Centre for Reproductive Medicine, Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium
1 To whom correspondence should be addressed. Email: stratis.kolibianakis{at}otenet.gr
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Abstract |
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Key words: endometrium biopsy/GnRH antagonists/hCG/recombinant FSH
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Introduction |
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As expected, the criteria used for hCG administration in GnRH antagonist cycles have also not been evidence-based, while their importance for the probability of pregnancy was unknown until recently. A large randomized controlled trial (Kolibianakis et al., 2004) evaluated the effect of prolongation of the follicular phase by delaying hCG administration on IVF outcome. In that study, stimulation was performed with recombinant (r)FSH for IVF and hCG was administered either as soon as
3 follicles
17 mm were present on ultrasound (early-hCG group) or 2 days after this criterion was met (late-hCG group). A significantly lower ongoing pregnancy rate was observed in the late-hCG group in which follicular phase was prolonged (Kolibianakis et al., 2004
). This was not accompanied by differences in fertilization rates or in the morphological quality of the embryos transferred and cryopreserved. It was therefore hypothesized that the decreased probability of pregnancy might be due to changes in endometrial receptivity, which could be detected at the level of histology. The importance of endometrial histology on the day of oocyte retrieval for the achievement of pregnancy has been recently shown in GnRH antagonist cycles. Extreme endometrial advancement has been associated with a poor pregnancy outcome (Kolibianakis et al., 2002
).
The hypothesis tested in the current study was that prolongation of the follicular phase, by delaying hCG administration, results with a higher incidence of endometrial advancement on the day of oocyte retrieval in donors stimulated with rFSH and GnRH antagonists.
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Materials and methods |
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Ovarian stimulation
Stimulation was performed with rFSH (Puregon; NV Organon, Oss, The Netherlands) starting in the afternoon of day 2 of the cycle with 200 IU of rFSH, which remained constant during stimulation. Daily GnRH antagonist 0.25 mg (Orgalutran; NV Organon, Oss, The Netherlands) was used to inhibit premature LH surge and was always started on the morning of day 6 of stimulation. Final oocyte maturation was triggered by the administration of 10 000 IU of hCG (Pregnyl; Organon) in accordance with the criteria described above. Steroid levels were measured, but were not taken into consideration for administration of hCG, which was based exclusively on follicular development.
Hormonal measurements and ultrasound assessment
Hormonal assessment was performed at initiation of stimulation, on day 6 of rFSH stimulation and on the day of hCG administration. Additional blood samples were taken as necessary between antagonist initiation and hCG administration. Serum LH, FSH, hCG, E2 and progesterone levels were measured by means of the automated Elecsys immunoanalyser (Roche Diagnostics, Mannheim, Germany). Intra- and inter-assay coefficients of variation were <3 and <4% for LH, <3 and <6% for FSH, <5 and <10% for E2 and <3 and <5% for progesterone respectively. Ultrasound was performed on day 6 of stimulation and thereafter as necessary to ensure that hCG was injected on the first day the patient had 3 follicles of
17 mm, or 2 days later if she was in the late-hCG group.
Endometrial biopsy and histological assessment
Aspiration biopsy of the endometrium was performed using the Pipelle de Cornier (Laboratoire CCD, Paris, France) on the day of oocyte retrieval. Endometrial samples were fixed immediately in neutral buffered formalin 10% solution and sent for histopathological examination. Endometrial histology was analysed prospectively using the criteria of Noyes et al. (1950) by one observer who was blinded to the group to which the donor was allocated, to the hormonal data and to the stimulation outcome. For assessment of endometrial maturation, the day of oocyte retrieval was considered the day of ovulation (day 0).
Statistical analysis
Assuming that no secretory changes are present in the early-hCG group while all samples are advanced in the late-hCG group, it was calculated that four patients who reach oocyte retrieval in each group would be necessary, at an alpha of 0.05 and beta of 0.2 using a Fisher's exact test. Twelve oocyte donors were randomized to compensate for inadequate endometrial sampling or possible cycle cancellation.
