1 Section of Endocrinology, Andrology and Internal Medicine, Department of Biomedical Sciences, and Master in Andrological and Human Reproduction Sciences,, 3 Department of Human Anatomy, University of Catania and 2 Unit of Cardiology, Garibaldi Hospital, Catania, Italy
4 To whom correspondence should be addressed at: Sezione di Endocrinologia, Andrologia e Medicina Interna, Dipartimento di Scienze Biomediche, Università di Catania, Ospedale Garibaldi, Piazza S. Maria di Gesù, 95123 Catania, Italy. Email: acaloger{at}unict.it
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: chromosomes 12/multicolour fluorescence in situ hybridization/oligo-astheno-teratozoospermia/sex chromosomes/aneuploidy/morphology
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ICSI bypasses all natural sperm selection processes, since the embryologist chooses the spermatozoon to be injected into the oocyte. Only motile and morphologically normal spermatozoa will be selected. However, these criteria will not exclude the presence of an abnormal karyotype, since the altered intratesticular environment may disrupt the fine-tuned mechanisms of chromosome segregation during spermatogenesis. In this case, spermatozoa with a normal morphology may also be aneuploid, cancelling out the careful selection of a normally shaped spermatozoon aimed at reducing the risk of transmitting aneuploidy to the ICSI offspring. The present study was undertaken to assess the sperm aneuploidy rate of spermatozoa with normal and abnormal morphology. To accomplish this, we evaluated the sperm aneuploidy rate for chromosomes 12, X and Y in single spermatozoa identified according to their morphology in patients with secretory OAT. Six normozoospermic men were selected as a matched control group.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
A triple-colour FISH was carried out on each patient and control, using -centromeric probes for chromosomes 12, X and Y. Alpha centromeric probes were obtained from plasmids containing a specific
6 kb DNA insert: pBR12 for chromosome 12, pDMX1 for chromosome X and pLAY5,5 for chromosome Y. Escherichia coli with the specific plasmid were grown in a Petri dish with LB medium containing 50 µg/ml ampicillin, and incubated overnight at 37°C. Subsequently, a colony was selected and amplified in 10 ml of LB medium containing 50 µg/ml ampicillin overnight at 37°C. The extraction of the plasmid was carried out by miniprep, using the alkaline lysis method, as previously reported (Sambrook et al., 1989
). Plasmids containing the 12 and X chromosome probes (DNA 1 µg) were labelled with Fluor X-dCTP, whereas plasmids containing the 12 and Y chromosome probes were labelled with Cy3-dUTP, using a nick-translation kit purchased from Pharmacia Biotech (Milan, Italy). The size of the fragments obtained was checked in a 1% agarose gel.
Each slide was denatured with a solution of 70% formamide/2x SSC (pH 7.5) at 80°C for 150 s. The slides were 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 the coverslip was removed and the slides were immersed in a post-hybridization wash of 50% formamide/2x SSC three times at 37°C for 5 min, 2x SSC three times at 42°C for 5 min and 2x SSC/0.1% Tween-20 once at room temperature for 5 min. The slides were then mounted in 4',6-diamidino-2-phenylindole (DAPI) counterstain and antifade and stored in the dark at 4°C until microscope observation was carried out.
The slides were observed on a Leica DMRXA2 fluorescent microscope with the appropriate set of filters: single band DAPI, fluorescein isothiocyanate (FITC) and Cy3. Previously identified intact spermatozoa bearing a similar degree of decondensation and clear hybridization signals were scored; disrupted or overlapping spermatozoa were excluded from 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. 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. Spermatozoa were scored as nullisomic if they showed no signal for a given chromosome, whereas 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 in cases where the tail as well as the normal oval shape of a sperm head were evident. No FISH signals in a spermatozoon head showing DAPI stain were considered a case of no hybridization. The hybridization efficiency was >99%.
Statistical analysis
Results are shown as median and range throughout the study, unless otherwise indicated. The data were analysed with the MannWhitney test. SPSS 9.0 for Windows was used for statistical calculation. A significant statistical difference was accepted when the P-value was <0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Sperm aneuploidy rates for chromosomes 12, X and Y are reported in Table I. OAT patients had a slight, but significantly (P<0.05), lower number of morphologically normal and abnormal spermatozoa carrying the X chromosome, compared with normozoospermic men. They also exhibited increased XY and XX disomy rates (P<0.01), whereas YY disomy, sex chromosome nullisomy and chromosome 12 disomy and nullisomy rates were similar. Spermatozoa with an abnormal sperm head from normozoospermic men also had a significantly (P<0.05) higher XX disomy rate compared with morphologically normal spermatozoa obtained from the same men.
The total aneuploidy rates for chromosomes X, Y and 12 in spermatozoa with a normal or abnormal head from normozoospermic men and OAT patients is shown in Figure 2. Morphologically abnormal spermatozoa of normozoospermic men had an aneuploidy rate 4.4-fold higher than that found in spermatozoa with a normal morphology (P<0.001). The total aneuploidy rates of spermatozoa with normal or abnormal head shape from OAT patients were similar to each other and to that of abnormally shaped spermatozoa from normozoospermic men, but they were significantly higher than the rate found in normally shaped spermatozoa of normal men (P<0.001). In particular, normally and abnormally shaped spermatozoa from OAT patients had an aneuploidy rate 3.6- and 4.7-fold higher, respectively, than that found in normally shaped spermatozoa of normozoospermic men.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of the present study are in close agreement with those of Ryu et al. (2001). The authors studied the frequency of aneuploidy for chromosomes 18, X and Y in morphologically normal spermatozoa of eight infertile patients having <4% normal forms according to Kruger's strict criteria. The aneuploidy rate of these spermatozoa was compared with that of normally shaped spermatozoa of six normal donors of proven fertility. The FISH analysis showed that the sperm aneuploidy rate of the infertile teratozoospermic patients was 3.3% (range 1.85.5%). This rate was nearly 3-fold greater than that found in morphologically normal spermatozoa of fertile men who had an aneuploidy rate of 1.3% (range 02.6%). The sex chromosome aneuploidy rate was 2-fold higher in the infertile group as compared with the control group. The authors did not show the aneuploidy rate of morphologically abnormal spermatozoa (Ryu et al., 2001
).
