Cumulative reproductive outcome after preimplantation genetic diagnosis: a report on 1498 couples

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1 Human Reproduction, Vol.24, No.11 pp , 2009 Advanced Access publication on August 3, 2009 doi: /humrep/dep272 ORIGINAL ARTICLE Reproductive genetics Cumulative reproductive outcome after preimplantation genetic diagnosis: a report on 1498 couples W. Verpoest 1,5, P. Haentjens 2, M. De Rycke 3, C. Staessen 3, K. Sermon 4, M. Bonduelle 3, P. Devroey 1, and I. Liebaers 3 1 Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, 101 Laarbeeklaan, B-1090 Brussel, Belgium 2 Centre for Outcomes Research and Laboratory for Experimental Surgery, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussel, Belgium 3 Centre for Medical Genetics, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussel, Belgium 4 Department of Embryology and Genetics, Vrije Universiteit Brussel, Brussel, Belgium 5 Correspondence address. willem.verpoest@uzbrussel.be background: Couples undergoing preimplantation genetic diagnosis (PGD) have a different background and set of treatment characteristics to couples undergoing regular IVF or ICSI. The aim of this study was to analyse the cumulative reproductive outcome of a large cohort of couples undergoing PGD in relation to a number of explanatory variables potentially affecting the prognosis. methods: Prospective cohort study, Kaplan Meier analysis was performed to estimate real (observed) and expected (calculated) cumulative delivery rates, and Cox proportional hazard regression analysis was used to assess the effect of age, number of cumulus oocyte complexes collected, fertility status, parity, genetic category and method of pituitary suppression. results: Between 1993 and 2005, 2753 unselected consecutive cycles of ICSI and PGD were carried out in 1498 couples. The cumulative observed delivery rate overall per couple with a maximum of six treatment cycles of ICSI and PGD performed was 29%. The expected cumulative delivery rate (max six cycles) overall was 62%. There were no significant differences in cumulative delivery rates between the different genetic categories (i.e. availability of transferable embryos after PGD of 50 or 75%, chromosomal translocations or aneuploidy screening). The cumulative reproductive outcome in this PGD cohort was also not significantly affected by the fertility status of the couple, their parity or the method of pituitary suppression. However, the age of the patient and the number of oocytes contributed significantly to the reproductive results. conclusion: This prospective observational study demonstrates that age has a significantly negative effect on outcome of PGD, due to poor reproductive performance of female partners 40 of age and older. The number of oocytes collected has a significant and independent effect. The other factors studied did not affect the cumulative reproductive outcome in this PGD cohort. Key words: ICSI / PGD / pregnancy / cumulative delivery rate / life table analysis Introduction Preimplantation genetic diagnosis (PGD) is a procedure used by fertile or infertile couples at high risk of transmitting a genetic condition that allows diagnosis of single gene disorders, chromosomal abnormalities or HLA typing in embryos prior to transfer and implantation. Preimplantation genetic screening (PGS) for aneuploidy is a related procedure that is offered to infertile couples with advanced age of the female partner, previously failed in vitro fertilization (IVF) treatment or unexplained repeated miscarriage, with the aim of improving the success rate of IVF. It allows the enumeration of chosen chromosome pairs and can be considered as an early form of prenatal aneuploidy screening. Ever since the first application of the technique in 1990 (Handyside et al., 1990; Verlinsky et al., 1990), the number of indications for PGD has risen considerably, as has the number of couples, and over cycles of PGD/PGS have been registered in Europe alone, resulting in over 2000 births (Sermon et al., 2004; Goossens et al., 2008). Preselection of embryos by PGD potentially reduces the reproductive prognosis due to embryo manipulation and biopsy, whereas the fact that these couples often do not suffer from a fertility problem may improve their chances of conceiving when