Fisher's exact test was used to analyse nominal variables in the form of frequency tables. Metric variables were analysed by the MannWhitney U-exact test. Values are expressed as median (interquartile range, IQR).
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Results |
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Patient characteristics and stimulation data are shown in Table I. Due to the study design, duration of stimulation was significantly longer in the late-hCG group in which significantly more follicles of 15 and
17 mm were observed on the day of hCG administration. Hormonal levels on the day of hCG and on day hCG +1 are also shown in Table I. Significantly higher progesterone levels were observed 1 day after hCG administration in the late-hCG as compared to the early-hCG group.
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Discussion |
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Although the effect of the prolongation of the follicular phase on pregnancy achievement has been examined in the literature previously (Clark et al., 1991; Dimitry et al., 1991
; Tan et al., 1992
; Kolibianakis et al., 2004
), this is the first study to evaluate its impact on endometrial histology. In GnRH antagonist cycles, prolongation of follicular phase has resulted in a lower ongoing pregnancy rate that could not be attributed to differences in the number and quality of the embryos transferred (Kolibianakis et al., 2004
). The lower probability of pregnancy reported by Kolibianakis et al. (2004)
might be explained by the significantly higher incidence of endometrial advancement that was observed with delayed hCG administration in the present study. An adverse effect of endometrial advancement on the day of oocyte retrieval on pregnancy rates in GnRH antagonist cycles has recently been demonstrated (Kolibianakis et al., 2002
).
The explanation as to why delaying hCG administration leads to an increased incidence of endometrial advancement is not clear. It has been previously shown that ovarian stimulation results in an abnormal steroid receptor profile at the end of the follicular phase, which resembles that present in the early luteal phase (Papanikolaou et al., 2004). It can thus be hypothesized that the delaying of hCG administration intensifies the receptor changes induced by ovarian stimulation even further, thus resulting by the time of oocyte retrieval in endometrial advancement. This is further supported by the significantly higher exposure of endometrium to supraphysiological E2 levels in the late-hCG group.
In addition, significantly higher progesterone levels on day hCG + 1 were observed in the late-hCG as compared to the early-hCG group (median level 9.1 versus 4.8 ng/ml respectively). Progesterone values >6 ng/ml on day hCG + 1 have been associated with an earlier down-regulation of progesterone receptor expression, as well as with accelerated glandular development and pinopode expression within the implantation window (Develioglu et al., 1999). Moreover, it has been previously shown that the timing of steroid receptor down-regulation in the endometrial epithelium marks the establishment of endometrial receptivity (Lessey et al., 1996
). Thus advanced endometrial maturation induced by high progesterone levels on day hCG + 1 might be associated with an earlier closure of the implantation window and decreased pregnancy rates in stimulated cycles (Develioglu et al., 1999
).
In the present study, endometrium was evaluated by using Noyes criteria. Although these criteria have been criticized for several reasons (Murray et al., 2004), they remain a time-honoured method for evaluating endometrium quality (Myers et al., 2004
). The present study suggests that the variability present in the literature regarding the proportion of patients with endometrial advancement on the day of oocyte retrieval (Table III) might be explained by the different timing of hCG administration used. Moreover, in the studies published, the criteria used for triggering final oocyte maturation were not strict. This means that hCG was administered when
x follicles
y mm were present at ultrasound, but not necessarily on that day. hCG could also be delayed for 1 or 2 days without violating the study criteria. Consequently, patients in the same study might have been treated differently despite using the same criteria for hCG administration. This makes it more difficult to detect the effect of an intervention and might contribute to the presence of heterogeneity in a meta-analysis.
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Acknowledgements |
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References |
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Submitted on December 20, 2004; resubmitted on February 14, 2005; accepted on April 7, 2005.