schoolof Medicine, New Haven, CT, USA and 2 Department of Histology and Embryology, Faculty of Medicine, Akdeniz University,07070 Antalya, Turkey

4 To whom correspondence should be addressed at: Gabor Huszar, Sperm Physiology Laboratory, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA. Email: gabor.huszar{at}yale.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: We hypothesize that the potential relationship between abnormal sperm morphology and increased frequency of numerical chromosomal aberrations is based on two attributes of diminished sperm maturity: (i) cytoplasmic retention and consequential sperm shape abnormalities; and (ii) meiotic errors caused by low levels of the HspA2 chaperone, a component of the synaptonemal complex. Because sperm morphology and aneuploidies were assessed in semen, but not in the same spermatozoa, previous studies addressing this relationship were inconclusive. We recently demonstrated that sperm shape is preserved following fluorescence in situ hybridization (FISH). Thus, we examined the shape and chromosomal aberrations in the same sperm. METHODS: We performed phase contrast microscopy and FISH, using centromeric probes for chromosomes X, Y, 10, 11 and 17 in 15 men. The fluorescence and respective phase contrast images were digitized using the Metamorph program. We studied 1286 sperm (256 disomic, 130 diploid and 900 haploid sperm) by three criteria: head and tail dimensions, head shape and Kruger strict morphology. Furthermore, in each analysis, we considered whether disomic or diploid sperm may be distinguished from haploid sperm. RESULTS: There was an overall, but not discriminative, relationship between abnormal sperm dimensions or shape and increased frequencies of numerical chromosomal aberrations. However, ~68 of the 256 disomic, and four of 130 diploid sperm showed head and tail dimensions comparable with the most normal, lowest tertile of the 900 haploid spermatozoa. Considering all 1286 sperm, among those with the most regular, symmetrical shape (n=367), there were 63 and five with disomic and diploid nuclei, respectively. In line with these findings, among the 256 disomic sperm, 10% were Kruger normal. CONCLUSIONS: Sperm dimensions or shape are not reliable attributes in selection of haploid sperm for ICSI.

Key words: FISH/ICSI/maturity/morphology/morphometry


    Introduction
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Acknowledgements
 References
 
The inception of the assisted reproduction technique ICSI provided a new approach in the treatment of men with very low motile sperm concentrations. However, ICSI as it is presently performed is far from an ideal solution because the selection of sperm is based on the judgement of an embryologist, who is looking for the most normal appearing sperm available. Thus, sperm selected for ICSI may have fragmented DNA or chromosomal impairments. Accordingly, interest in ICSI has encouraged studies directed to the association between sperm shape and chromosomal aneuploidies. The results of such studies were inconclusive because sperm morphology and aneuploidy were assessed in the same semen samples, but the two attributes were not examined in the same spermatozoon.

A solid basis for an expected relationship between sperm head shape, tail length and frequency of chromosomal aneuploidies stems from earlier studies on objective biochemical markers of sperm maturity and function. In measurements of sperm creatine kinase B isoform (CK), we found significantly higher sperm CK content in oligospermic men (Huszar et al., 1988aGo,bGo; Huszar and Vigue, 1993Go). Immunocytochemical visualization indicated that the high sperm CK content was related to increased CK and cytoplasmic protein concentrations. We proposed that the high cytoplasmic content is related to a maturational defect in terminal spermiogenesis, when the cytoplasm normally is extruded and left in the adluminal area as ‘residual bodies’. Further, there was a relationship between diminished sperm maturity, as detected by cytoplasmic retention, increased size and roundness of sperm heads and a high incidence of amorphous sperm. In diminished maturity sperm with larger heads, tail sprouting is also retarded; thus, the tail length/long head axis ratio, evaluated in these studies, is lower, and is a marker of spermiogenetic maturity (Huszar and Vigue, 1993Go; Gergely et al., 1999Go, Cayli et al., 2004Go).

