Prognostic role of preimplantation genetic diagnosis for aneuploidy in assisted reproductive technology outcome

A.P. Ferraretti1,3, M.C. Magli1, L. Kopcow2 and L. Gianaroli1

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


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
BACKGROUND: Preimplantation genetic diagnosis (PGD) for aneuploidy is recommended to couples at risk of generating chromosomally abnormal embryos. The aim of this study was to demonstrate that PGD for aneuploidy has an important role in the prognosis of subsequent treatments. METHODS: A total of 389 couples underwent their first PGD for aneuploidy due to either female age ≥38 years (n = 266) or ≥3 previous unsuccessful cycles (n = 123). After the first PGD followed by an unsuccessful treatment cycle, 141 couples underwent 175 subsequent PGD cycles. These patients were divided into three groups depending on the number of euploid embryos available for transfer in their first PGD cycle: group A included patients where no euploid embryos were diagnosed; group B included patients who had only one euploid embryo; and group C included patients with at least two normal embryos resulting from chromosomal analysis. RESULTS: In subsequent cycles, group A patients underwent significantly fewer transfers (45%) compared with group B (69%, P < 0.05) and group C patients (85%, P < 0.001). The pregnancy rate per transfer was significantly decreased in group A (15%) compared with group B (36%; P < 0.02) and group C (30%; P < 0.03). Accordingly, the live birth rate per patient was significantly lower in group A compared with group C (8.5% versus 30%; P < 0.005). CONCLUSIONS: The outcome of the first PGD for aneuploidy may have a predictive role for subsequent attempts.

Key words: aneuploidy/fluorescence in situ hybridization (FISH)/IVF/poor prognosis/preimplantation genetic diagnosis (PGD)


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Preimplantation genetic diagnosis (PGD) is a technique developed for the screening of monogenic diseases and chromosomal abnormalities in preimplantation embryos. PGD for aneuploidy screening has been available in our centre since 1996 for couples presenting one of the following indications: female age ≥38 years, ≥3 previous unsuccessful embryo transfers with conventional assisted reproductive technology (ART), altered karyotype or recurrent miscarriage (Gianaroli et al., 2002Go). These couples are especially at risk of generating chromosomally abnormal embryos (Verlinsky et al., 1995Go; Gianaroli et al., 1997Go; Magli et al., 1998Go, 2001b; Munné et al., 2002Go). It has been reported that the transfer of chromosomally normal embryos increases the implantation rate and diminishes the risk of abortion and trisomic pregnancies (Gianaroli et al., 1999aGo; Munné et al., 1999Go).

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., 1984Go; Sharma et al., 1988Go; Hull et al., 1992Go; Mackenna et al., 1992Go; Tan et al., 1992Go; Roseboom et al., 1995Go; Templeton et al., 1996Go). 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., 2002Go). 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., 1999aGo; Munné et al., 1999Go). 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., 2002Go), 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.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
From September 1996 to June 2002, 389 couples underwent their first PGD for aneuploidy at SISMeR Reproductive Medicine Unit, due to female age ≥38 years (n = 266) or ≥3 previous unsuccessful cycles (n = 123). Couples who repeated at least one PGD cycle after the first unsuccessful PGD treatment, which included those with no euploid embryos available for transfer and those who did not obtain a term pregnancy after the transfer of at least one euploid embryo, were included in the study. These patients were divided into three groups depending on the number of euploid embryos which were diagnosed after chromosomal analysis in their first PGD cycle: group A included patients with no euploid embryos diagnosed at the first attempt; group B included patients with only one euploid embryo; and group C included patients with at least two euploid embryos.

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., 1996Go). At 34–36 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., 1999bGo). 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., 2001aGo).

In all cycles, embryo transfer was performed on day 4 (Gianaroli et al., 1999bGo). 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 {chi}2 analysis and Fisher’s exact test for proportions. Differences between groups have been evaluated with a non-parametric test (Mann–Whitney U-test). The critical value (5%) approximated to the normal was calculated as follow: U{alpha}=0.05 = n1n2/2 – z{alpha}[(n1n2)(n1n2 + 1)/12]–2 (Camussi et al., 1995Go).


    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Table I shows the results of the 389 couples who underwent their first PGD for aneuploidy treatment and the percentage of couples who repeated PGD for aneuploidy. According to FISH diagnosis, the replacement of at least one euploid embryo was possible for 244 couples, yielding 59 viable pregnancies. In the remaining 145 cycles, embryo transfer was cancelled as no chromosomally normal embryos were diagnosed. Out of the 330 couples who failed the first PGD cycle, 141 (43%) entered a second PGD attempt and 34 a third PGD attempt, accounting for 175 subsequent PGD cycles (study group). In detail, the study group included 35 patients from group A who underwent 44 cycles, 40 patients from group B who underwent 48 cycles and 66 patients from group C who underwent 83 cycles. Patients in the study group from groups A, B and C were homogeneous in terms of female age (group A, 37.84 ± 4.6; group B, 37.54 ± 3.54; group C, 36.64 ± 3.88 years), cause of infertility and reproductive history.