The increased aneuploidy rate found in normally shaped spermatozoa of OAT patients may also explain the lower success rate of ICSI in patients with OAT (for a review see Calogero et al., 2003). However, the abnormal sperm parameters found in these patients may as well have a negative effect on the ICSI outcome. For instance, a recent study showed that ICSI clinical results correlate negatively with the degree of abnormal sperm morphology. Implantation and pregnancy rates were 9.6 and 20.2%, respectively, in patients with teratozoospermia, whereas they were 18.7 and 36.7% in patients with normal sperm morphology (De Vos et al., 2003
). However, we have shown recently that an increased sperm aneuploidy rate has a negative impact on ICSI outcome. Unselected patients undergoing ICSI were sorted into two groups according to their sperm aneuploidy rate: one with a normal and the other with an elevated sperm aneuploidy rate. Although fertilization and cleavage rates were similar between the two groups, patients with a normal sperm aneuploidy rate had higher pregnancy and implantation rates compared with patients who had an elevated aneuploidy rate, whereas the abortion rate was significantly higher in the latter. Other factors that may have a negative outcome on the clinical ICSI outcome, such as female partner age, abnormalities of conventional semen parameters or female factor of infertility, were similar in both groups (Burrello et al., 2003
).
In conclusion, the present study showed that morphologically normal spermatozoa of OAT patients carry an abnormal chromosomal constitution with the same frequency as that found in spermatozoa with an abnormal head shape. Therefore, the risk of selecting aneuploid spermatozoa to be used for oocyte injection is high in OAT patients. These findings further strengthen the needs for genetic counselling before carrying out ICSI, to decrease the risk of transmitting genetic disease to the offspring. This may be achieved, for example, by establishing methods to identity spermatozoa with a normal chromosomal complement (Cayli et al., 2003).
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bonduelle M, Van Assche E, Joris H, Keymolen K, Devroey P, Van Steirteghem A and Liebaers I (2002) Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod 17, 26002614.
Burrello N, Vicari E, Shin P, Agarwal A, De Palma A, Grazioso C, D'Agata R and Calogero AE (2003) Lower sperm aneuploidy frequency rates in ICSI programmes. Hum Reprod 18, 13711376.
Calogero AE, De Palma A, Grazioso C, Barone N, Romeo R, Rappazzo G and D'Agata R (2001) Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters. Hum Reprod 16, 11721179.
Calogero AE, Burrello N, De Palma A, Barone N, D'Agata R and Vicari E (2003) Sperm aneuploidy in infertile man. Reprod Biomed Online 6, 310317.[Medline]
Cayli S, Jakab A, Ovari L, Delpiano E, Celik-Ozenci C, Sakkas D, Ward D and Huszar G (2003) Biochemical markers of sperm function: male fertility and sperm selection for ICSI. Reprod Biomed Online 7, 462468.[Medline]
De Vos A, Van De Velde H, Hubertjoris MT, Verheyen G, Devroey P and Van Steirteghem A (2003) Influence of indiviual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection. Fertil Steril 79, 4248.[CrossRef][ISI][Medline]
Gole LA, Wong PF, Ng PL, Wang XQ, Ng SC and Bongso A (2001) Does sperm morphology play a significant role in increased sex chromosomal disomy? A comparison between patients with teratozoospermia and OAT by FISH. J Androl 22, 759763.
Harkonen K, Suomimen J and Lahdetie J (2001) Aneuploidy in spermatozoa of infertile men with teratospermia. Int J Androl 24, 197205.[CrossRef][ISI][Medline]
Meschede D and Horst J (1997) Genetic counselling for infertile male patients. Int J Androl 20, 2030.[ISI][Medline]
Palermo G, Joris H, Devroey P and Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of a single spermatozoon into an oocyte. Lancet 340, 1718.[ISI][Medline]
Pang MG, Hegerman SF, Cuticchia AJ, Moon SY, Doncel GF, Acosta AA and Kearns WG (1999) Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescent in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum Reprod 14, 12661273.
Ryu HM, Lin WW, Lamb DJ, Chuang W, Lipshultz LI and Bischoff FZ (2001) Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for intracytoplasmic sperm injection. Fertil Steril 76, 879883.[CrossRef][ISI][Medline]
Sambrook J, Fritsh EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Ushijima C, Kumasako Y, Kihaile PE, Hirotsuru K and Utsunomiya T (2000) Analysis of chromosomal abnormalities in human spermatozoa using multi-colour fluorescence in-situ hybridization. Hum Reprod 15, 11071111.
Van Steirteghem A, Bonduelle M, Devroey P and Liebaers I (2002) Follow-up of children born after ICSI. Hum Reprod Update 8, 1111116.
Vegetti W, Van Assche E, Frias A, Verheyen G, Bianchi MM, Bonduelle M, Liebaers I and Van Steirteghem A (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men. Hum Reprod 15, 351365.
World Health Organization (1999) Laboratory Manual for the Examination of the Human Semen and Sperm-Cervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge.
Submitted on January 2, 2004; resubmitted on February 12, 2004; accepted on April 2, 2004.