In a further development related to sperm maturation, we identified HspA2, the testis-specific 70 kDa chaperone in human sperm (Huszar et al., 2000Go). The rodent homologue of HspA2, HSP70-2, was shown to be a component of the synaptonemal complex that supports chromosomal crossing-over during the meiotic process (Dix et al., 1996Go; Eddy, 1999Go). There is a two-wave expression pattern of HspA2 in human spermatogenesis. The chaperone first appears in the primary and secondary spermatocytes, most probably as a component of the synaptonemal complex. The second wave of HspA2 family expression occurs in elongating spermatids at the border of the adluminal compartment, simultaneously with cytoplasmic extrusion and plasma membrane remodelling (Huszar et al., 1997Go, 2000Go).

The expression of HspA2 during meiosis and terminal spermiogenesis suggested that sperm immaturity and low levels of constituent HspA2 may be associated with both increased frequencies of chromosomal aneuploidies and cytoplasmic retention. Indeed, in studies with the combined methods of CK immunocytochemistry and fluorescence in situ hybridization (FISH), diminished sperm maturity was related to increased frequencies of chromosomal disomies. The correlation between the proportion of immature sperm versus X, Y and 17 chromosome disomies or Y disomy was r=0.7 and 0.78, respectively (P<0.001 in both, 142 000 sperm evaluated; Kovanci et al., 2001Go). Thus, increased frequencies of chromosomal aneuploidies, sperm immaturity and abnormal sperm head shape are related (Huszar and Vigue, 1993Go; Gergely et al., 1999Go; Kovanci et al., 2001Go). The present study of head shape and numerical chromosomal aberrations in the same sperm was facilitated by our recent work showing that spermatozoa maintain their shape following the decondensation and denaturation steps that are necessary for FISH (Celik-Ozenci et al., 2003Go).

The issue of diminished sperm maturity has a major impact on sperm selection for ICSI, because severely oligospermic men have an increased proportion of immature sperm, and show a higher incidence of chromosomal aneuploidy compared with normospermic fertile men (Bernardini et al., 1997Go, 1998Go; In't Veld et al., 1997; Storeng et al., 1998Go; Templado et al., 2002Go). Due to the deficiencies of the current ‘visual’ selection of sperm, several studies have also demonstrated 4- to 5-fold increased incidences of sex chromosome aberrations in ICSI offspring (Bonduelle et al., 1998Go; Gianaroli et al., 2000Go; Van Steirteghem et al., 2002Go).

In order to examine further whether disomic or diploid sperm have some special dimensional or shape attribute features that would distinguish them from normal haploid sperm and would thus facilitate the selection of haploid sperm, we applied three approaches. (i) We performed FISH and evaluated the sperm nuclei as haploid, disomic or diploid. We sorted the haploid sperm by dimension and divided the cells into tertiles as ‘small head’, ‘intermediate head’ and ‘large head’, as well as ‘long’, ‘intermediate’ and ‘short’ tail length/long head axis ratios. Subsequently, we examined the distributions of disomic and diploid sperm in the three head and tail dimension categories. (ii) The sperm were divided into four shape groups as ‘symmetrical’, ‘asymmetrical’, ‘irregular’ and ‘amorphous’ (Celik-Ozenci et al., 2003Go), and the numbers of haploid, disomic and diploid sperm were determined within each of the groups. (iii) We determined the proportions of sperm with Kruger normal morphology within the haploid, disomic and diploid sperm cells.


    Methods and materials
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Acknowledgements
 References
 
Patient population and experimental design
Semen samples of 15 men (mean±SEM sperm concentration: 21.9±2.8 x 106 sperm/ml range 8.9–45.5; motility 45.6±1.9% range 30.7–59.8) who presented for semen analysis at the Sperm Physiology Laboratory of the Department of Obstetrics and Gynecology at Yale University School of Medicine were studied. The sperm concentrations and motilities of the semen were determined by computer-assisted semen analysis (Hamilton–Thorne Scientific Co., Beverly, MA). All studies were approved by the Human Investigation Committee of Yale School of Medicine.

The purpose of this study is to address the question of whether or not the shape properties of sperm are related to the haploid, disomic or diploid nuclei status of sperm, and whether it is possible to predict the lack of numerical chromosomal aberration in a sperm from its shape. In order to address the potential relationship between sperm shape and chromosomal aneuploidies, we pursued the following strategy. First, we performed FISH following our methods (Kovanci et al., 2001Go; Jakab et al., 2003Go). In all, 30 slides from the 15 different patients (7000 sperm from each man, a total of 210 000 sperm) were studied. We evaluated the sperm nuclei, and categorized them as haploid, disomic or diploid.

