Hum. Reprod. Advance Access originally published online on June 6, 2006
Human Reproduction 2006 21(9):2390-2395; doi:10.1093/humrep/del177
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Does PGD for aneuploidy screening change the selection of embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men?
1 Centre for Reproductive Medicine and 2 Centre for Medical Genetics, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Brussels, Belgium
3 To whom correspondence should be addressed at: Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail: pdonoso{at}alemana.cl
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Abstract |
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BACKGROUND: An increased incidence of aneuploid embryos has been recently described from azoospermic men. The aim of this study was to assess if embryo selection on day 5, based on morphological criteria, would be different from the selection based on PGD for aneuploidy screening (AS) in couples undergoing ICSI for male azoospermia. METHODS: Sixty-two cycles of testicular sperm extraction (TESE)-ICSI with PGD-AS were included in the analysis. Two embryologists, blinded to the PGD-AS results, retrospectively reviewed the available embryology data from day 5 embryos and selected one, two or three embryos to be transferred. These results were compared with the selected embryos based on PGD-AS. RESULTS: A total of 39 cycles from non-obstructive azoospermia (NOA) and 23 cycles from obstructive azoospermia (OA) were retrospectively analysed. If single embryo transfer (SET) had been performed, in 64.8% of the NOA cycles and 54.5% of the OA cycles, no difference in embryo choice would have occurred compared to PGD-AS and in 10.8 and 36.6% of the cycles, respectively, an aneuploid embryo would have been chosen. If double ET (DET) had been performed, in 72.9% of the NOA cycles and 86.5% of the OA cycles, no difference in embryo choice would have occurred compared to PGD-AS and in 2.7 and 4.5% of the cycles, respectively, an aneuploid embryo would have been chosen. If triple ET (TET) had been performed, the outcome would have been the same as for DET. DISCUSSION: Our results suggest that under the terms of an SET policy, the performance of PGD-AS in azoospermia would result in a higher chance of success, as the possibility of selecting a euploid embryo is enhanced.
Key words: azoospermia/embryo selection/FISH/PGD/TESE
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Introduction |
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The use of surgically retrieved testicular sperm for ICSI has become the standard treatment both for non-obstructive (NOA) and obstructive azoospermia (OA) (Devroey et al., 1996
; Osmanagaoglu et al., 2003). However, concerns have been raised regarding the genetic constitution of the retrieved spermatozoa from infertile men and their implication on the ART outcome and health status of their offspring (Bonduelle et al., 2002).
An increased aneuploidy rate has been found on retrieved testicular and epididymal sperm from NOA and OA patients compared to ejaculated spermatozoa from both oligo-astheno-teratospermic and normozoospermic men (Levron et al., 2001; Calogero et al., 2003). Furthermore, chromosome abnormalities limited to spermatogenic cells and not detectable in the somatic cells (Karyotype analysis) have been reported in 
6% of patients (Egozcue et al., 2000). This could be due to an altered intratesticular environment or gene mutations that disrupt the chromosome segregation process during meiosis (Lange et al., 1997; Egozcue et al., 2000).
Extending embryo culture to day 5 enhances the embryo selection process by allowing the best embryos to reach this advanced developmental stage (Papanikolaou et al., 2005). Nevertheless, some chromosomally abnormal embryos can still be developed until the blastocyst stage (Sandalinas et al., 2001; Staessen et al., 2004). Under the new Belgian legislation, single embryo transfer (SET) should be performed at the first two IVF cycles in women under 36 years of age, so that, selecting which embryo is to be transferred is crucially important. The new legislation particularly concerns patients with azoospermia (NOA and OA), as it was recently shown by means of preimplantation genetic screening [(PGD-aneuploidy screening (AS)] that the embryos derived from NOA and OA men carried a higher aneuploidy rate compared to embryos derived from fertile men (52.5 versus 40.5% and 60 versus 40.5%, respectively) irrespective of the young age of their female partners (Platteau et al., 2004). Considering these data and the fact that PGD-AS is not currently being performed as a routine procedure in azoospermic men undergoing infertility treatment, it is possible that to reduce the number of transferred embryos might dramatically affect the probability of pregnancy in these couples undergoing ICSI without PGD-AS. Ultimately, the only way of confirming this hypothesis would be to conduct a randomized controlled trial, which is difficult, however, as it would take a long time to reach a suitable number of patients because of the low prevalence of azoospermia (2%) (Hull et al., 1985).
