Sperm aneuploidies and low progressive motility

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1 Human Reproduction Vol.22, No.7 pp , 2007 doi: /humrep/dem099 Sperm aneuploidies and low progressive motility G.Collodel 1,2,4, S.Capitani 2,3, B.Baccetti 1,2, A.Pammolli 1 and E.Moretti 1,2 1 Department of Surgery, Biology Section, Siena University, Policlinico Le Scotte, Viale Bracci 14, Siena, Italy; 2 Interdepartmental Centre for Research and Therapy of Male Infertility, University of Siena, Siena, Italy; 3 Department of Physiopathology, Experimental Medicine and Public Health, University of Siena, Siena, Italy 4 Correspondence address. Tel: ; Fax: ; collodel@unisi.it BACKGROUND: Patients with poor semen quality show increased sperm disomy and diploidy rates. Oligozoospermia and teratozoospermia are known to influence sperm aneuploidy, but there is still a debate about whether aneuploidies are associated with reduced motility. METHODS: Ejaculates from a large group of patients were examined by light microscopy to evaluate sperm concentration, motility and morphology, and by fluorescence in-situ hybridization (FISH) to analyse the presence of aneuploidies. Statistical analysis was performed to compare differences and to evaluate the relationship between sperm aneuploidy rate and semen quality. RESULTS: Five groups were established following the motility parameter, and total aneuploidy rates were statistically significantly higher in the groups where motility was <30% compared to the controls. A homogeneous group of men with asthenozoospermia showed higher FISH values compared to control data, although the difference was not statistically significant. Motility and sperm morphology were each found to be statistically related to aneuploidy using a multiple linear regression analysis, whereas sperm concentration was only related to aneuploidy by the equation of a hyperbolic curve. CONCLUSIONS: In conclusion, biological and statistical data from the present research support the idea that the presence of aneuploidies could also be associated with reduced sperm motility. Keywords: aneuploidies; FISH; sperm motility; semen quality Introduction The analysis of sperm motility plays a central role in the evaluation of male fertility, as it is known that a high percentage of poorly motile or immotile sperm will not be able to fertilize. Several conditions are known to impair human sperm motility, such as antisperm antibodies, infections of genital tract, anatomical pathologies, metabolic defects and structural phenotypic and genotypic defects (Baccetti et al., 2001; Chemes and Rawe, 2003). Asthenozoospermia negatively influences fertility prognosis both in spontaneous conditions and with the use of assisted reproductive techniques (ART). Intracytoplasmic sperm injection (ICSI) was a revolutionary advance in the treatment of male infertility, until it became clear that abnormal and immotile spermatozoa could successfully fertilize oocytes, and questions were raised about the suitability of using them in ART. The literature has already verified that patients with poor semen quality show an increased rate of sperm disomy and diploidy (Shi and Martin, 2001; Gianaroli et al., 2005; Baccetti et al., 2006). The influences of both severe oligozoospermia and teratozoospermia on sperm aneuploidy are generally accepted (Egozcue et al., 2000; Templado et al., 2002; Machev et al., 2005; Miharu, 2005); whether sperm aneuploidy might also occur in cases of isolated asthenozoospermia is still being debated (Vegetti et al., 2000; Hristova et al., 2002; Bernardini et al., 2005; Rives, 2005). An increased presence of aneuploidy in cases of severe asthenozoospermia was found by Baccetti et al. (2005) in spermatozoa from a group of patients affected by the genetic sperm defect of dysplasia of the fibrous sheath and in a man with Kartagener syndrome (Rives et al., 2005). These data add to the growing evidence that sperm anomalies of flagella occur along with increased rates of sperm aneuploidies (Sun et al., 2006). However, in phenotypic sperm alterations it is often difficult to correlate the presence of aneuploidy with reduced motility because asthenozoospermia is often concomitant with oligo and/or teratozoospermia. These conditions are frequently associated with infertility and ICSI is the treatment of choice, and for this reason many concerns have been raised about the potential increased risk of transmission of chromosomal aneuploidy to offspring (Bonduelle et al., 1998, 2002). In this study, a large-scale investigation was designed to contribute to elucidating the association between motility and sperm aneuploidies. During the years , the ejaculates from patients recruited at the Interdepartmental Centre for Research and # The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 1893