undergoing IVF. With increasing numbers of PGD performed, as well as expanding diagnostic possibilities and rapidly growing numbers of children born following PGD, the need for accurate & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 2952 Verpoest et al. outcome analysis is required, in order to correctly counsel couples, and guide them in their decision whether or not to proceed to PGD treatment. In addition, couples need to receive full information on a number of key points including options relating to prenatal diagnostic testing and their associated hazards as well as the risk of embryo transfer cancellation due to unavailability of transferable embryos (The Practice Committee of the Society for Assisted Reproduction and the Practice Committee of the American Society for Repoductive Medicine, 2007). Here we present the cumulative reproductive outcome of a large cohort of couples undergoing PGD and/or PGS (PGD/PGS), in relation to factors potentially influencing reproductive outcome including age, number of oocytes collected, fertility status, parity, genetic category and method of pituitary suppression. Most reports on the reproductive outcome of PGD/PGS have not gone beyond the descriptive analysis of indications, the number of PGD/PGS cycles performed and the number of children born (Verlinsky et al., 2005; Goossens et al., 2008), except for a number of randomized trials of the effectiveness of PGD for aneuploidy screening (PGS) (for overview, see Donoso and Devroey, 2007). Life table analysis, which was used in this study, has both strengths and weaknesses in reproductive medicine (Hull, 1992; Osmanogaoglu et al., 1999; Stolwijk et al., 2000; Daya, 2005) but, in combination with multivariate analysis for a number of relevant explanatory variables, it may give a more realistic view on reproductive outcome in an unselected population when prospective randomized studies are not feasible. The aim of this observational study is to provide a basis for counselling couples on the reproductive prognosis of PGD/PGS, both per cycle and cumulative rates, and the factors contributing to that prognosis. Materials and Methods The study design was a prospective longitudinal cohort study at a tertiary referral centre for ICSI and PGD. Patients The initial outpatient consultation consisted of combined genetic and reproductive assessment and counselling, as well as psychological advice as and when required. Subsequently eligibility of the couple for PGD was assessed for the indication concerned. Treatment with ovarian stimulation and IVF/ICSI plus PGD/PGS was initiated upon completion of the genetic test development. This study did not include results of cycles where frozen-thawed embryos after PGD/PGS were transferred. The couples were categorized according to age, fertility status (documented infertility versus documented absence of infertility), parity (nulliparity versus multiparity), the method of pituitary suppression [gonadotrophin releasing hormone (GnRH) agonist versus GnRH antagonist] and genetic categories according to availability of transferable embryos after PGD (50% transferable embryos versus 75%, Robertsonian translocations, reciprocal translocations, PGD for HLA typing, other chromosomal abnormalities and PGS). The category of 50% genetically transferable embryos includes X-linked dominant conditions in which carrier female embryos may result in affected offspring, and hence have not been transferred. This category also includes X-linked recessives with sexing and transfer of XX embryos only. The category of PGD with HLA typing was assessed separately in view of the small sample size, and regardless of an association with a genetically inherited disorder. The category of other chromosomal abnormalities includes structural or numerical chromosomal abnormalities other than Robertsonian or reciprocal translocations, and may show diverse patterns of inheritance and therefore availability of genetically transferable embryos. The effect of the number of oocytes was assessed by continuous variable analysis. Ovarian stimulation and oocyte retrieval Pituitary desensitization was carried out in an agonist protocol, using GnRH analogues (buserelin, Suprefact; Hoechst, Frankfurt, Germany), in combination with ovarian stimulation with human menopausal gonadotrophins (hmg) (Menopur, Ferring Pharmaceuticals A/S, Copenhagen, Denmark) or recombinant FSH (Puregon, NV Organon, Oss, The Netherlands) (Van de Velde et al., 1998), or in an antagonist protocol with a GnRH antagonist (ganirelix, Orgalutran, NV Organon) combined with recombinant FSH or hmg (Kolibianakis et al., 2004). The starting dose of gonadotrophins was based on the female partner s age and/or previous response to ovarian stimulation (range IU). Human chorionic gonadotrophin (hcg) ( IU, Pregnyl; NV Organon or Profasi, Serono, Geneva, Switzerland) was administered for final oocyte maturation. Transvaginal ultrasound-guided oocyte collection (OC) was scheduled 36 h after hcg administration. OC was carried out under premedication with pethidine 1 mg/kg IM and paracervical block with mepivacaine hydrochloride, or under general anaesthesia as and when indicated (Van de Velde et al., 1998; Kolibianakis et al., 2004). ICSI, embryo culture and biopsy The details of the IVF and ICSI procedure have been described previously (Van Landuyt et al., 2005). In the event of PGD/PGS, ICSI was the method of choice rather than classical IVF to prevent contamination with residual sperm DNA in case of polymerase chain reaction (PCR)-based PGD (Liebaers et al., 1998) and to maximize the fertilization rate in PGS. Fertilization was assessed h after ICSI. Further development was evaluated in the morning of Day 2 and again on Day 3, when embryos were evaluated before biopsy. According to the number of anucleate fragments, the embryos were subdivided into grades A, B, C and D as described previously (Vandervorst et al., 1998). From the 5-cell stage onwards for Fluorescence In Situ Hybridization (FISH) analysis and from the 6-cell stage onwards, embryo biopsy of grade A, B and C performed on Day 3 of culture. In the initial stages of PGD at our centre, an acid solution (Tyrode s solution) was used to breach the zona pellucida. Laser assisted biopsy has been applied since June 1999 as previously described (De Vos and Van Steirteghem, 2001; Sermon, 2002). Overall the aspiration method was used to remove one or two blastomeres from the embryo. For PCR analysis, each blastomere was placed in a solution that lyses the cell and releases the DNA (Sermon et al., 2004). For FISH purposes, a blastomere was spread on a slide using the HCl/ Tween 20 method (Coonen et al., 1994). Genetic diagnosis The PCR procedures were performed as previously described (Sermon et al., 2001; Sermon and De Rycke, 2007). Multiplex PCR is the simultaneous amplification of two or more DNA sequences, and over the it has become the standard method of DNA amplification at single cell level, reducing both the risk of undetected contamination and allele drop out by the use of linked markers alone, or linked markers combined with the detection of a specific mutation (Sermon, 2002). Numerical chromosomal analysis was performed using a FISH procedure, allowing analysis of chromosomes X and Y, and depending on the availability of the fluorochromes at the time of analysis, for chromosome 18, for chromosomes 13 and 21, and at even later stages also for chromosomes 16 and 22 in a second round of hybridization (Staessen et al., 1999, 2004). For reciprocal translocations, the direct labelled and

3 Cumulative reproductive outcome after PGD 2953 commercially available probes (from Vysis or Cytocell) consisted of a combination of three probes, one telomeric and two centromeric, or two telomeric and one centromeric of the chromosomes involved. For Robertsonian translocations, we use a combination of either two subtelomeric or two locus-specific probes or a combination of a subtelomeric and a locus-specific probe. By this approach, the embryos carrying normal or balanced chromosomes can be differentiated from the embryos carrying unbalanced chromosomes. Embryo transfer procedure One or more unaffected embryos were transferred into the uterus on Days 3 5 post-insemination, subject to availability and (legal) restrictions on the number of embryos for transfer. As in regular IVF cycles, the age of the female partner, the rank of trial and embryo quality determined the number of embryos transferred. For Belgian patients, the number of embryos for transfer was restricted according to age and rules laid out by federal law from July 2003 onwards (Van Landuyt