Secondly, focusing upon sperm in which the head and tail features were in the same plane and fully evaluable for dimension and shape determinations, we captured both phase-contrast images and their corresponding FISH images, and selected all conforming disomic and diploid sperm, and also haploid sperm in the same microscopic fields. In the 15 samples, 256 disomic and 130 diploid sperm were identified, and 900 haploid sperm were randomly selected (n=60 sperm from each of 15 men: n=30 probed with 17, X and Y chromosome markers, and n=30 probed for chromosomes 10 and 11).

For determination of a head dimensional relationship, the haploid sperm were sorted, and divided into three categories as ‘small head’, ‘intermediate head’ and ‘large head’. Subsequently, we determined the proportion of the disomic and diploid sperm that showed dimensions comparable with the most normal ‘small head’ group. In order to evaluate whether the tail length/long head axis ratio can be helpful in distinguishing haploid sperm, we sorted the haploid sperm into tertiles as ‘long tail ratio’, ‘intermediate tail ratio’ and ‘short tail ratio’ groups, and determined the proportion of disomic or diploid sperm that were comparable with the tail length/long head axis ratios of that of these three groups. Finally, in the study of a potential shape relationship, we classified each sperm as symmetrical, asymmetrical, irregular or amorphous (Celik-Ozenci et al., 2003Go), and determined the distributions of haploid, disomic and diploid sperm within the four sperm shape categories.

Thirdly, using the phase contrast images, two investigators have independently assessed the 1286 sperm for Kruger strict morphology scores.

FISH experiments
Preparation of slides. Aliquots (7–10 µl) of liquefied neat semen were smeared on slides, which were then fixed with methanol:acetic acid (3:1 ratio) for 10 min, air-dried, dehydrated in a series of 70, 80 and 100% ethanol, and stored at –20°C until FISH was performed.

Preparation of sperm nuclei for FISH. For decondensation, the sperm slides were warmed to room temperature and, in order to render the sperm chromatin accessible to DNA probes, were first treated with 10 mmol/l dithiothreitol (DTT; Sigma) in 0.1 mol/l Tris–HCl pH 8.0, for 30 min, and then with 10 mmol/l lithium diiodosalicylate (LIS; Sigma) in Tris–HCl for 1–3 h.

DNA probes. The FISH studies were carried out using five probes: (i) a 20 kb repeated family probe assigned to the Xp11–Xp21 region of chromosome X (pXBR-1; Yang et al., 1982Go); (ii) microdissected probes for the Y chromosome (Guan et al., 1996Go); (iii) (3–5) {alpha}-satellite sequence-specific centromeric probes for chromosome 17 (p17H8; Waye and Willard, 1986Go); (iv) chromosome 10 (p{alpha}10RP8; Devilee et al., 1988Go); and (v) chromosome 11 (pLC11A; Waye et al., 1987Go). The DNA probes were labelled indirectly with hapten-conjugated nucleotide (biotin-11-dUTP for chromosome 10, 17 and X probes, or digoxigenin-11-dUTP for chromosome 11, 17 and Y probes) by nick translation (Rigby et al., 1977Go), and added to metaphase chromosome spreads to develop optimal conditions for hybridization.

FISH. Since three probes are necessary to study the frequencies of disomy and diploidy in the sex chromosomes, multicolour FISH was performed when chromosomes X, Y and 17 were hybridized together (X-Y-17 assay). In order to detect the frequency of autosomal disomy and diploidy using chromosome 10 and 11 probes, two-colour FISH was utilized (10-11 assay). The hybridization mixture containing the probes of either multicolour or two-colour assays was denatured at 75–80°C for 8 min and applied to the slide specimens previously denatured in 70% formamide, 2 x SSC for 8 min at 70°C. Hybridization was carried out at 37°C in a moist chamber for 12–14 h. Post-hybridization washes were performed with 50% formamide/2 x SSC three times at 42°C and another three times with 0.1 x SSC at 60°C in order to remove excess probe reagents. After a blocking step in 4 x SSC/3% bovine serum albumin/0.1% Tween-20 for 30 min at 37°C, the sperm nuclei were incubated for 30 min at 37°C with avidin–fluorescein isothiocyanate (FITC) (fluorescence green, Roche Biochemicals, Indianapolis, IN) for biotin-labelled probes and anti-digoxigenin–rhodamine (fluorescence red) for digoxigenin-labelled probes. After 5 min staining with 4,6' diamidino-2-phenylindole (DAPI; Sigma), slides were mounted with an antifade solution (Vectashield; Vector Laboratories).

