1 SISMeR, Reproductive Medicine Unit, Via Mazzini 12, 40138 Bologna, Italy and 2 IFER, Reproductive Medicine Unit, Buenos Aires, Argentina
3 To whom correspondence should be addressed. e-mail: sismer{at}sismer.it
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Abstract |
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Key words: aneuploidy/fluorescence in situ hybridization (FISH)/IVF/poor prognosis/preimplantation genetic diagnosis (PGD)
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Introduction |
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Here we present evidence of a prognostic role for PGD for aneuploidy on subsequent treatment cycles. There are several predictive factors described in the literature regarding the success of IVF, such as maternal age (recognized as the most important factor affecting the outcome of fertility treatments), number of retrieved oocytes, number of available embryos, embryo quality, fertilization rate, duration of infertility, previous pregnancies and live births, and number of previous cycles (Edwards et al., 1984; Sharma et al., 1988
; Hull et al., 1992
; Mackenna et al., 1992
; Tan et al., 1992
; Roseboom et al., 1995
; Templeton et al., 1996
). In young couples with a normal response to hormonal stimulation and a normal karyotype, the probability of achieving a pregnancy is the same during the first three attempts (Sharma et al., 2002
). However, the delivery rate is dramatically reduced when ART is performed in young women who had already experienced several unsuccessful transfers or in women of advanced reproductive age. It is not clear whether one major, or multiple causes contribute to this fertility decline, but aneuploidy has been demonstrated to play an important role in determining embryo viability (Gianaroli et al., 1999a
; Munné et al., 1999
). Prognostic criteria are needed to counsel these couples appropriately regarding their decision either to continue therapy to have a biological child or to cease treatment.
In a previous study (Gianaroli et al., 2002), the possible role of the first PGD for aneuploidy in the prognosis of subsequent attempts had already been suggested. The aim of this study was to confirm the validity of this proposal by adding more data and performing a rigorous analysis after the elimination of any possible variable that could affect the conclusions.
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Material and methods |
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The percentage of transferred cycles, pregnancy rate per embryo transfer and live birth rate (LBR) per patient in subsequent cycles were evaluated in each group. Analysis of cumulative LBR from subsequent cycles using the life table approach was also performed, assuming that those who dropped out would have the same probability of pregnancy as those who continued.
Controlled ovarian stimulation was performed as previously described (Ferraretti et al., 1996). At 3436 h after HCG administration, oocytes were collected transvaginally via ultrasound guidance; IVF or ICSI was performed depending on semen characteristics. Embryo biopsy was performed on day 3 embryos having at least five blastomeres and a percentage of fragmentation not greater than 50%. The biopsied cells were fixed and analysed by multicolour fluorescence in situ hybridization (FISH) as previously described (Gianaroli et al., 1999b
). Up to November 1997, chromosomes X, Y, 13, 16, 18 and 21 were analysed in a one-step FISH protocol; subsequently, a second panel was also screened in a two-step protocol with probes specific for chromosomes 14, 15 and 22. Beginning in December 1998, the panels were revised: the first was specific for chromosomes 13, 16, 18, 21 and 22, and the second panel probes detected chromosomes X, Y, 15 and 17. In July 2001, the probe for chromosome 17 was replaced by a telomeric probe specific for chromosome 21 that, since then, has been screened twice, in both the first and the second panels (Magli et al., 2001a
).
In all cycles, embryo transfer was performed on day 4 (Gianaroli et al., 1999b). A maximum of three embryos were transferred until 1998 and a maximum of two embryos were transferred from 1999. Every patient received luteal phase support with progesterone in oil (50 mg/day). Couples with an altered karyotype were not included in the study, neither were poor responder patients (
3 collected oocytes). Written informed consent was obtained from all couples. This study was approved by our institutional review board.
Pregnancies were defined by confirmation of an intrauterine sac at ultrasound or the excision of an ectopic pregnancy. The implantation rate represents the number of gestational sacs with fetal heart beat divided by the total number of embryos transferred. Live birth is defined as any birth event in which at least one baby is born alive and survives for >1 month.
Statistical analysis
Data were analysed by 2 analysis and Fishers exact test for proportions. Differences between groups have been evaluated with a non-parametric test (MannWhitney U-test). The critical value (5%) approximated to the normal was calculated as follow: U
=0.05 = n1n2/2 z
[(n1n2)(n1n2 + 1)/12]2 (Camussi et al., 1995
).