et al., 2006). Supernumerary unaffected embryos were cryopreserved subject to consent by the couple. Outcome parameters The embryological data, treatment characteristics, clinical pregnancy rate and delivery rate were analysed on a per cycle basis. The baseline characteristics and cumulative delivery rate were analysed on a per patient basis. A preclinical abortion was defined as an abortion that takes place before clinical or ultrasound evidence of pregnancy. A clinical pregnancy was defined as a gestational sac seen at transvaginal ultrasound scan at least 5 weeks after embryo transfer. If and when the pregnancy proved to be ongoing, and without any exception, the couple was advised to undergo prenatal diagnosis in order to confirm the preimplantation diagnosis. The clinical pregnancy rate was defined as the number of clinical pregnancies expressed per 100 initiated cycles, aspiration cycles or embryo transfer cycles. The delivery rate was defined as the number of live birth deliveries expressed per 100 initiated cycles, aspiration cycles or embryo transfer cycles. A live birth was defined as a birth in which a live fetus is delivered beyond 20 completed weeks of gestational age. A stillbirth was an intrauterine or intrapartum death of a child born at a gestation of 20 weeks or with a birthweight of 500 g (Zegers-Hochschild et al., 2006). An early neonatal death was defined as the death of a live born infant within 7 days after birth. The reproductive outcome parameters in this study are reported per oocyte collection cycle (OCC) and per embryo transfer. The real cumulative delivery rate is the observed number of deliveries born at a gestation of 20 weeks or with a birthweight of 500 g. The expected cumulative delivery rate using Kaplan Meier analysis takes into account drop out patients over a maximum of six treatment cycles, and calculates the cumulative delivery rate had they not discontinued the treatment (Kaplan and Meier, 1958; Hull, 1992; Osmanogaoglu et al., 1999; Malizia et al., 2009). Statistical analysis The cumulative delivery rates were calculated by life table analysis using the Kaplan Meier procedure (Kaplan and Meier, 1958; Hull, 1992; Osmanogaoglu et al., 1999), and are expressed as cumulative percentage probabilities with 95% confidence intervals (CIs). The maximum number of cycles included per couple was six. Transfers of frozen-thawed embryos were not included in the analysis. The effect of different explanatory variables (often called prognostic factors or covariates) including age, fertility status, parity, genetic categories in terms of rate of availability of genetically transferable embryos after PGD, number of oocytes retrieved and method of pituitary suppression were assessed and adjusted for the effects of the other variables using Cox regression analysis. The relationship between the number of oocytes retrieved and the delivery rate was assessed by receiver operating characteristic (ROC) analysis. Exact binomial 95% CIs were computed using STATA 10.0 for Windows (Statacorp, College Station, TX, USA). All other data analyses were performed using SPSS version 17.0 for Windows (SPSS, Inc., Chicago, IL, USA). Results Patient characteristics Between 1993 and 2005, 2753 cycles of ICSI and PGD were carried out in 1498 couples. The mean age overall was 34.4 (SD 5.0). The overall documented infertility rate was 35.3%. Of the female partners, 74% were nulliparous i.e. 36% had had at least one delivery prior to starting PGD treatment. The mean age, documented infertility ratio and parity status in the different genetic categories are illustrated in Table I. The list of indications for PGD performed is available in Supplementary Table A. Treatment characteristics The mean number of treatment cycles per couple overall was 2.1 (SD 1.7). The treatment characteristics in terms of mean number of oocytes collected, the mean number of metaphase II oocytes, the mean number of embryos fertilized, the mean number of embryos biopsied and the mean number of embryos transferred and cryopreserved per OCC are illustrated in Table II, overall as well as per genetic subcategory. Treatment outcome The delivery rate and clinical pregnancy rate, per OCC and per embryo transfer overall and per genetic subcategory, are shown in Table III and Supplementary Table