1 Section of Endocrinology, Andrology and Internal Medicine, Department of Biomedical Sciences, University of Catania, Catania, Italy and 2 Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Urological Institute, Cleveland Clinic Foundation, Cleveland, OH, USA
3 To whom correspondence should be addressed at: Cattedra di Endocrinologia II, Dipartimento di Scienze Biomediche, Università di Catania, Ospedale Garibaldi, Piazza S. Maria di Gesù, 95123 Catania, Italy. e-mail: acaloger{at}unict.it
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Abstract |
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Key words: abortion rate/ICSI/male infertility/pregnancy rate/sperm aneuploidy
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Introduction |
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Materials and methods |
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Fifteen patients were azoospermic. Of these, nine had congenital bilateral absence of the vas deferens and underwent percutaneous epididymal sperm aspiration. The other six had non-obstructive azoospermia and underwent testicular sperm aspiration (n = 5) or microsurgical testicular sperm extraction (n = 1).
To define the reference values of sperm aneuploidy rate in the ejaculate, we studied 14 healthy men with a median age of 25 years (range 1935), normal semen parameters and normal somatic karyotypes. Their median sperm aneuploidy rate was 0.91% (range 0.301.55). The upper range in the normal men was established as the upper limit of normal sperm aneuploidy rate. The patients subsequently were divided into two groups. Group A had sperm aneuploidy rates ≤1.55% and group B had rates >1.55%. The age of the female partners of the patients of groups A and B was not statistically different [31 years (range 2039) and 33 years (range 2041) respectively].
Ovulation induction was achieved with a GnRH analogue and FSH in all the female partners according to our previously described protocol for ICSI (Calogero et al., 2001a). We confirmed biological pregnancy by measuring serum
-HCG on at least two occasions 14 days after embryo transfer.
FISH analysis, DNA hybridization and scoring
Sperm were prepared for FISH analysis as reported previously, using an aliquot of the swim-up preparation or of the sperm retrieved from the epididymis or testis (Calogero et al., 2001a; b
). Likewise, we have previously reported our methods for sperm head decondensation, DNA hybridization and visual scoring of the sperm. Sperm were fixed and spread on slides washed in 2x standard saline citrate solution and incubated in dithiothreitol. Sperm structure, including the tail, was preserved, allowing for morphological differentiation between the sperm and other cells present in the ejaculate.
Alpha-centromeric probes for chromosomes 8, 12, 18, X and Y were used for both patient and control samples. The probe mixture for triple FISH consisted of a repetitive DNA sequence of centromeric probes for chromosome X (pDMX1), labelled FITC, for chromosome Y (pLAY5.5), labelled Cy3 and for chromosome 12 (pBR12), labelled FITC and Cy3. The probe mixture for the double-colour FISH also consisted of a repetitive DNA sequence of centromeric probes for chromosome 8 (pZ8.4) and chromosome 18 (2Xba), labelled FITC or Cy3, respectively. The probes were provided by Professor M.Rocchi, (University of Bari, Bari, Italy). The slides were scored using an Axiophot fluorescence microscope (C.Zeiss, Oberkochen, Germany) with single-band DAPI, FITC and Cy3 filters.
Sperm were scored as reported elsewhere (Calogero et al., 2001a; b
). Only intact sperm with clear hybridization signals were scored. We excluded disrupted or overlapping sperm. Sperm were considered polysomic if they presented two or more distinct hybridization signals of equal intensity separated by at least one signal domain. Diploid sperm displayed two signals for each tested chromosome with normal head and tail morphology.
Statistical analysis
We analysed the data with SPSS 9.0 for Windows statistical software. The MannWhitney or 2-test determined significance as appropriate. A P-value <0.05 was considered statistically significant. Results are shown as median and range.
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Results |
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Discussion |
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The two groups of patients did not differ regarding the main parameters known to affect ICSI outcome. Maternal and paternal age as well as semen analysis parameters were similar in the two groups. Because epididymal and testicular sperm have a greater sperm aneuploidy rate (Burrello et al., 2002) and negatively affect the ICSI outcome (Vicari et al., 2001
), we re-evaluated the data excluding these patients, and there were no significant changes. We therefore conclude that the only difference that affected the pregnancy rate was the greater number of genetically abnormal sperm in the group B patients, which was >2-fold that found in group A patients. It is noteworthy that sperm examined in this study were taken from the swim-up fraction used for oocyte injection, thus confirming that swim-up did not eliminate aneuploidy sperm (Pfeffer et al., 1999
). The embryologist may therefore unknowingly select aneuploid sperm for oocyte injection. However, similar to what has been reported previously (Pfeffer et al., 1999
), the diploidy rate found in sperm recovered after swim-up was lower than that found in the whole semen of OAT patients (Calogero et al., 2001b
).
Other authors have suggested a negative trend of gamete chromosomal abnormalities on implantation rate (Int Veld et al., 1997; Pang et al., 1999
; Rubio et al., 2001
), pregnancy rate (Pang et al., 1999
; Pfeffer et al., 1999
; Rubio et al., 2001
), and fetal survival (Rubio et al., 2001
). We have previously observed a higher percentage of men with elevated sperm aneuploidy rate among infertile couples with pregnancy failure after ICSI (Calogero et al., 2001a
). That study and the present one therefore support a negative impact of aneuploidy in achieving a pregnancy after ICSI. In addition, Gianaroli and colleagues have shown that pregnancy success after IVF is influenced significantly by embryo chromosome aneuploidy (Gianaroli et al., 1997
).
The pregnancy rate observed in patients of group B is commensurate with ICSI results reported by IVF centres worldwide. The pregnancy rate of a similar subset of infertile patients selected with normal sperm chromosomal constitution (group A) was more than optimal. This high value may be due to the relatively low number of patients found with an normal sperm aneuploidy rate, since the vast majority of the patients requiring ICSI have an abnormal sperm chromosomal complement (Calogero et al., 2001a; Rubio et al., 2001
). Sperm aneuploidy may play a major role in explaining the suboptimal pregnancy rates in many infertile couples undergoing ICSI, but our findings need confirmation in larger series.
Normal morphology and good motility are two of the main criteria for selecting sperm to be injected during micromanipulation. Therefore, the overall likelihood that an embryologist may select a genetically abnormal spermatozoon for ICSI would be low. However, Pfeffer et al. (1999) and the results in this study demonstrate that the swim-up preparation does not completely eliminate genetically abnormal sperm. In addition, a recent study by Ryu and colleagues suggests that morphologically normal sperm in infertile patients with <4% normal forms (Krugers strict criteria) have 23-fold higher aneuploidy rates compared with normally shaped sperm from normozoospermic men (Ryu et al., 2001
). Based on these results, even morphologically normal spermatozoa may have an abnormal chromosome asset.
In conclusion, this study showed that higher total sperm aneuploidy rates are associated with lower implantation and pregnancy rates, and higher rates of miscarriage in patients undergoing ICSI. Future development of methods to identify genetically abnormal sperm may allow for better selection and improved ICSI outcomes. Studies in a larger series of patients are needed to confirm these findings.
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Acknowledgements |
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References |
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Submitted on December 17, 2002; accepted on April 4, 2003.