1 Centre for Reproductive Medicine, Middelheim Hospital, Lindendreef 1, 2020 Antwerp and 2 Department of Microbiology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
3 To whom correspondence should be addressed. e-mail: Diane.deneubourg{at}zna.be
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Abstract |
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Key words: assisted reproduction/early pregnancy/single embryo transfer/top quality embryo
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Introduction |
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We have been interested to know what happens to highly competent embryos after implantation in the first trimester, particularly because a direct link to embryo quality can be made.
What happens to the embryo after it has been transferred in the uterus remains largely unknown. The only outcome variables after embryo transfer are detection of HCG in serum and urine, and ultrasonography later in pregnancy. The predictive value of a single serum HCG level has been shown after assisted conception (Zayed et al., 2001; Homan et al., 2002
).
In this study, we used single embryo transfer of a top quality IVF/ICSI embryo as a model to calculate the predictive value of variables such as HCG measurements with regard to early pregnancy outcome.
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Materials and methods |
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Treatment protocol
Patients were treated with the long GnRH agonist desensitization protocol, starting in the midluteal phase with 6 x 100 µg of buserelin (Suprefact, Hoechst, Germany) intranasally for a period of 3 weeks. Thereafter, gonadotrophin stimulation (Metrodin HP or Gonal-F; Serono, Geneva, Switzerland) was initiated. When at least three mature follicles with a diameter of 18 mm were present, 10 000 IU of HCG (Profasi; Serono, Geneva, Switzerland) was given. The oocyte pick up was performed exactly 37 h later under vaginal ultrasound guidance.
Embryo quality assessment
Approximately 1619 h after insemination/injection, normal fertilization was checked. Two pronuclear oocytes were cultured in Ménézo B2 medium. The next day (4043 h after insemination/injection), embryos were separated and transferred to Medi-Cult M3 medium for a further 24 h culture. Every embryo was scored for the total number of cells, and the presence of anuclear fragments and multinucleated blastomeres on day 2 and day 3.
An embryo was considered a top quality embryo if there were four or five blastomeres on day 2 and seven or more blastomeres on day 3 with <20% of fragments and the total absence of multinucleated blastomeres at any stage of early cleavage (Van Royen et al., 1999). Supernumerary embryos were frozen.
Embryo transfer technique
All transfers were performed on an out-patient basis using a Wallace embryo transfer catheter (Sims Portex Ltd, Hythe, Kent, UK) consisting of an inner and outer catheter.
Luteal phase
In all cycles, the luteal phase was supported with 3 x 200 mg of micronized natural progesterone (Utrogestan, Besins, Belgium) administered vaginally.
HCG analysis
HCG analysis in serum was performed routinely on day 8 and day 12 after embryo transfer. There were 192 conception cycles; in 173 cases, HCG values on both day 8 and 12 after embryo transfer were obtained. HCG was measured in serum with an immunometric assay (Vitros ECI, Ortho-clinical diagnostics NV, Beerse, Belgium). Calibration of the standards was performed against the WHO third international standard 75/537.
Definitions
Pregnancy was defined as two increasing values of HCG >5 IU/l. First-trimester pregnancy loss (FTPL) was a pregnancy leading to pregnancy loss prior to 13 weeks of gestation (i.e. biochemical pregnancy, clinical miscarriage and ectopic pregnancy). Ongoing pregnancy was defined as a pregnancy reaching beyond 12 weeks of pregnancy.
Statistical analysis
Statistical analysis was performed using SPSS software. Differences between continuous variables were analysed using the Students t-test; differences between percentages were analysed using the 2 test. The predictive value of HCG was determined using receiver operator curves (ROCs) to calculate sensitivity and specificity. Multiple regression analysis was used to investigate the contribution of age and fertilization method in the outcome. A P-value <0.05 was considered statistically significant.
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Results |
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We analysed whether pregnancy rate and FTPL were related to age (Table I). The pregnancy rate after single top quality embryo transfer was independent of age. FTPL, however, occurred significantly less often in women 30 than in patients >30 years of age (P = 0.01).
