1 Centre for Reproductive Medicine and 2 Centre for Medical Genetics, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, 1090 Brussels, Belgium
3 To whom correspondence should be addressed. e-mail: peter.platteau{at}az.vub.ac.be
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Abstract |
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Key words: azoospermia/chromosomal aneuploidy/FISH/PGD/TESE
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Introduction |
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A further explanation of the differences in pregnancy rate could be the higher aneuploidy frequency in embryos where testicular sperm was used from NOA men. Several sperm studies support an association between morphological (Int Veld et al., 1997; Bernardini et al., 1998
; Viville et al., 2000b
) or motility aberrations (Vegetti et al., 2000
) and sperm chromosome aneuploidy. Moreover, several studies (Bernardini et al., 2000
; Martin et al., 2000
; Viville et al., 2000a
; Levron et al., 2001
; Burrello et al., 2002
; Mateizel et al., 2002
; Palermo et al., 2002
; Sukcharoen et al., 2003
) report that the aneuploidy frequency of testicular sperm from azoospermic men suffering from severe testicular failure is higher compared to sperm from azoospermic patients with a normal spermatogenesis. The same studies also report that the aneuploidy frequency from the latter group is significantly higher than ejaculated sperm from donors.
From May 2001 until September 2003, we therefore conducted a prospective cohort study, offering couples with an OA or NOA partner ICSI in combination with preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Our aim was to compare the aneuploidy frequency of the screened embryos between the two groups. We also included a group of fertile patients who underwent the same PGD treatment for gender selection because of an X-linked disease as a historical control group.
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Materials and methods |
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Testicular sperm recovery
In patients with a clinical diagnosis of NOA, open excisional testicular biopsy samples were obtained under general anaesthesia or occasionally under loco-regional anaesthesia, as described previously (Tournaye et al., 1997). The testicular samples were processed by mechanical shredding (Verheyen et al., 1995
); microscopic examination of the wet preparations was carried out at x400 magnification under an inverted microscope. When no sperm were found after 1 h of initial searching, enzymatic digestion of the testicular tissue with collagenase type IV was performed (Crabbé et al., 1998
) in order to digest the tissue and release the few sperm that might be present. During surgery, if no histological diagnosis was yet available, a single, randomly taken biopsy of each testis was sent for histological examination. In patients with OA, sperm were obtained by fine needle aspiration (FNA) or (if no histological diagnosis was yet available) testicular biopsy under local anaesthesia.
Ovarian stimulation and ICSI procedure
All female partners underwent ovulation induction using a GnRH analogue suppression protocol (short or long) or a GnRH antagonist protocol and human menopausal gonadotrophins or recombinant FSH. Oocytecumulus complexes (OCC) were recovered 36 h after the administration of 10 000 IU of hCG. The surrounding cumulus and corona cells were then removed and the nuclear maturation of the oocytes was assessed under an inverted microscope. Only metaphase II oocytes were injected with, preferably, morphologically normal motile sperm into the ooplasm. Fresh as well as frozenthawed testicular sperm were used. These procedures have been described previously (Van Steirteghem et al., 1993; Joris et al., 1998
).
Assessment of fertilization, embryo development and biopsy
Further culture of injected oocytes was performed in 25 µl microdrops of culture medium under lightweight paraffin oil. Fertilization was confirmed after 1618 h by the observation of two distinct pronuclei (2PN). Oocytes with 2PN were assessed for embryonic development on day 2 and day 3 after injection, and the embryos reaching at least the 5-cell stage on day 3 of development were biopsied. The selection criteria for embryo biopsy were similar to those used to decide whether an embryo was transferable on day 3 in the regular ICSI programme without PGD. Before biopsy, the blastomeres were checked for the presence of a nucleus. From the 6-cell stage onward, two blastomeres per embryo were removed (Van De Velde et al., 2000; De Vos et al., 2001
).
FISH procedure
The individually biopsied blastomeres were spread onto a Superfrost Plus glass slide (Kindler GmbH, Freiburg, Germany) using 0.01 M HC1/0.l% Tween 20 solution (Coonen et al., 1994; Staessen et al., 1996
). Both blastomeres from the same embryo were fixed on the same slide in very close proximity.