Scoring criteria and data collection. For each patient, two slides of the initial sperm fractions were scored [one for the evaluation of 17, X and Y chromosomes (triple-probe FISH) and the other for the evaluation of 10 and 11 chromosomes (double-probe FISH)]. The overall hybridization efficiency in these experiments was >98%. Sperm nuclei were scored according to published criteria (Martin and Rademaker, 1995Go). A spermatozoon was considered disomic when it showed two fluorescent domains of the same colour, comparable in size and brightness in approximately the same focal plane, clearly positioned inside the edge of the sperm head and at least one domain apart. Diploidy was recognized by the presence of two double fluorescence domains with the above criteria. Scoring was performed on an Olympus AX70 epifluorescence microscope primarily with the triple pass filter for DAPI, FITC and rhodamine signals together (Chroma Technologies Co., Brattleboro, VT). For improved signal resolution and distinction, the signals on sperm were also scored with monochrome filters for DAPI, FITC and rhodamine, separately. Disomic or diploid spermatozoa were always examined with all filters, and also with a phase-contrast objective in order to verify the presence of the tail and to exclude apparent diploidy in two spermatozoa in close proximity.

Sperm shape determinations
Computerized morphometry measurements. Morphometry studies were carried out by the computer-based MetamorphTM program (Universal Imaging Corp., Downingtown, PA). These methods were described in detail recently (Celik-Ozenci et al., 2003Go).

Briefly, phase-contrast and fluorescent images of the FISH-performed sperm slides were digitized, and then the phase-contrast images were used to carry out morphometry. After digitizing the images, MetamorphTM overlay tools were used to delineate the head versus tail regions of individual spermatozoa in order to measure head and tail parameters separately. For the assessment of sperm head parameters, we measured area, perimeter, long head axis and short head axis. The sperm tail parameters were assessed by measuring tail length. In addition, the sperm parameter tail length/long head axis ratio, which closely reflects sperm cellular maturity, was also evaluated (Gergely et al., 1999Go). Calibration of the dimensions was performed by viewing an objective micrometer scale (OB-M 1/100) at 100 x magnification and digitizing the images with the MetamorphTM program. The automated, computerized conversion of pixels to µm was 0.13 µm/pixel.

Kruger strict morphology. In each phase-contrast image, haploid, disomic and diploid sperm were scored by two different investigators in a blinded manner, according to the Kruger criteria (Menkweld et al., 1990Go; WHO Laboratory Manual, 1999Go). Subsequently, the results were averaged for each of the sperm nuclei categories. We evaluated the sperm head, neck, midpiece and tail. Sperm that were considered Kruger normal displayed oval-shaped heads, symmetrical tail insertions and no visible indications of neck or midpiece defects.

Statistical analysis
In order to compare the various sperm morphometry attributes between the haploid, disomic and diploid sperm, we used the SigmaStat statistical program (Jandel Scientific Corporation, San Rafael, CA) and performed one-way ANOVA analysis on normally distributed data, and the one-way ANOVA on ranks test on data that were not normally distributed. Following ANOVA tests, post hoc Dunn' or Tukey tests of all pair-wise comparisons were applied to distinguish between the multiple comparisons. The {chi}2 test was used to compare the distributions of haploid, disomic and diploid sperm in the different dimensional categories. All data are presented as mean±SEM. Level of significance was selected as P<0.05.