B, respectively. The overall clinical pregnancy rate per OCC was 22.2% (95%CI %) and 30.7% per embryo transfer (95%CI %) (Supplementary Table B). The overall delivery rate per OCC was 17.5% (95%CI %) and 24.2% per embryo transfer (95%CI %) (Table III). There were 481 deliveries, 386 (80.2%) of which were singleton deliveries, 91 (18.9%) were twin deliveries and 4 (0.8%) were triplet deliveries. There have been 581 children born, of which 554 were live born (95.4%), 18 were stillborn (3.1%) and nine were neonatal deaths (1.5%). The cumulative observed delivery rate overall per couple with a maximum of six treatment cycles of ICSI and PGD performed was 29%. The calculated probability including information on censored cases gives an expected cumulative delivery rate of 62% over six cycles. The observed and expected cumulative delivery rates for the different age categories are illustrated in Figs. 1 and 2, respectively (number of patients per cohort, Table IV). As the drop out rate was 100% after three cycles in the group,25 of age, life table analysis was not feasible beyond the third cycle. Cox regression analysis revealed that age and the number of oocytes contribute significantly and independently to the cumulative reproductive outcome in this study, also when simultaneously adjusted for genetic category, fertility status, parity and method of pituitary suppression (Table V). However, the genetic category, expressed as the degree of availability of genetically transferable embryos, did not contribute significantly or independently to the cumulative reproductive outcome of the PGD

4 2954 Verpoest et al. Table I Baseline characteristics of patients according to genetic category Baseline characteristics N patients Mean age Documented infertility (%) Nulliparity (%)... Overall (SD 5.0) % Inheritance (SD 4.3) % Inheritance (SD 4.1) Robertsonian translocations (SD 5.0) Reciprocal translocations (SD 4.5) HLA typing (SD 4.4) Other chromosomal abnormalities (SD 5.6) PGS (SD 4.8) Table II Treatment characteristics of patients according to genetic category Treatment Mean # Mean # Mean # Mean # Mean # Mean # embryo Mean # characteristics treatment cycles COC MII 2PN biopsy transfer cryo... Overall 2.1 (SD 1.7) 12.6 (SD 6.7) 10.4 (SD 6.0) 7.8 (SD 4.8) 5.6 (SD 3.8) 1.3 (SD 1.0) 0.4 (SD 1.3) 50% Inheritance 2.2 (SD 1.4) 12.7 (SD 7.1) 10.7 (SD 6.4) 8.1 (SD 5.0) 5.3 (SD 3.7) 1.3 (SD 0.9) 0.3 (SD 0.9) 25% Inheritance 2.2 (SD 1.5) 13.1 (SD 7.0) 10.8 (SD 5.8) 8.0 (SD 4.8) 5.7 (SD 3.7) 1.5 (SD 1.1) 0.7 (SD 2.1) Robertsonian 1.7 (SD 1.0) 14.8 (SD 7.5) 12.0 (SD 7.1) 8.8 (SD 5.6) 6.1 (SD 4.8) 1.1 (SD 0.9) 0.6 (SD 1.5) translocations Reciprocal 2.0 (SD 1.2) 13.7 (SD 5.6) 10.8 (SD 5.5) 8.3 (SD 4.5) 6.0 (SD 4.0) 0.8 (SD 0.9) 0.1 (SD 0.5) translocations HLA typing 2.6 (SD 1.6) 13.1 (SD 8.3) 10.3 (SD 7.9) 7.9 (SD 6.2) 5.5 (SD 5.5) 0.6 (SD 0.8) 0.3 (SD 0.8) Other chromosomal 1.8 (SD 1.0) 12.6 (SD 6.7) 10.3 (SD 5.7) 7.2 (SD 4.5) 5.3 (SD 3.3) 1.3 (SD 0.9) 0.4 (SD 1.2) abnormalities PGS 1.6 (SD 0.9) 12.0 (SD 6.3) 10.0 (SD 5.8) 7.5 (SD 4.6) 5.6 (SD 3.8) 1.3 (SD 1.1) 0.4 (SD 1.3) COC, cumulus-oocyte complex; Mll, metaphase ii; 2PN, 2 pronucleus. Table III Delivery rate per OCC and per embryo transfer according to genetic category Delivery rate Per OCC (n ) 95% CI Per embryo transfer (n ) 95% CI... Overall 17.5% (481/2753) % 24.2% (481/1987) % 50% 17.2% (109/635) % 22.5% (109/485) % 25% 20.6% (73/355) % 26.1% (73/280) % Robertsonian translocations 25.5% (24/94) % 38.1% (24/63) % Reciprocal translocations 11.6% (22/190) % 23.2% (22/95) % HLA typing 7.8% (4/51) % 17.4% (4/23) % Other chromosomal abnormalities 21.1% (26/123) % 27.1% (26/96) % PGS 17.1% (223/1305) % 23.6% (223/945) % Percentages with exact binomial 95% CIs. cohort studied (Table V, Supplementary Table C). The cumulative reproductive outcome in this PGD cohort was also not significantly affected by the fertility status of the couple, their parity, or the method of pituitary suppression (Table V). ROC analysis confirmed an influence of the number of oocytes as an independent and significant factor on success rate, with an area under the curve (AUC) of 63%. In 393 cycles (14.3%), one or more embryos were cryopreserved after PGD/PGS, totalling a number of 1052 embryos (mean 2.7 per cycle). One hundred and fifty-four thawing cycles have been performed, resulting in 62 frozen-thawed embryo transfers (40.3% per thawing cycle). There were 12 cycles which resulted in a pregnancy, of which four were biochemical, one was an early miscarriage, one was an extrauterine pregnancy and six were singleton live births