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Analysis of the cohort of embryos from which the top quality embryo was selected is summarized in Table III. The total number of embryos, the number of top quality embryos and the number of cryopreserved embryos was calculated per cycle. The number of embryos did not differ between cycles which resulted in a pregnancy and those which did not. However, there were significantly more top quality embryos and cryopreserved embryos in the pregnant patient group than in the patient group that did not become pregnant. Further analysis (Table IV) showed significantly more top quality embryos and cryopreserved embryos in cycles ending in FTPL than in cycles resulting in ongoing pregnancy.
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We analysed whether HCG follow-up could predict the risk of FTPL after transfer of a top quality embryo. In 184 patients receiving a top quality embryo, the HCG value on day 8 after transfer could be analysed; the mean value was 20.5 IU/l (±10.7 SD). For 180 patients, the mean HCG value on day 12 was 153.3 IU/l (±90.8 SD). To evaluate the level of HCG rise the HCG index was calculated as HCG on day 12/HCG on day 8. The mean value for this parameter was 8.0 (±4.5 SD).
The predictive value for ongoing pregnancy after transfer of one top quality embryo was calculated using ROCs. A cut-off value for HCG 45 IU/l predicts a sensitivity of 75.6% for ongoing pregnancy after transfer of a top quality embryo with 100% specificity (Figure 1). A cut-off value for the HCG index (HCG day 12/day 8)
3.5 predicts a sensitivity of 72.3% for ongoing pregnancy after transfer of a top quality embryo with 100% specificity (Figure 2).
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Discussion |
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Analysis of the cohort of embryos (number of embryos, number of top quality embryos and number of cryopreserved embryos) from which the top quality embryo for transfer was selected shows that there are significantly more top quality and cryopreserved embryos in the pregnant patient group. This means that the availability of more excellent quality embryos in itself can serve as a good predictive factor for pregnancy and can be taken into account when the number of embryos for transfer is discussed with the patient. This has already been described by Staessen et al. (1993), Coetsier and Dhont (1998
) and Strandell et al. (2000
). The patient population that receives a single top quality embryo transfer is a selected group of patients which represents 22.6% of all transfers. This top group of patients seems to produce a homogenous cohort of embryos after ovarian stimulation.
The implantation potential of a top quality embryo can be derived from analysis of HCG levels in serum. Both the individual value of HCG level on the twelfth day after embryo transfer and the dynamics of the HCG increase, calculated by the HCG index (day 12/day 8), are excellent predictors for ongoing pregnancy. The combination of both parameters does not improve the predictive value. Poikkeus et al. (2002) evaluated HCG levels on day 12 after fresh day 2 embryo transfers and frozen transfers; a cut-off value for HCG of 76 IU/l predicts a viable pregnancy with a sensitivity of 80% and specificity of 82%.
It is becoming more and more clear that not only the genetic make-up of the oocyte but also the integrity of the meiotic spindle is pivotal to early embryogenesis. Recent hypotheses point to the presence of two distinct mechanisms of embryo wastage in early embryo development. The first is chaotic mosaicism which can be considered a non-nuclear (mitochondrial) mechanism of early embryo wastage. The second is non-disjunction which affects early embryo development through a nuclear (chromosomal) mechanism (Wilding et al., 2003). Therefore, improvement of the success rate in IVF/ICSI should be directed to making the best possible embryo selection in order to improve the pregnancy rate and to decrease fetal wastage. It has been shown by Munné et al. (1999
) that aneuploidy screening in preimplantation embryos does not improve the implantation rate but reduces the embryo loss after implantation.
The question of whether subfertile couples are more prone to pregnancy loss remains unsolved. No such association has been reported despite the chance of over-representation in this group (Macklon et al., 2002).
A history of FTPL following IVF has been reported to be a positive factor to predict future success with IVF treatment (Levy et al., 1991; Templeton et al., 1996
; Croucher et al., 1998
). We have also shown that patients experiencing FTPL had significantly more top quality and cryopreserved embryos than patients with an ongoing pregnancy and thus should not be considered a poor prognosis group.
It is likely that the events following the onset of implantation are determined predominantly by the genetic quality of the embryos. We have shown that with the use of morphological embryo selection criteria, a pregnancy rate of 51.9% can be achieved with top quality embryos. However, post-implantation wastage is still 15.4%. These data indicate that embryo selection gives an excellent estimation of the chance of becoming pregnant but cannot predict FTPL.
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References |
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Submitted on November 11, 2003; accepted on April 1, 2004.