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Results |
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A total of 1672 embryos were analysed by FISH: 344 in group A, 426 in group B and 902 in group C. Due to the higher number of oocytes collected in group C, the mean number of embryos analysed/cycle was significantly higher in this group compared with the others (Figure 1b). The results of the PGD test are shown in Figure 2 where the cumulative proportion in the subsequent cycles of FISH-normal (i.e. euploid) embryos is given. Adding the second and third PGD cycle results, group A patients, who by definition had no euploid embryos in the first PGD cycle, maintained in the following cycles a significantly lower incidence of chromosomally normal embryos compared with group B and group C. Only 29 embryos out of 344 (8.4%) were diagnosed normal by FISH in group A, 95 out of 426 (22.3%) in group B and 292 out of 902 (32.4%) in group C (A versus B and C, P < 0.001).
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Discussion |
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Any predictive factor of pregnancy is extremely valuable for patients, especially those who belong to apparently homogeneous categories defined as having a poor prognosis. Estimation of the success rate is also important for the social costs of treatments.
Before being admitted to PGD for aneuploidy, the couples in the present study represented a group of normal responder patients who shared the common definition of poor prognosis ART population due to advanced maternal age or 3 previous unsuccessful cycles. Despite a higher number of oocytes collected in group C, an average of eight oocytes was retrieved from group A and B who, in relation to their age and considering their poor prognosis condition, cannot be considered as poor responders. Fertilization, cleavage rate and embryo morphology were consistent with a normal gametes competence (similar among the groups) to produce in vitro viable embryos. However, when these couples underwent their first PGD for aneuploidy, the detection of a significant difference in the proportion of euploid embryos enabled the identification of different subpopulations; this difference remained unchanged in the subsequent PGD attempts. Couples with no euploid embryos in the first cycle had a lower probability of reaching embryo transfer in subsequent attempts compared with the other groups. Accordingly, the prognosis of term pregnancy was significantly decreased (8.5% LBR in group A versus 26.4% in group B + C, P < 0.05). The present findings suggest that the performance in the first PGD for aneuploidy is strongly predictive of the clinical outcome in subsequent ART attempts.
When the cumulative LBR for subsequent cycles was analysed using the life table approach, patients with two or more euploid embryos in the first PGD for aneuploidy had almost a 70% cumulative term pregnancy rate after two additional cycles. On the other hand, when no euploid embryos were detected in the first PGD for aneuploidy, only 15% of the patients obtained a term pregnancy in the following attempts, confirming that these patients have a very poor prognosis for pregnancy. Despite the limitations in the life table analysis (Engmann et al., 1999) and the numbers generated in the present study, the clinical implications seem to be relevant. The prognostic assessments based on cumulative conception rates of a specific programme permits one to advise patients on the basis of the provisions made in the framework of PGD experience. The data generated from this analysis demonstrate that the chances of pregnancy are high in women with at least two euploid embryos diagnosed in the first PGD for aneuploidy. Therefore, further attempts can be encouraged in these patients.
These findings support our preliminary data (Gianaroli et al., 2002) and strongly suggest that the transfer of FISH-selected embryos using PGD for aneuploidy in poor prognosis patients is not only associated with a high implantation rate and a low incidence of spontaneous abortions (Table I) (Ferraretti et al., 1999
; Gianaroli et al., 1999a
; Munné et al., 1999
), but also represents a favourable prognostic index.
In conclusion, the current results strongly suggest that PGD for aneuploidy has a predictive role on the outcome of subsequent cycles, at least in couples with a poor prognosis of pregnancy. Patients who develop only aneuploid embryos have the tendency to generate the same performance in subsequent treatments. Accordingly, the prognosis of pregnancy is very poor (<10%). Conversely, when euploid embryos are detected in the first PGD cycle, the prognosis is highly favourable.
In infertile couples diagnosed with a poor prognosis by conventional ART, PGD for aneuploidy can be regarded as a tool for estimating the ability of each couple to generate euploid embryos. This information may be useful in predicting the outcome of subsequent cycles by recognizing the subpopulation of poor prognosis patients for which further attempts are still recommended. As represented in the life table calculated after PGD (Figure 3), there is a constant increase in the percentage of full-term pregnant patients per cycle irrespective of the low number of embryos transferred. This is opposite to what happens in conventional IVF/ICSI cycles (Azem et al., 1995; Meldrum et al., 1998
) and should stimulate physicians to support couples with positive predictions to persist in their quest for children. On the other hand, patients with a negative prognosis could be assisted in the difficult decision of refraining from further attempts or, after undergoing chromosomal analysis of the couples female and male gametes, considering the possibility of resorting to gamete donation.
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Submitted on May 6, 2003; resubmitted on September 23, 2003; accepted on November 7, 2003.