5 Cumulative reproductive outcome after PGD 2955 Figure 1 Observed cumulative delivery rates after PGD in different age groups. Figure 2 Expected cumulative delivery rates after PGD in different age groups.

6 2956 Verpoest et al. Table IV Number of patients entered in each cohort per oocyte retrieval cycle, according to the woman s age Number of Treatment patients in cycle number cohort by woman s age... Group, Group Group Group Group 40þ (delivery rate 4.5% per thawing cycle, 15.4% per frozen-thawed embryo transfer). Discussion Over a period of 12, we assessed the outcome of 2753 ICSI-PGD cycles in 1498 couples. The main findings of this study were (i) that age and the number of oocytes significantly affect the reproductive outcome in PGD and (ii) that the rate of availability of genetically transferable embryos after PGD, fertility status and parity do not have a significant effect. The strength of this study is that it provides reliable cumulative probabilities of successful reproductive outcome in a large cohort of consecutive ICSI and PGD cycles at a single centre, in relation to a number of relevant explanatory variables. The effect of genetic selection of embryos is unfavourable as the expected cumulative delivery rate of 62% is much lower than the 79% expected cumulative delivery rate reported in a non-pgd ICSI population at our centre (Osmanogaoglu et al., 1999). However, the mode of inheritance hence the rate of genetically transferable embryos did not have a significant effect as an independent factor on cumulative reproductive outcome. Although the delivery rate per OCC is lower in some groups, particularly in the reciprocal translocations group, as is to be expected as a result of a higher rate of unbalanced and therefore untransferable embryos (Table III), the reproductive performance in terms of cumulative delivery rate is not significantly lower. The clinical pregnancy rate and delivery rate per embryo transfer are similar in all genetic subgroups, suggesting an isolated effect of genetic embryo selection especially in some subgroups such as the reciprocal translocations group where the availability of transferable embryos after PGD as low as 30%. The lack of a significant influence of the HLA typing group results on the cumulative reproductive outcome, as well as the lack of effect of number of oocytes in the HLA group, is presumably attributable to underpowering. The number of oocytes collected at retrieval significantly contributes to the cumulative reproductive outcome as an independent Table V Contribution of factors to cumulative reproductive outcome based on logistic regression modelling OR (95% CI)... Unadjusted odds Adjusted odds ratio ratio*... Age,25 reference category ( ) 0.92 ( ) ( ) 0.61 ( ) ( ) 0.53 ( ) ( ) 0.22 ( ) Number of COC 1.02 ( ) 1.02 ( ) Genetic category 50% Reference category 75% Available embryos 1.20 ( ) 0.90 ( ) for transfer Robertsonian 1.39 ( ) 1.24 ( ) translocations Reciprocal 0.72 ( ) 0.75 ( ) translocations HLA typing 0.56 ( ) 0.73 ( ) Other chromosomal 1.11 ( ) 1.13 ( ) abnormalities PGS 0.98 ( ) 1.36 ( ) Fertility status 1.04 ( ) 1.00 ( ) Parity 0.93 ( ) 1.09 ( ) Mode of pituitary 0.93 ( ) 1.07 ( ) suppression *Odds ratio simultaneously adjusted for all other variables listed in the first column of Table 5. factor with an OR of 1.02, however, ROC analysis reveals that the AUC at 63% does not allow setting a threshold below which the prognosis is significantly impaired. This is in contrast with previous studies reporting a threshold level of nine oocytes below which a significant reduction in reproductive outcome could be expected (Vandervorst et al., 1998). Very few publications have addressed this issue in PGD. A recent study by Tur-Kaspa et al. showed that the availability of normal/unaffected embryos and the chances of pregnancy increase with increasing numbers of oocytes retrieved, but that a lower number of oocytes (,8) is still associated with a fair chance for normal/unaffected blastocyst transfer, especially in young patients, implying that routine cancelling of cycles with an anticipated low number of oocytes is to be reconsidered (Tur-Kaspa et al., 2007). The fact that the fertility status did not contribute significantly to the cumulative reproductive prognosis is to be attributed to the bias effect of ICSI. Previous studies showed that the cumulative probability of an ongoing pregnancy was higher in women with secondary subfertility than in those with primary subfertility (Stolwijk et al., 2000). This was not confirmed in our study, where multiparity did not have a significant independent effect on cumulative reproductive outcome of