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Table I. Results of the first PGD for aneuploidy
 
Figure 1a shows the number of collected oocytes in the first, second and third PGD cycles. Group C patients were higher responders (P < 0.05) compared with group A and B although all studied patients were considered to be ‘good responders’ in relation to their age and the number of collected oocytes (≥8 oocytes in all groups).




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Figure 1. Mean number of oocytes retrieved (a) and mean number of embryos analysed by FISH (b) in the first PGD for aneuploidy and subsequent attempts in groups A, B and C. Numbers with the same superscript are significantly different (P < 0.05).

 
The cumulative fertilization rate did not differ among the groups (67, 69 and 68%, respectively) as well as the cleavage rate to 5–8 cell embryos on day 3 (73, 76 and 75%). According to these results, a comparable oocyte and sperm competence to generate morphologically normal embryos seemed to be present in all couples.

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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Figure 2. Percentage of euploid embryos generated in groups A, B and C in the first PGD for aneuploidy and subsequent attempts. Numbers with the same superscript are significantly different.

 
Table II shows the clinical outcome resulting from the repeated PGD cycles in the study group. Significantly fewer patients in group A underwent embryo transfer (45%) compared with group B (69%, P < 0.05) and group C (85%, P < 0.001). Although the implantation rate did not differ, the pregnancy rate per transfer was lower in group A (15%) when compared with group B (36%, P < 0.02) and group C (30%, P < 0.03). Similarly, the LBR per patient was significantly lower in group A in relation to group C (8.5% versus 30%, P < 0.005).


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Table II. Results of subsequent PGD for aneuploidy cycles
 
In Table III, the data are subdivided according to the indication for PGD, female age ≥38 years or ≥3 previous unsuccessful cycles. In both cases, patients in group A maintained a significantly lower probability of undergoing embryo transfer compared with group C, while the difference in the LBR lost statistical significance. Nevertheless, a trend towards an increased LBR in group C was detected in both indications.


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Table III. Clinical outcome according to PGD for aneuploidy indications
 
Figure 3 demonstrates the cumulative LBR for subsequent cycles using the life table approach. According to this analysis, the cumulative chances of pregnancy were 70% at the third cycle for group C patients, 50% for group B patients and only 15% in group A patients.



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Figure 3. Life table representing the cumulative percentage of term pregnancy calculated per patient. Numbers with the same superscript are significantly different.

 

    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
IVF is widely used to treat infertility. Several factors have contributed to a substantial improvement in the success rate of this therapy during the last 10 years, offering young women a high cumulative pregnancy rate in the first three cycles (Engmann et al., 1999Go). Nevertheless, many couples with a poor prognosis for full-term pregnancy face the difficult decision of whether to continue therapy or desist from unfruitful attempts. In addition, women approaching infertility treatment in advanced reproductive age have a low chance of pregnancy due to the physiological decline in their oocyte quality.

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 gamete’s 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., 1999Go) 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., 2002Go) 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., 1999Go; Gianaroli et al., 1999aGo; Munné et al., 1999Go), 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., 1995Go; Meldrum et al., 1998Go) 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 couple’s female and male gametes, considering the possibility of resorting to gamete donation.


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Azem F, Yaron Y, Amit A, Yovel I, Barak Y, Peyser MR, David MP and Lessing JB (1995) Transfer of six or more embryos improves the success rate in patients with repeated in vitro fertilization failures. Fertil Steril 63,1043–1046.[ISI][Medline]

Camussi A, Moller F., Ottaviano E and Sari Gorla M (1995) Metodi Statistici per la Sperimentazione Biologica, 2nd edn. Zanichelli, Bologna.

Edwards RG, Fishel SB, Cohen J, Fehilly CB, Purdy JM, Slater JM, Steptoe PC and Webster JM (1984) Factors influencing the success of in-vitro fertilization for alleviating human infertility. J In Vitro Fertil Embryo Transf 1,3–23.[Medline]

Engmann L, Maconochie N, Bekir JS, Jacobs HS and Tan SL (1999) Cumulative probability of clinical pregnancy and live birth after a multiple cycle IVF package: a more realistic assessment of overall and age-specific success rates? Br J Obstet Gynaecol 106,165–170.[ISI][Medline]

Ferraretti AP, Magli MC, Feliciani E, Montanaro N and Gianaroli L (1996) Relationship of timing agonist administration in the cycle phase to the ovarian response to gonadotropins in the long down-regulation protocols for assisted reproductive technologies. Fertil Steril 65,114–121.[ISI][Medline]