The aim of this study was to retrospectively review all the embryology data available from azoospermic patients undergoing ICSI with PGD and to examine whether the embryo selection on day 5, based only on the developmental and morphological criteria, would have been different from the selection based on PGD-AS results.
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Materials and methods |
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Patients, testicular sperm retrieval, ovarian stimulation, ICSI procedure, assessment of fertilization, embryo development, biopsy, FISH procedure and FISH results were previously described in Platteau et al. (2004)
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Two senior embryologists retrospectively reviewed all the embryology data from azoospermic patients in whom PGD-AS had been performed (previously included in Platteau et al., 2004) and selected the one, two or three best embryos, blinded to the PGD-AS information, according to the morphological evaluation for transfer on day 5. These results were then compared with the embryos that were eventually transferred and for which the PGD-AS information was known.
Subjects, ovarian stimulation, testicular sperm recovery and ICSI procedure
All patients included in the study were shown to be azoospermic on the basis of at least two semen analyses and had histological confirmation of their normal or deficient spermatogenesis. An extended clinical and laboratory work up had also been performed. Cases with abnormal karyotype or Yq microdeletions were excluded.
Both a GnRH-agonist protocol and a GnRH-antagonist protocol, along with recombinant FSH or urinary gonadotrophins, were used (Papanikolaou et al., 2005). Urinary HCG (Pregnyl®) of 10 000 IU was used to trigger final oocyte maturation, and cumulus–oocyte complexes were recovered 36 h later. The surrounding cumulus and corona cells were then removed, and the nuclear maturation of the oocytes was assessed under an inverted microscope.
In patients with a clinical diagnosis of NOA, open excisional testicular biopsy was performed under general anaesthesia or loco-regional anaesthesia, as described previously (Tournaye et al., 1997). Testicular samples were processed by mechanical shredding. When no spermatozoa were found after 1 h of searching, enzymatic digestion with collagenase type IV was added. In patients with OA, spermatozoa were obtained by fine needle aspiration (FNA) or testicular biopsy under local anaesthesia if no histological diagnosis was available. Only metaphase II oocytes were injected with preferably morphologically normal motile spermatozoa into the ooplasm. Fresh as well as frozen thawed testicular sperm was used (Van Steirteghem et al., 1993). Fertilization was confirmed after 16–18 h by the observation of two distinct pronuclei. On the third day of embryo culture, embryo evaluation was performed, and, from the six-cell stage onward, two blastomeres per embryo were biopsied (Van de Velde et al., 2000).
FISH procedure
The individually biopsied blastomeres were spread onto a Superfrost Plus glass slide (Kindler GmbH, Freiburg, Germany) using 0.01 N 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 fluorescence in-situ hybridization (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).
Embryo scoring
The following classification of embryos based on the biopsy of two blastomeres was adopted: (i) when both blastomeres had two copies of each analysed chromosome, the embryo was classified as euploid; (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 euploid and the second blastomere had one chromosome with an abnormal number of copies, the embryo was classified as discordant and (v) when at least one blastomere had more than one chromosome with an abnormal number of copies, the embryo was classified as complex abnormalities.
The blastocyst quality was assessed according to the criteria of Gardner and Schoolcraft (1999). Only euploid embryos were transferred on day 5.
Results
A total of 62 cycles from azoospermic patients undergoing ICSI with embryo biopsy were included in the analysis: 39 cycles from 23 NOA men and 23 cycles from 13 OA men. The results are summarized in Tables I, II and III. The mean age of the female partners was 30.6 ± 4.6 years in the non-obstructive group and 33.5 ± 3.9 years in the obstructive group. In the NOA group, there were two cycles without transfer, as all embryos were not morphologically suitable to replace (cycles 23 and 36) leaving 37 cycles to be analysed. In the OA group, there was only one cycle without transfer, as all embryos were not morphologically suitable to replace, leaving 22 cycles to be analysed.
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The ongoing clinical pregnancy rate per ET was 14.3% in the NOA group and 20% in the OA group. Two twin pregnancies occurred in OA cycles (Tables I and II).