7 Cumulative reproductive outcome after PGD 2957 PGD, most probably due to the particular population with a low documented infertility ratio (35.3%). Consistent with reports in regular assisted reproductive treatment (Templeton et al., 1996), advanced age of the female has a significant and independent negative effect on reproductive outcome, and those couples in which the female partner is aged 40 and over should be counselled very clearly on the very limited prognosis (Figs. 1 and 2). Within the group over 39 of age, there are no significant differences. Although a trend is observed towards a highly reduced prognostic outcome at age 42 and beyond, the sample size within this age group is too small to allow significant conclusions. Cumulative life table analysis is based on the assumption that dropout patients have the same probability as patients proceeding to a next attempt for IVF or ICSI after failed treatment. Previous studies have established the validity of life table analysis as a prognostic tool, in reproductive medicine (Hull, 1992; Osmanogaoglu et al., 1999; De Vries et al., 1999) even though the expected cumulative delivery rate potentially overestimates the true reproductive outcome over a large number of treatment cycles, due to informative censoring as well as a selection bias of good responders. In our study, the observation that the mean number of cycles per couple overall is 2.1 (SD 1.7) and the consequent finding that the observed cumulative delivery rate barely increases in treatment cycles 4 6 indicate that figures of cumulative reproductive outcome beyond three cycles should be interpreted with caution. In spite of a statistical increase in reproductive outcome in cycles 4 6 in all age groups, the clinical relevance is limited due to both informative and non-informative censoring. Although the data did not allow statistical comparison between genetic categories of the mean number of cycles performed, the latter does not seem differ in the Robertsonian or the reciprocal translocation group compared with the other groups, indicating that censoring did not occur more often in these indications. The same conclusions therefore apply with regard to clinical relevance of cumulative outcome in higher order cycles for couples undergoing PGD for chromosomal translocations. Previous studies have confirmed that there is no significant difference in background characteristics and/or the occurrence of prognostic indicators of poor treatment) of couples who continued treatment versus those who dropped out i.e. there is no informative censoring in the first two (Roest et al., 1998) to three treatment cycles (Croucher et al., 1998). The proportion of couples in our study who discontinued treatment remained almost constant over the cycles observed, with overall proportions varying between 42 and 50%, and perhaps most importantly, the proportions did not increase over the cycles observed. This is consistent with dropout rates of 23 45% for regular IVF/ICSI reported in literature (Gleicher et al., 1996), but is in contrast to reports of dropout rates increasing with each successive cycle (Land et al., 1997). We were not able to establish the reasons for dropout, but the mean number of cycles performed is similar to that reported in other studies (Osmanogaoglu et al., 1999 (mean 1.91); Malizia et al. (2009) (mean 2.3). The mean number of cycles performed in the HLA typing group of couples is remarkably higher at 2.6 (SD 1.6), indicating a higher level of compliance, in spite of limited success rates secondary to a highly reduced mean number of transferable embryos and a higher mean female partner age (Van De Velde et al., 2008). Attempts have been made at designing more