    Results
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Acknowledgements
 References
 
Classification by shape: dimensions of haploid, disomic and diploid spermatozoa
We performed objective morphometry using the various Metamorph probes on 900 haploid, 256 disomic and 130 diploid spermatozoa, and compared the similarities and differences of the haploid, disomic and diploid sperm using the one-way ANOVA on ranks and post hoc Dunn' tests (Table I). There were significant differences in nearly all pair-wise comparisons of total head area, perimeter, long axis, short axis and the tail length/long head axis ratios among the haploid, disomic and diploid sperm. Mean values for tail length were not significantly different between haploid and disomic sperm; however, the tails of diploid sperm were significantly shorter than those of haploid sperm. Similar patterns of smaller differences between haploid and disomic as compared with between haploid and diploid sperm were also evident in the head measurements of area, perimeter, long axis and short axis. This indicated that disomic and diploid sperm have larger heads than the haploid sperm. However, these differences also suggest that, although one cannot reliably distinguish between disomic and haploid sperm in ICSI sperm selection based on head size, there is a higher likelihood of avoiding diploid sperm.


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Table I. Dimensions of the haploid, disomic and diploid spermatozoa (all values mean±SEM, n=15 men, 1286 sperm)

 
Dimensional characteristics of haploid, disomic and diploid spermatozoa
In the next approach, we sorted and divided the 900 haploid sperm into three tertiles according to the area of the sperm as ‘small head’, ‘intermediate head’ and ‘large head’ groups. Further, we examined the 256 disomic and 130 diploid sperm, and determined the proportion of sperm with disomies and diploidies that are similar in dimensions to that of the haploid sperm within each of the three size groups (Table II). There were disomic sperm similar in dimensions to all three ‘small’, ‘intermediate’ and ‘large’ sperm head categories (68, 59 and 129). Diploid sperm were also present in all three groups (four, 10 and 116). The frequencies of both disomies and diploidies were significantly higher in the ‘large head’ category compared with the ‘small’ and ‘intermediate’ heads in any of the four sperm head attributes (P<0.001). Head size reflects diminished maturity and cytoplasmic retention, thus there is an overall maturity-related association in sperm shape and disomies or diploidies. However, sperm of any head size or shape may have chromosomal aberrations. Furthermore, ~68 sperm with disomy and four sperm with diploidy were comparable in head size with the ‘small head’ haploid group.


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Table II. Distribution of the 256 disomic and 130 diploid sperm within the sperm head dimension tertiles

 
Classification by tail/long head axis ratio
Because we have established previously that the sperm tail length/long head axis ratio reliably reflects sperm maturity (Gergely et al., 1999Go), we sorted and divided the 900 haploid sperm into three tertiles as ‘long tail ratio’, ‘intermediate tail ratio’ and ‘small tail ratio’ and compared the tail length/long head axis ratios of disomic and diploid sperm in order to determine whether sperm with disomies and diploidies would occur preferentially within one of the groups of haploid sperm (Table III). There were disomic and diploid sperm with ratios comparable with all of the three tail length/long head axis ratio groups of haploid sperm, with a distribution of 60, 67 and 129 for disomies, and six, seven and 117 for diploidies.


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Table III. Distribution of the 256 disomic and 130 diploid sperm within the shape categories according to their tail length/long head axis ratio

 
Shape characteristics of the haploid, disomic and diploid spermatozoa
In yet another approach, we classified the 1286 sperm according to their sperm head shape characteristics as symmetrical (n=367), asymmetrical (n=368), irregular (n=504) and amorphous (n=47), a classification system we have used previously (Celik-Ozenci et al., 2003Go). As with the dimensional categories, disomic and diploid sperm were present in all four shape groups, with increasing proportions from the smaller to the larger classifications (Table IV). Thus, frequencies of disomies and diploidies were reflected by the severity of sperm shape abnormality, with a significantly higher frequency of aneuploidies in the irregular and amorphous sperm groups as compared with those in the symmetrical and asymmetrical categories (P<0.001). However, even the most ‘normal appearing sperm’ in the symmetrical category of 367 sperm included 68 sperm (of the total 386 disomic and diploid sperm) with numerical chromosomal aberrations. We can conclude that neither size or shape assessment is a reliable method for selection of haploid sperm.