NOA group (37 cycles with ET)
Anticipating SET
If only one embryo had been transferred, in 64.8% of the cycles (n = 24), there was no difference in embryo choice and in 10.8% of the cycles (n = 4), an aneuploid embryo would have been chosen, possibly leading to two babies with Turner syndrome (cycles 11 and 13), one baby with mosaic Edward syndrome (cycle 29) and one missed abortion (cycle 19), and 24.3% of the transfers (n = 9) would have been worthless, giving false hope to the patients by replacing only chromosomally abnormal embryos, possibly even leading to seven miscarriages (Table III).
Anticipating double ET (DET)
If two embryos had been transferred, in 72.9% of the cycles (n = 27), at least one euploid embryo would have been transferred, in three of the cycles, however, one embryo would have been chromosomally abnormal, adding an extra risk of a Turner syndrome baby (cycle 7) and two miscarriages (cycles 2 and 24), and in 2.7% of the cycles (n = 1), an aneuploid embryo would have been chosen, possibly leading to a Klinefelter syndrome baby (cycle 13), and 24.3% of the transfers (n = 9) would have been worthless, giving false hope to the patients.
Anticipating triple ET (TET)
If three embryos had been transferred, the outcome would not have differed from the DET policy. Only in cycle 13 would an aneuploid embryo have been chosen, possibly leading to a mosaic Turner/XXXXY baby.
OA group (22 cycles with ET)
Anticipating SET
If only one embryo had been transferred, in 54.5% of the cycles (n = 12), there would have been no difference in embryo choice and in 36.6% of the cycles (n = 8), an aneuploid embryo would have been chosen, possibly leading to a baby with mosaic Edward syndrome and five missed abortions (cycles 1, 2, 4, 9, 18, 20, 21 and 23), and 9.1% of the transfers (n = 2) would have been worthless, false hope transfers, without chromosomally normal embryos (cycles 12 and 17).
Anticipating DET
If two embryos had been transferred, in 86.5% of the cycles (n = 19), at least one euploid embryo would have been transferred, in three of the cycles, however, one embryo would have been chromosomally abnormal, adding an extra risk of a Turner syndrome baby (cycle 7) and two miscarriages (cycles 2 and 24), and in 2.7% of the cycles (n = 1) an aneuploid embryo would have been chosen (cycle 18), and 9.1% of the transfers (n = 2) would have been worthless, giving false hope to the patients.
Anticipating TET
If three embryos had been transferred, the outcome would have not differed from the DET policy.
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Discussion |
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The findings of the present study highlight the importance of PGD-AS in azoospermic patients, especially when SET is to be performed. According to our results, if a policy of SET was followed in NOA and OA men based only on morphological criteria on day 5, then, an aneuploid embryo would have been chosen instead of the normal embryos in 10.8 and 36.3% of the cycles, respectively. Adding the negative psychological effects (Newton et al., 1990
) of false hope transfers (24.3 and 9.1% respectively), the routine use of PGD-AS in these patients might be more appropriate. Nevertheless, the transfer of two or three embryos can increase the probability of replacing euploid embryos in both NOA and OA (72.9 and 86.5%, respectively), thereby reducing the advantages of PGD-AS. Although this strategy seems reasonable when there is no limitation on the number of embryos that can be replaced, the achievement of comparable pregnancy rates would also be accompanied by the adverse effect of raising the multiple pregnancy rates and miscarriage rates. On the contrary, SET intends to reduce the incidence of multiple pregnancies after IVF treatment. Considering that SET is imposed by law and reimbursed by the health insurance system in some countries including Belgium, the selection of the embryo with the higher implantation potential becomes crucial.
Although the embryo morphology evaluation on day 5 allows for the selection of embryos with a superior chance of implantation as the activation of the embryonic genome has occurred (Gardner et al., 1998; Papanikolaou et al., 2005), its main disadvantage is that it does not ensure a normal chromosomal constitution, given that it has been reported that 37% of trisomic embryos reach the blastocyst stage (Sandalinas et al., 2001). Furthermore, it has been observed that up to 35% of top quality blastocysts were chromosomally abnormal (Staessen et al., 2004). Therefore, the information provided by PGD-AS on embryos derived from azoospermic men diminishes the probability of receiving chromosomally abnormal embryos and eventually increases the chance of implantation.