A two-round FISH procedure, as described previously (Staessen et al., 2003), allowed us to detect the chromosomes X, Y, 13, 18, 21 (round 1) and 16, 22 (round 2).
In short an aliquot (0.2 µl) of the probe solution (DXZ1, Spectrum Blue; DYZ3, SpectrumGold; LSI13, SpectrumRed; D18Z1, SpectrumAqua; LSI21 SpectrumGreen; Multivision PGT Probe Panel; Vysis, Inc.) was added to the nuclei, covered with a round cover slip (4 mm diameter), denatured for 5 min at 75°C and left to hybridize for between 4 h and overnight at 37°C in a moist chamber. After washing in 0.4xstandard saline citrate solution (SSC)/0.3% Nonidet P40 at 73°C for 5 min and 2xSSC/0.1% Nonidet P40 for 60 s at room temperature. Antifade solution (Vectashield) was added and fluorescence signals were evaluated. The nuclei were then examined using a Zeiss Axioskop fluorescence microscope with the appropriate filter sets. The FISH images were captured with a computerized system.
Following the analysis of the first set of probes, the cover slips were gently removed and the slides rinsed in 1xphosphate-buffered saline at room temperature, denaturated in 0.0625xSSC for 7 min at 75°C, and then dehydrated (70, 90, 100 and 100% ethanol at 18°C, 60 s each). The second hybridization solution was prepared by mixing a probe for chromosome 16 (Satellite II DNA/D16Z3 probe, spectrum Orange; Vysis) and a probe for chromosome 22 (LSI 22, 22q11.2, SpectrumGreen; Vysis). The probes were denaturated separately in a hot water bath at 75°C for 5 min. An aliquot (0.2 µl) of the probe solution was then added to the nucleus, covered with a round coverslip (4 mm diameter), sealed with rubber cement and then hybridized overnight in a water bath at 37°C. Finally, the slides were washed for 2 min in 0.4xSSC solution at 73°C and 2xSSC/0.1% Nonidet P40 for 60 s at room temperature. The washed slides were then mounted with DAPI in antifade solution and analysed, and the results interpreted by two independent observers.
Embryo scoring and selection for transfer
The subsequent classification of embryos based on the biopsy result of two blastomeres was followed: (i) when both blastomeres had two copies of each chromosome analysed, the embryo was classified as normal; (ii) when both blastomeres had one chromosome with an abnormal number of copies, the embryo was classified as aneuploid; (iii) when both blastomeres had one, three or more copies of each chromosome, the embryo was classified as haploid or polyploid; (iv) when one blastomere was normal and the second blastomere had one chromosome with an abnormal number of copies, the embryo was classified as mosaic; (v) when at least one blastomere had more than one chromosome with an abnormal number of copies, the embryo was classified as complex abnormal. Only chromosomally normal embryos were transferred on day 5.
Definition of endpoint
A rise in serum hCG on two consecutive occasions from 11 days after transfer indicated pregnancy. A clinical pregnancy was defined as at least one fetus with a positive heartbeat revealed by vaginal ultrasound 5 weeks after embryo transfer. The implantation rate was defined as the number of viable fetuses, as assessed by ultrasound at 7 weeks gestation, divided by the number of embryos transferred for each subject.
Statistical analysis
The mean age of the female partner at the time of oocyte retrieval for each cycle and the mean number of retrieved oocytes, metaphase II oocytes, normally fertilized oocytes and the mean number of abnormal embryos of each cycle was first calculated per couple. In a second step, the mean of all previous parameters of all the couples was calculated. The comparison of the variables was performed by means of two-way analysis of variance, with Bonferroni t-test to perform pairwise comparison of the three groups. P < 0.05 was considered statistically significant.
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Results |
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In the embryos, no differences in aneuploidy frequency were observed between the TESE groups; there were, however, significantly more abnormal embryos in the OA group compared to the control group. Subanalysing the chromosomal abnormal embryos (Table III) into the different categories (described in Materials and methods) and for each chromosome did not reveal any significant differences between the three groups, probably due to the small numbers.