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Table IV. Proportion of disomic and diploid sperm within each morphological category

 
Results of Kruger strict morphology and FISH
The normal strict morphology scores of the haploid (n=900), disomic (n=256) and diploid (n=130) sperm were 24, 10 and 1%, respectively. These values are also in accordance with the morphometric results, which indicate that the haploid, disomic and diploid sperm are different from each other, not only in genetic or morphometric aspects but also in morphology. In another approach, we also substantiated our sperm shape classification method with Kruger strict morphology classification. The Kruger normal scores in the symmetrical, asymmetrical, irregular and amorphous groups were 26, 3, 1 and 0%, respectively.


    Discussion
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Acknowledgements
 References
 
The potential relationship between sperm shape and genetic integrity has became very pertinent with the introduction of ICSI, because ICSI sperm selection is not based on sperm–zona pellucida interaction as in conventional conception, but on visual assessment of sperm shape by the embryologist. There are concerns about ICSI conception because immature spermatozoa are prevalent in men who are candidates for ICSI. The concerns were justified further as men with oligozoo/asthenozoo/teratozoospermia were shown to have significantly elevated levels of sperm numerical chromosomal aberrations, reactive oxygen species (ROS) production and DNA chain fragmentation (Huszar and Vigue, 1993Go, 1994Go; Aitken et al., 1994Go; Bernardini et al., 1997Go, 1998Go; Rubes et al., 1998Go; Twigg et al., 1998Go; Sakkas et al., 1999Go; Griffin et al., 2003Go). Because spermatozoa of diminished fertility and genetic integrity are not part of the conventional fertilization pool, but may be selected for ICSI, several investigators, but primarily the Van Steirteghem group, initiated follow-up studies on children born after ICSI. ICSI conception caused an increase in the rate of de novo abnormalities, autosomal chromosomal aneuploidies and structural aberrations (Bonduelle et al., 1994Go, 1998Go, 2003Go; Gianaroli et al., 2000Go; Van Steirteghem et al., 2002Go).

Several recent papers have dealt with semen samples of severe oligozospermia and/or high incidence of abnormal sperm morphology, both of which are indications of sperm immaturity. Storeng et al. (1998)Go studied aneuploidy rates in 19 men who were assigned to IVF or ICSI, based on their higher and lower sperm concentrations. The overall disomy rates, although different in absolute values, were ~20-fold higher in the ICSI group than in the IVF group. The extremely high rate of aneuploidies in men who have few sperm, and thus are likely to have a high proportion of immature sperm, was also documented by In't Veld et al. (1997)Go and, in oligozoospermic samples (higher incidence of immature sperm), there were significantly higher frequencies of sperm aneuploidies (Bernardini et al., 1998Go). Indeed, directly related to this issue are the data from our laboratory that indicate a close correlation between sperm immaturity, as detected by cytoplasmic retention, and frequency of chromosomal disomies (r=0.7–0.78; Kovanci et al., 2001Go). Further, we also found a relationship between arrested sperm maturation and infertility, as low levels of HspA2 expression predicted lack of pregnancy success after intrauterine insemination or IVF treatments (Huszar and Vigue, 1990Go; Huszar et al., 1992Go; Ergur et al., 2002Go).

The present experiments were carried out along four lines of assessment. First, we studied the sperm head dimensions that gradually increased from haploid to diploid sperm, as size reflects immaturity and cytoplasmic retention. Secondly, there was a relationship between disomic, and particularly diploid sperm, showing a higher preponderance of these abnormal sperm in the ‘large group’. However, sperm with numerical chromosomal aberrations were present within all three groups. Indeed, from the perspective of ICSI sperm selection, it is of interest that 68 of the disomic and four of the diploid sperm were in the ‘small head’ category, which would be most likely to include those selected for ICSI. Conversely, the tail length/long head axis ratio sperm maturity marker has also failed to discriminate. In the ‘long tail’ sperm group of >8.7 tail length/long head axis ratio, there were 67 disomic and six diploid sperm.

In the third approach, we classified the 1286 sperm according to their shape, and examined the distribution of disomic and diploid spermatozoa (Celik-Ozenci et al., 2003Go). As with the size classification above, all four, symmetrical, asymmetrical, irregular and amorphous, sperm shape groups included disomic and diploid nuclei, and the incidences of sperm nuclei with disomy and diploidy within each morphological category increased from normal to amorphous, reflecting the relationship between sperm shape and maturity. Indeed, even in the normal symmetrical group, we identified 63 disomic and five diploid spermatozoa. Fourthly, the evaluation of sperm by strict criteria also failed to show a relationship between sperm shape and numerical chromosomal aberrations. Although the fact that shape properties are not characteristic for haploid or aneuploid sperm is likely to be generally true, it is of note that the data and values of this report are specific for the population of these particular 15 men, 1286 sperm and five chromosome probes.