2 Collodel et al. Therapy of Male Infertility were examined. Semen samples were analysed by light microscopy to evaluate sperm concentration, motility and morphology. Finally, an association between reduced motility and meiotic chromosome segregation was investigated by fluorescence in-situ hybridization (FISH), performed directly on sperm nuclei. Materials and Methods Patients Between January 2000 and April 2006, 254 patients were recruited at the Interdepartmental Centre for Research and Therapy of Male Infertility for semen analysis and FISH was performed on their sperm nuclei. We selected 131 men (aged years) using the following exclusion criteria: patients with severe anatomical pathologies related to fertility, hormonal imbalance, uro-genital infections and carriers of genetic sperm defects. The ultrastructural genetic sperm defects were confirmed by transmission electron microscopy analysis. Only patients with an apparently normal 46, XY karyotype were included in this study. In patients with a sperm concentration lower than , PCR analysis on DNA extracted from blood lymphocytes was carried out to exclude the presence of Y chromosome microdeletions. The patients had no history of radiotherapy, chemotherapy, chronic illness or medication, and gave informed consent for this research. Semen analysis Semen samples of patients were collected by masturbation after 4 days of sexual abstinence and examined after liquefaction for 30 min at 378C. Volume, ph, concentration, motility and morphology were evaluated according to WHO guidelines (1999). Sperm morphology was evaluated by the Papanicolaou test modified for spermatozoa. Ejaculated human spermatozoa were washed two times in PBS buffer, smeared on pre-cleaned glass slides, air dried and fixed in equal volume of ethanol 95% and ether, for 5 15 min. The fixed slides were stained using the protocol reported by WHO guidelines (1999). FISH analysis In order to evaluate aneuploidy frequency, FISH was performed according to Baccetti et al. (2003) on the sperm nuclei of the 131 selected patients. A mix of a-satellite DNA probes (CEP, Chromosome Enumeration Probes, Vysis, IL, USA) for chromosomes 18, X and Y directly labelled with different fluorochromes was used. Sperm nuclei were scored according to published criteria (Martin and Rademaker, 1995; Baccetti et al., 2003). No slide was scored unless the hybridization efficiency was greater than 98%. Sperm nuclei were scored only if they were intact, non-overlapped and had a clearly defined border. In the case of aneuploidy, the presence of a sperm tail was confirmed. A spermatozoon was considered disomic if the two fluorescent spots were of the same colour, comparable in size, shape and intensity and positioned inside the edge of the sperm head at least one domain apart. Diploidy was recognized by the presence of two double fluorescent spots following the above criteria. All samples were analysed by a highly trained examiner. A minimum of 5000 sperm was scored for each man, with a total of sperm analysed. Observation and scoring were performed using a Leitz Aristoplan Optical Microscope equipped with a fluorescence apparatus with a triple bandpass filter for aqua, orange and green fluorochromes 1894 (Vysis) and a monochrome filter for 4 0,6-Diamidino-2-phenylindole (DAPI). Controls Semen samples from seven fertile men (aged years), who had fathered a child in the past 1 2 years, were analysed and used as controls (Baccetti et al., 2003). The fertile men showed a normal karyotype and they did not present any anatomical problems, uro-genital infections or hormonal imbalances. Statistical analysis Statistical analysis was performed using the StatGraphicsPlus (vers. 5.0) statistical package. Two sides P-values of a, 0.05 were considered significant. All groups of quantitative variables were checked for normal distribution by standardized skewness and kurtosis, and the F-test was used to compare variances between the groups. The Mann Whitney test, rather than the t-test, was performed to evaluate the statistical differences between the variables when the conditions of normality of distribution and of homogeneity of variance were not satisfied. A multiple regression