It is difficult to compare the clinical outcome of azoospermic men undergoing ICSI with or without PGD-AS as the number of performed cycles is limited. Nonetheless, the ongoing clinical pregnancy rate in this study was similar to previously reported results without performing PGD-AS (clinical pregnancy rate per ET of 17.9% in NOA and 26% in OA) (Vernaeve et al., 2003). Although it would be expected to obtain a higher pregnancy rate after performing PGD-AS, a beneficial effect might not become obvious by limiting the number of embryos transferred. However, a potential advantage of PGD-AS in azoospermic patients is a reduction in the probability of high-order pregnancies, as a lower number of embryos can be transferred. In the present series of patients who underwent AS, a mean of 1.9 embryos were replaced in the NOA group and 1.6 in the OA group compared to 2.7 and 2.8, respectively without PGD-AS (Vernaeve et al., 2003). Nevertheless, only the performance of a multicentre prospective randomized study where the same number of embryos are replaced in both groups will enable a definitive conclusion on this regard.
Since the introduction of PGD-AS, multiple efforts (Gianaroli et al., 1999; Werlin et al., 2003; Staessen et al., 2004; Munne et al., 2005; Platteau et al., 2005) have been made to identify which group of patients can benefit from this strategy. It has been proposed that azoospermic men could benefit from the addition of PGD-AS as the reported increased incidence of sperm aneuploidy in these men might lead to a higher chance of developing embryos with numerical chromosome abnormalities or mosaicism (Silber et al., 2003; Platteau et al., 2004). This hypothesis has been confirmed in clinical trials by Platteau et al. (2004), who found that around 50% of the embryos derived from NOA and OA men revealed numerical chromosomal abnormalities compared to 40% in the control group.
For NOA, there is sufficient evidence with regard to the aetiology of the high incidence of genetic abnormalities in the retrieved spermatozoa (Lange et al., 1997; Gianaroli et al., 2005). On the contrary, in the case of OA, many possible explanations can be offered: disturbed testicular environment (Mroz et al., 1999), tubular degeneration (Lohiya et al., 1987) or the use of unselected spermatozoa that would not reach the ejaculate. Recently, McVicar et al. (2005) have also found a significant decrease in spermatogenesis in OA men due to either vasectomy or other causes of obstruction, probably as a result of oxidative stress, interstitial fibrosis or increased apoptosis.
The follow-up of children born after ICSI has revealed an increased incidence of de-novo chromosomal abnormalities (sex chromosomes and structural anomalies) compared to the general population (1.6 versus 0.45%). This has been attributed to spermatozoa defects associated with severe male factor infertility (Bonduelle et al., 2002). In our series of patients, the incidence of de-novo sex chromosome abnormalities was 5.6% in the NOA men and 1.7% in the OA men. Therefore, the implementation of PGD-AS in these patients could constitute an effective tool to reduce the augmented frequency of de-novo sexual chromosomal disorders.
One important disadvantage of the use of PGD-AS is mosaicism, which has been estimated to be present in up to 50% of cleavage embryos (Baart et al., 2006) as, theoretically, some mosaic embryos could change into a euploid status by means of apoptosis, overgrowth of euploid cells or displacement towards trophectoderm lineage (contributing to the development of extra-embryonic tissues) (Voullaire et al., 2000). Moreover, it has been observed that nearly 25% of the embryos rejected for transfer after PGD-AS have normal cells (Staessen et al., 2004). Additionally, only a cytogenetical confirmation of 32% has been found after re-analysis on day 5 in embryos with either one or two blastomeres biopsied (Baart et al., 2004). The use of PGD-AS could therefore lead to the loss of normal embryos for transfer and eventually jeopardize the chances of success.
In conclusion, our results suggest that under the terms of a SET policy, the implementation of PGD-AS in couples undergoing ICSI because of azoospermia would result in a higher chance of success as the possibility of selecting a euploid embryo is enhanced.
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The authors thank the clinical, paramedical and laboratory staff of the Centre for Reproductive Medicine and Medical Genetics, especially Mrs Marleen Carlé, Sylvie Mertens and Griet Meersdom, who work in the FISH laboratory, Mr Ronny Janssens and Hubert Joris, who carried out the biopsies, and Mr Michael Whitburn for correcting this text.
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Submitted on January 9, 2006; resubmitted on February 10, 2006; resubmitted on March 24, 2006; resubmitted on April 14, 2006; accepted on April 21, 2006.
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