Another consideration is the fact that the proportions of sperm with diminished maturity is variable from man to man. In general, this proportion declines as the sperm concentration rises. However, the data in the present paper, in agreement with previous cytoplasmic retention and aneuploidy frequency findings (Kovanci et al, 2001Go), indicate that there is no sperm concentration or total motile sperm range that would ensure that no immature sperm would occur in a semen sample.

The association between abnormal sperm shape and increased frequency of aneuploidies has been studied previously by others, with inconsistent conclusions, most probably because the sperm attributes were evaluated in the same semen sample, but not in the same sperm. Bernardini et al. (1998)Go suggested a relationship between increased frequencies of aneuploidy and diploidy in semen samples containing spermatozoa with enlarged heads. Several other studies have concluded that morphologically abnormal sperm may also have a significantly increased risk for being aneuploid (Colombero et al., 1999Go; Calogero et al., 2001Go; Rubio et al., 2001Go; Yakin and Kahraman, 2001Go; Templado et al., 2002Go). An interesting report, based on examination of sperm injected into mouse oocytes, suggested that in semen samples with high incidences of amorphous, round and elongated sperm heads, there was an increased proportion of structural chromosome abnormalities, such as chromosome and chromatid fragments, dicentric and ring chromosomes, but no increase in numerical chromosomal aberrations (Lee et al., 1996Go). Further, Ryu et al. (2001)Go studied ~120 Kruger normal and abnormal sperm in eight men each, and concluded that normal morphology is not a valid indicator for selection of sperm with haploid nuclei. Rives et al. (1999)Go showed that although the disomy frequencies of infertile males were directly related to the severity of oligospermia, there was no relationship between aneuploidy frequency and abnormal morphology. In men with increased levels of globozoospermia, shortened flagella syndrome or sperm with acrosomal abnormalities, no association was found between sperm shape and numerical chromosomal aberration (Viville et al., 2000Go).

Our laboratory is developing a new approach for ICSI sperm selection based on the relationship between sperm maturation and plasma membrane remodelling in spermiogenesis (Huszar et al., 1997Go, 2003Go). Because normal sperm cellular maturation and plasma membrane remodelling facilitate the formation of the receptors for hyaluronic acid, along with those for the zona pellucida, sperm that bind to immobilized hyaluronic acid may be considered normally developed. Indeed, both the frequency of chromosomal aberrations and DNA integrity (lack of DNA chain fragmentation) in such hyaluronic acid-selected sperm were within the range of normal men (Kovanci et al., 2001Go; Cayli et al., 2003Go; Jakab et al., 2003Go).

In summary, we studied the relationship between sperm shape and numerical chromosomal aberrations in individual spermatozoa, using FISH, objective morphometry, sperm dimension and shape assessment, along with Kruger strict morphology. The results indicate that numerical chromosomal aberrations can be present in sperm heads of any size or shape, but the risk is greater with amorphous sperm. Even the most normal appearing sperm with normal head and tail size could be disomic or diploid, although diploidy is less prevalent with normal sperm dimensions and shape. Thus, we concluded that visual assessment is an unreliable method for ICSI selection of sperm. More specific methods for sperm selection, such as hyaluronic acid binding, will alleviate the problem of fertilization with sperm of diminished maturity and genetic integrity.


    Acknowledgements
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Acknowledgements
 References
 
The authors gratefully acknowledge the help of G.Leyla Sati, MS, in the preparation of the manuscript. This study was a part of C.C.-O.'s PhD thesis. Part of this research was presented at the 18th Annual Meeting of ESHRE, 2002, Vienna, Austria. This work was supported by the NIH (OH-04061, HD-19505)


    References
 Top
 Abstract
 Introduction
 Methods and materials
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on April 21, 2004; resubmitted on May 11, 2004; accepted on May 18, 2004.





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