analysis, using interaction terms, was carried out to explain the behaviour of the aneuploidy variable in terms of predictor variables: motility and sperm concentration (continuous variables), sperm morphology (dummy variable). The normal probability plot of the deviations from linear regression and the residual plot confirmed the normality and the homogeneity of variance of the examined variables. The results of the multiple regression analysis were evaluated by removing the outliers from the model, as indicated by the Durbin Watson statistic, and using Cp Mallow statistics for the best subset. Moreover, since by simple linear regression the scatter plot and the F-value did not indicate a linear relationship between sperm concentration (x variable) and aneuploidy (y variable), the variable sperm concentration was linearized using the equation for a hyperbolic curve. The x variable was transformed into a new variable, k ¼ 1/x. After this transformation we again applied a simple linear regression. Results The seminological features of 131 selected male patients were analysed by light microscopy over a seven-year period. Rapid and slow progressive motility (a þ b) were lower compared to WHO parameters in 109 men out of 131. FISH analysis for chromosomes 18, X and Y was performed in all sperm samples in order to evaluate meiotic segregation. Based on sperm motility, we decided to establish five groups. Aneuploidy values and semen types referring to the five groups are reported in Table 1. Group 1 was composed of 9 patients with a progressive sperm motility value of between 0 5%, and of these, 5 patients showed asthenoteratozoospermia (AT) and 4 were oligoasthenoteratozoospermia (OAT). Regarding FISH analysis, an increase in the mean percentages of values of all examined chromosomes compared to controls was observed; in particular, there were statistically significant for disomies 18XX, 1818X, 18YY and for 1818XX and 1818XY diploidies (Table 1). Group 2 was composed of 11 patients showing a sperm motility ranging from 5 10%; 5 patients were AT and 6 were OAT. FISH screening was very similar to that observed in the first group with almost all disomies and diploidies out of the normal range (Table 1). Significant differences in mean percentages were reached for

3 Asthenospermia and sperm aneuploidies Table 1: Mean + SD of FISH data and semen types of 131 patients divided into five groups according to motility parameters Group Motility, % 1818XX 1818YY 1818XY 1818X 1818Y 18XX 18YY 18XY Semen types * * * ** * AT n ¼ 9 4OAT ** * ** ** ** * ** 5 AT n ¼ 11 6OAT ** ** ** ** * ** ** 23 AT n ¼ OAT 12 A 1OA ** ** ** ** ** 10 AT n ¼ 37 9OAT 17 A.50 1OA 5 n ¼ * ** O 20 N 1OA a Controls n ¼ N a Healthy men of proven fertility; WHO parameters (1999): O (oligozoospermia, sperm concentration, ); A (asthenozoospermia, motility,50%); T (teratozoospermia, morphology,30%), N (normal); *P, 0.05 and **P, XX, 1818YY and 1818XY diploidies, and 1818X, 18XX, 18YY and 18XY disomies. Group 3 consisted of 52 patients with a motility value of 10 30%. There were 23 patients who were AT and 16 were OAT, 1 patient was oligoasthenozoospermic (OA) and 12 were only asthenozoospermic (A). There was a statistically significant increase in mean percentages of 1818X disomy, sex chromosome disomies and all diploidies compared to controls (Table 1). Group 4 included 37 patients with motility ranging from 30 50%. There were 10 patients who were AT, 9 were OAT, 1 patient was OA and 17 were only A. Statistically significant increases in the mean percentages of 1818XX and 1818XY diploidies was observed, and 1818X and sex chromosome disomies was also significantly out of the normal range, except for 18YY mean percentages (Table 1). Group 5 included 22 patients with normal motility (.50%). There were 20 patients who also showed sperm concentration and morphology (N) within the ranges recommended by WHO guidelines (1999), while 1 patient was teratozoospermic (T) and 1 oligozoospermic (O). FISH screening did not highlight any statistically significant increase in the mean percentages of diploidy and disomy (Table 1) compared to controls. It was particularly interesting to find a consistent population of patients (29 individuals) showing only asthenozoospermia. Comparing the FISH mean percentages of this group with those from fertile controls, the values for the asthenozoospermic group were significantly higher for all the aneuplodies, except for 1818YY diploidy and 1818Y disomy (Table 2). We then grouped all