Ferraretti AP, Gianaroli L, Magli MC, Feliciani E, Gergolet M and Fortini D (1999) The impact of preimplantation genetic diagnosis for aneuploidy on the implantation and abortion rates in patients over 37 years old. Hum Reprod 14,99–100.[Medline]

Gianaroli L, Magli MC, Munné S, Fiorentino A, Montanaro N and Ferraretti AP (1997) Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum Reprod 12,1762–1767.[Abstract]

Gianaroli L, Magli MC, Ferraretti AP and Munné S (1999a) Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 72,837–844.[CrossRef][ISI][Medline]

Gianaroli L, Magli MC, Munné S, Fortini D and Ferraretti AP (1999b) Advantages of day 4 embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy. J Assist Reprod Genet 16,170–175.[CrossRef][ISI][Medline]

Gianaroli L, Magli MC, Ferraretti AP, Tabanelli C, Trombetta C and Boudjema E (2002) The role of preimplantation diagnosis for aneuploidies. Reprod BioMed Online 4,31–36.[Medline]

Hull MG, Eddowes HA, Fahy U, Abuzeid MI, Mills MS, Cahill DJ, Fleming CF, Wardle PG, Ford WC and McDermott A (1992) Expectations of assisted conception for infertility. Br Med J 304,1465–1469.[ISI][Medline]

Mackenna AI, Zegers-Hochschild F, Fernandez EO, Fabres CV, Huidobro CA, Prado JA, Roblero LS, Altieri EL, Guadarrama AR and Lopez TH (1992) Fertilization rate in couples with unexplained infertility. Hum Reprod 7,223–226.[Abstract]

Magli MC, Gianaroli L, Munné S and Ferraretti AP (1998) Incidence of chromosomal abnormalities from a morphologically normal cohort of embryos in poor prognosis patients. J Assist Reprod Genet 15,297–301.[CrossRef][ISI][Medline]

Magli MC, Sandalinas M, Escudero T, Morrison L, Ferraretti AP, Gianaroli L and Munné S (2001a) Double locus analysis of chromosome 21 for preimplantation genetic diagnosis of aneuploidy. Prenat Diagn 21,1080–1085.[CrossRef][ISI][Medline]

Magli MC, Gianaroli L and Ferraretti AP (2001b) Chromosomal abnormalities in embryos. Mol Cell Endocrinol 183,29–34.[CrossRef][ISI][Medline]

Meldrum DR, Silverberg KM, Bustillo M and Stockes L (1998) Success rate with repeated cycles of in vitro fertilization–embryo transfer. Fertil Steril 69,1005–1009.[CrossRef][ISI][Medline]

Munné, S., Magli C, Cohen J, Morton P, Sadowy S, Gianaroli L, Tucker M, Marquez C, Sable D, Ferraretti AP et al. (1999) Positive outcome after preimplantation diagnosis of human embryos. Hum Reprod 14,2191–2199.[Abstract/Free Full Text]

Munné, S., Cohen J and Sable D (2002) Preimplantation genetic diagnosis for advanced maternal age and other indications. Fertil Steril 78,234–236.[ISI][Medline]

Roseboom TJ, Wermeiden JPW, Schoute E, Lens JW and Schats R (1995) The probability of pregnancy after embryo transfer is affected by the age of the patients, cause of infertility, number of embryos transferred and the average morphology score, as revealed by multiple logistic, regression analysis. Hum Reprod 10,3035–3041.[Abstract]

Sharma V, Riddle A, Mason BA, Pampiglione J and Campbell S (1988) An analysis of factors influencing the establishment of a clinical pregnancy in an ultrasound-based ambulatory in vitro fertilization programme. Fertil Steril 49,468–478.[ISI][Medline]

Sharma V, Allagar V and Rajkhowa M (2002) Factors influencing the cumulative conception rate and discontinuation of in vitro fertilization treatment for infertility. Fertil Steril 78,40–46.[ISI][Medline]

Tan SL., Royston P, Campbell S, Jacobs HS, Betts J, Mason B and Edwards RG (1992) Cumulative conception and livebirth rates after in-vitro fertilization. Lancet 339,1390–1394.[CrossRef][ISI][Medline]

Templeton A, Morris JK and Parslow W (1996) Factors that affect outcome of in-vitro fertilization treatment. Lancet 348,1402–1406.[CrossRef][ISI][Medline]

Verlinsky Y, Cieslak J, Freidine M, Ivakhnenko V, Wolf G, Kovalinskaya L, White M, Lifchez A, Kaplan B, Moise J et al. (1995) Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescence in situ hybridization. Hum Reprod 10,1923–1927.[Abstract]

Submitted on May 6, 2003; resubmitted on September 23, 2003; accepted on November 7, 2003.