accurate statistical tools to assess cumulative reproductive outcome in reproductive medicine, including selective dropout exclusion (Stolwijk et al., 1996; Land et al., 1997) and multiple imputation (Soullier et al., 2008). Whereas in conventional IVF/ICSI, time to conception is the ideal standard, this is difficult to apply in PGD due to several factors. The time of intake is not usually the start of the treatment, i.e. there is usually a significant time lag needed for establishing the details of the genetic condition and preparing the PGD test. This time period until the actual start of the PGD treatment varies according to the genetic condition tested for, and can vary from 2 to 24 months. The interval between different treatments is often shorter due to a higher embryo transfer cancellation rate and a higher degree of priority, especially with those disorders that require PGD associated with HLA matching. The optimum time scale criterion for PGD outcome analysis therefore seems to be related to the treatment cycle. Despite the limitations of life table analysis for PGD in terms of time scale criterion and informative censoring to some degree, as well as a number of modifications to diagnostic and therapeutic modalities over the that are studied in this article, subjects included towards the latter part of the study group are not likely to differ systematically from those who underwent PGD earlier in the study. However, secular trends (Daya, 2005) are to some degree inevitable, due to changes in ovarian stimulation regimens (e.g. the introduction of antagonist regimens as from 1996), the application of single versus double cell biopsy (Goossens et al., 2008) and the changes in embryo transfer policy, but the changes have been relatively confined by the consistent genetic selection criteria and genetic diagnostic techniques, the limited number of ovarian stimulation protocols available and applied, as well as the consistent ICSI technique applied. Conclusion The results of our study in terms of cumulative reproductive outcome per age group, genetic and other categories as well as the influence of a number of explanatory variables on reproductive outcome of PGD presented here provide a basis for correct counselling of couples undergoing ICSI and PGD, and may serve as a guide in their decision whether or not to undergo this treatment, especially as PGD couples often do not suffer from an infertility problem. In view of the significant effect of age on reproductive outcome of PGD, consideration should be given to counselling couples in which the female partner is over 39 about their poor reproductive prognosis, especially in the light of genetic selection of embryos. The number of oocytes at retrieval contributes significantly to the reproductive outcome; however, a critical threshold level cannot be determined. The clinical value of cumulative reproductive analysis in relation to a number of relevant explanatory variables is more representative of true outcome than figures per cycle, however, the clinical value beyond the first three treatment cycles is limited. Supplementary data Supplementary data are available at Acknowledgements The authors thank the clinical, laboratory, technical, nursing and secretarial staff at the Centre for Reproductive Medicine and the

8 2958 Verpoest et al. Centre for Medical Genetics at UZ Brussel. We would like to thank Mr Pieter Verdyck and Mr Walter Meul for their significant contribution in data gathering and analysis. Funding This study was supported by The Fund for Scientific Research (FWO), Flanders and the Onderzoeksraad, Universitair ZIekenhuis Brussel, Vrije Universiteit Brussel. References Coonen E, Dumoulin JCM, Ramaekers FCS, Hopman AHN. Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescent in situ hybridisation. Hum Reprod 1994; 9: Croucher CA, Lass A, Margara R, Winston RM. Predictive value of the results of a first in-vitro fertilization cycle on the outcome of subsequent cycles. Hum Reprod 1998;13: Daya S. Life table (survival) analysis to generate cumulative pregnancy rates in assisted reproduction: are we overestimating our success rates? Hum Reprod 2005;20: De Vos A, Van Steirteghem A. Aspects of biopsy procedures prior to preimplantation genetic diagnosis. 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