meiotic abnormalities, as reported in Table 3. Comparing the total aneuploidy mean percentages for each category of patients versus the control group, we observed a statistically significant increase in aneuploidy values in the groups showing motility,30% (Table 3). Regarding the group with only asthenozoospermia, we found a significant increase in the mean percentages of total disomy compared to that of total diploidy (Table 3). We performed a multiple linear regression analysis of all patients in order to assess the behaviour of aneuploidy in terms of motility, sperm concentration and sperm morphology as predictor variables. We found that both motility and sperm morphology contributed to the statistically significant relationship between the aneuploidy rates and the multiple regression equation (respectively P ¼ 0.033; P ¼ 0.047), but that the sperm concentration did not (P ¼ 0.9). Cp Mallow statistics indicated a model with only two variables, corresponding to motility and sperm morphology. The F statistic was highly significant (P ¼ ) and the T statistic indicated the contribution of motility (P ¼ 0.032) and sperm morphology (P ¼ 0.036) to the statistically significant relationship between the aneuploidy rate and the multiple regression equation. The adjusted R 2 was 12.31%. Table 2: Mean + SD of FISH data from sperm of a homogeneous group of 29 patients with only altered motility (,50%) among the three sperm parameters (concentrations, motility and morphology) Group 1818XX 1818YY 1818XY 1818X 1818Y 18XX 18YY 18XY Asthenozoospermia ** * ** ** ** ** n ¼ 29 a Controls n ¼ a Healthy men of proven fertility; *P, 0.05 and **P,

4 Collodel et al. Table 3: Mean + SD of FISH data grouped as disomy, diploidy and total aneuploidies of 131 patients divided into five groups according to motility parameters, the group of selected men with isolated asthenozoospermia is also shown Group Motility, % Disomy Diploidy Total aneuploidies * * * Astheno, Controls *P, 0.05 total aneuploidies of the examined groups versus the control group and P, 0.05 total disomy versus total diploidy in the asthenospermic group. Consequently, the evaluation of the model indicated a significant linear relationship between the aneuploidy variable and the two variables of motility and sperm morphology. This implies that, given the constant sperm morphology values, the 1% increase in motility is associated with an average decrease in aneuploidy rate of 0.008% (P ¼ 0.032). Moreover, assuming that motility values did not change, the model suggested that when sperm morphology values are,30%, the aneuploidy values tend to be higher than when sperm morphology values are.30% (P ¼ 0.036). Since, from elaboration already described, a linear relationship between sperm concentration and aneuploidy was not highlighted, we used the equation of a hyperbolic curve. The transformation allowed for the linearization of the relationship between the new variable obtained and the aneuploidy variable. The subsequent application of the method of simple linear regression indicated a significant (P, 0.001), but not linear, relationship between the two variables (Fig. 1). Discussion There has been a recent increase in the literature of studies investigating whether asthenozoospermia could be associated with the presence of sperm aneuploidy (Rives, 2005). Some studies (Hristova et al., 2002; Bernardini et al., 2005) have been performed to explore whether low sperm motility is associated with increased sperm disomy and diploidy rates in Figure 1: Scatter plot of the observed data related to aneuploidy (%) and sperm concentration of 121 patients showing the number of sperm/ml Ten patients, showing a number of sperm/ml and normal values of aneuploidy, were not considered in this figure in order to highlight the data that influenced distribution 1896

5 Asthenospermia and sperm aneuploidies infertile patients, but unfortunately, up until now, it was impossible to isolate a large group of men with asthenozoospermia only. We report the results of a statistical analysis applied to a set of data obtained using FISH for chromosomes 18, X and Y in spermatozoa from 131 selected men, divided into five groups following the motility parameter, in order to highlight whether an association between motility and sperm aneuploidy could be present. Group 1 (motility 0 5%), group 2 (motility 5 10%), group 3 (motility 10 30%) and group 4 (motility 30 50%) showed similar aneuploidy values, with most of them out of range and significantly higher