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Discussion |
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An explanation for the high aneuploidy frequency in embryos derived from non-obstructive as well as obstructive sperm could therefore be the compromised testicular environment (obstruction, no natural selection in the epididymis, high FSH values, low testosterone values etc.). Some authors have described the degeneration of the testicular tubules and even a decrease in testicular volume after vasectomy (Lohiya et al., 1987). This environment could make these germ cells particularly susceptible to meiotic abnormalities. Earlier data from studies in XXY mice (Mroz et al., 1999
) have already suggested this. An alternative explanation could be that abnormal spermatozoids are usually sequestrated along the way between the testis and the tip of the urethra; we would therefore be comparing selected (through the epididymis, prostate etc.) with non-selected sperm.
It is known that aneuploidy appears to increase with maternal age and is related to defects in the oocyte resulting in meiotic disjunction errors. There is no difference between the groups in the embryos categorized as aneuploid, as the age of the female partners was not very different. The percentage of (autosomal and sex chromosomal) aneuploid embryos (calculated over the total of biopsied embryos with diagnosis: 21.0% in the NOA and 29.9% in the OA groups) is remarkably similar to the aneuploidy rate of patients of similar age, but with normozoospermic partners (Munné et al., 2002). There is a trend towards a higher aneuploidy frequency in the OA and control groups, most probably related to a higher mean age in these groups. It seems that the higher abnormality frequency in our two groups is mainly due to the high number of mosaic and complex abnormal embryos, which are mainly due to post-zygotic events. This points more to a sperm centriole defect, rather than sperm aneuploidy (Schatten et al., 1986
; Palermo et al., 1994
; Silber et al., 2003
). There is also a trend towards a higher rate of sex chromosomal abnormalities (especially in the NOA group), which fits with the prenatal testing results of ICSI pregnancies (Bonduelle et al., 2002
).
This group of patients seems to provide ideal candidates for PGD-AS: the women are relatively young and therefore produce a large number of oocytes and embryos to biopsy, combined with a very high aneuploidy frequency. However, before we advocate PGD-AS as a standard treatment for these patients, we need more data to confirm these findings and, even better, a prospective randomized study comparing PGD-AS with a control group.
Arguments against PGD-AS are that the embryo biopsy (the removal of one or two blastomeres) might affect the viability of these embryos, generated sometimes with extremely rare sperm. No study until now has demonstrated any difference. A few, potentially healthy, embryos are also lost due to technical difficulties during the biopsy (1%; C.Staessen, personal communication), or due to false negative results (
3.5%; C.Staessen, personal communication), where it was falsely thought that a healthy embryo was aneuploid. In
2% of all biopsied embryos, no FISH result is obtained due to the loss of the nucleus or difficulties in the interpretation of the FISH signals; these embryos are not lost as they can still be transferred, after informing the patient that the embryo was not screened. In 5% of the biopsies, no nucleus or multiple nuclei are found in the blastomeres, which are mostly embryos of poor morphology, which would not have been transferred anyway. A third argument is mosaicism; it could well be that although the blastomeres removed from the screened embryo are aneuploid, the rest of the embryo is fully euploid and would have led to a perfectly normal baby. So in total we have, in our hands, a real loss of ± 4.5% of potentially healthy embryos, plus the unknown loss of mosaic embryos, which could have led to a healthy child.
We expected a lower implantation rate in the group with the highest aneuploidy frequency (OA) compared with the NOA group, especially as the mean age in the OA group was older (not statistically significant); this could be due to the small groups or alternatively to other intrinsic factors in the NOA group which are not seen after PGD-AS screening.
In conclusion, we think that there is a place for PGD-AS for NOA as well as OA patients in view of the high aneuploidy frequency and the large pool of embryos that can be screened, to allow us to select one or two embryos for transfer. However, the ultimate confirmation that PGD-AS improves the pregnancy rate/live birth rate in this group of patients will only happen after a study that is randomized and probably multicentre (as the number of NOA men with embryos to biopsy is quite small).
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Acknowledgements |
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References |
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Submitted on December 22, 2003; accepted on April 16, 2004.