than controls. We found the presence of aneuploidies in all analysed groups showing motility of,50%, however, these patients did not show asthenozoospermia only, but also possibly teratozoospermia and/or oligozospermia. In group 5 (motility.50%), disomy and diploidy values were similar to those of controls. The only significant differences reported were related to 1818YY diploidy and 1818Y disomy that were significantly lower in group 5 compared to the controls. The large number of cases enrolled in this study allowed us to identify a homogeneous group composed of 29 men with asthenozoospermia only; in this group, all sex chromosomes disomies and 1818XX and 1818XY diploidies were significantly higher than control values. A later analysis of total pooled FISH data highlighted a significant increase in aneuploidy values in the groups with reduced motility (,30%) versus the control group, however the semen profile showed that, in these groups, asthenozoospermia appears frequently with oligo and teratozoospermia. When motility ranged between 30 and 50%, the total aneuploidy values were higher than controls but they did not reach statistical significance. In the selected group with asthenozoospermia only, we observed the same trend but the total disomy rates were significantly higher compared to the total diploidy rate. The clinical implication of this result could be an increased risk of sex chromosome hyperhaploidy, causing chromosomally abnormal conceptions, as observed during prenatal testing when paternally derived sex chromosome aberrations seemed to be more frequent after ICSI (Bonduelle et al., 2002). Our data are consistent with errors occurring during meiotic division in the case of asthenozoospermia, demostrating that low motility could be associated with altered sperm chromosome segregation. Although the risk of aneuploidy is higher in men with poor sperm motility, it still a low risk, as evident from aneuploidy values. It is not known whether motile/non-motile sperm are the same as those considered to be abnormal by morphology or containing aneuploid nuclei; through the use of an electronic microstage locator, it has been possible to demonstrate that normal morphology is not an absolute indicator for the selection of genetically normal sperm (Ryu et al., 2001; Burrello et al., 2004). The data obtained by this study allowed us to perform a multiple regression analysis considering aneuploidy as a dependent variable and sperm morphology, sperm concentration and sperm motility as independent variables. The data of these variables were obtained by semen analysis performed following WHO guidelines (1999). The results of multiple linear regression analysis indicated that motility and sperm morphology were significantly related to the aneuploidy rate, although the low value of the adjusted R 2 (12.31%) suggests that factors other than those included in the study also play a role in determining the altered segregation of chromosomes. Sperm concentration did not contribute to a statistically significant linear relationship with the aneuploidy rate. This result was apparently in contrast with data reported in the literature and recently revised (Miharu, 2005); patient selection criteria alone was not sufficient to explain these contradictory conclusions. Consequently, since the values of sperm concentration were distributed as a hyperbolic and not a linear curve, we transformed the variable to highlight a significant relationship between sperm concentration and presence of aneuploidy. Therefore, the conditions of teratozoospermia, oligozoospermia and asthenozoospermia, and above all the combination of these three factors, seem to condition the normal development of spermatogenesis. On the other hand, even the normally shaped spermatozoa of OAT patients showed an increased aneuploidy rate (Burrello et al., 2004), highlighting that if the spermatogenetic process is altered in any way, meiosis may also be affected due to testicular perturbation. In conclusion, biological and statistical data from the present research support the idea that higher sperm aneuploidies could be associated with altered sperm morphology and low sperm concentration, as well as with reduced motility. Acknowledgements Research supported by COFIN 2005, Italy. References Baccetti B, Bruni E, Capitani S, Collodel G, Mancini S, Piomboni P, Moretti E. Studies on varicocele III. Ultrastructural sperm evaluation and 18, X and Y aneuploidies. 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