Elevated progesterone at initiation of stimulation is associated with a lower ongoing pregnancy rate after IVF using GnRH antagonists

E.M. Kolibianakis1, K. Zikopoulos, J. Smitz, M. Camus, H. Tournaye, A.C. Van Steirteghem and P. Devroey

Centre for Reproductive Medicine, Dutch-Speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium

1 To whom correspondence should be addressed. e-mail: stratis{at}easynet.be


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The objective of this prospective study was to assess the impact of elevated serum progesterone levels on day 2 of the cycle on pregnancy rates in patients treated by IVF using GnRH antagonists. METHODS: Ovarian stimulation was started on day 2 of the cycle if progesterone levels were normal (normal-P group, n = 390). In the presence of elevated progesterone, initiation of stimulation was postponed for 1 or 2 days (high-P group, n = 20) and was started if repeat progesterone levels returned to normal range (n = 16). Stimulation was performed with recombinant FSH (rFSH) and GnRH antagonist was always started on day 6 of stimulation. RESULTS: A significantly higher exposure to progesterone and a significantly lower exposure to estradiol was present in the high-P as compared with the normal-P group from day 1 to day 8 of stimulation. In addition, a significantly lower ongoing pregnancy rate both per started cycle (5.0% versus 31.8%; P = 0.01) and per embryo transfer (6.3% versus 36.9%; P = 0.01) was present in the high-P compared with the normal-P group, respectively. CONCLUSIONS: The presence of elevated serum progesterone on day 2 of the cycle is associated with a decreased chance of pregnancy in patients treated with rFSH and GnRH antagonists.

Key words: GnRH antagonists/IVF/ongoing pregnancy rate/progesterone elevation/recombinant FSH


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Functional luteolysis, occurring at the end of the menstrual cycle, is characterized by the loss of the corpus luteum’s ability to produce progesterone and is followed by its structural regression (McCracken et al., 1999Go). Typically, progesterone levels reach their nadir at menstruation, although in a proportion of cycles they are still elevated, possibly as a result of inefficient luteolysis.

No information is available on IVF outcome regarding the role of elevated progesterone levels at the time stimulation should commence, probably due to the universal use of the long GnRH agonist protocol in IVF in the last decade. The long agonist protocol demands the suppression of gonadotropins and achievement of basal levels of steroid hormones prior to initiation of stimulation. Therefore, all patients treated under this scheme start stimulation with normal progesterone levels (Huang et al., 1996Go).

Under the short GnRH protocol, however, or by using GnRH antagonists for premature LH surge inhibition, elevated progesterone levels at the time stimulation should begin can still be observed. In GnRH antagonist cycles, delaying administration of gonadotropins for 1–2 days usually results in normalization of progesterone values in healthy patients. It is not known, however, if the chance of pregnancy after IVF is different from that in patients with normal progesterone levels on day 2 of the cycle.

In phase III comparative studies between GnRH antagonists and GnRH agonists, no information is available in the antagonist arm either for the incidence of abnormal steroid levels on the day stimulation should start or for the management and outcome of these patients (Albano et al., 2000Go; Borm and Mannaerts, 2000Go; Olivennes et al., 2000Go; European and Middle East Orgalutran Study Group, 2001Go; Fluker et al., 2001Go).

The purpose of this study was to prospectively compare the ongoing pregnancy rates in patients with normal or elevated progesterone levels on day 2 of the cycle treated by GnRH antagonist and recombinant FSH (rFSH) for IVF.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient population
Four hundred and ten patients treated by IVF at the Centre for Reproductive Medicine of the Dutch-Speaking Brussels Free University from May 2002 until July 2003 were included in the study. Patients could enter the study only once. Inclusion criteria were age <39 years and presence of both ovaries. None of the patients had received hormonal treatment in the cycle preceding treatment and all had normal hormonal levels at the initial evaluation prior to initiation of IVF treatment. The present study was approved by our Institutional Review Board.

Ovarian stimulation and IVF procedure
Stimulation was initiated on day 2 of the cycle when estradiol (E2) and progesterone levels were normal (E2 ≤80 pg/ml, progesterone ≤1.6 ng/ml; normal-P group).

When progesterone was >1.6 ng/ml on day 2 of the cycle, initiation of stimulation was postponed for 1–2 days and progesterone levels were followed daily (high-P group). If progesterone levels were normalized within 2 days, stimulation with rFSH was initiated. If however, progesterone remained elevated after 2 days, the cycle was cancelled.

Stimulation was performed with rFSH (Puregon; NV Organon, Oss, The Netherlands) at a dose determined according to patient’s age and/or previous response to ovarian stimulation (range 100–500 IU). The dose of rFSH remained constant during stimulation unless there was no increase in serum E2 levels after 5 days of stimulation. Daily GnRH antagonist 0.25 mg (Orgalutran; NV Organon) was used to inhibit premature LH surge and was always started on the morning of day 6 of stimulation.

Final oocyte maturation was achieved by 10 000 IU of HCG (Pregnyl; NV Organon) when at least three follicles of 17 mm were present at ultrasound.

Oocyte retrieval was carried out 36 h after HCG administration by transvaginal ultrasound-guided puncture of follicles. Conventional IVF was performed in 109 couples, ICSI in 277 couples and both conventional IVF and ICSI in 24 couples. ICSI and IVF procedures have been described in detail previously (Van Steirteghem et al., 1993Go; Devroey et al., 1995Go). As a matter of principle, two embryos were transferred on day 3 or day 5 after oocyte retrieval. Embryos were classified as top quality (score 1) medium quality (score 2) or low quality (score 3), as described previously (Staessen et al., 1992Go; Gardner et al., 1999Go). The mean score of the embryos transferred to each patient was used for the calculation of the mean quality score of all embryos transferred.

Luteal supplementation
The luteal phase was supplemented with vaginal administration of 600 mg natural micronized progesterone in three separate doses (Utrogestan; Besins, Brussels, Belgium), starting 1 day after oocyte retrieval and continued until 7 weeks of gestation if pregnancy was achieved.

Hormonal measurements
Hormonal assessment was performed on day 2 of the cycle, at initiation of stimulation, on day 6 and 8 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-assay and inter-assay coefficients of variation were <3% and <4% for LH, <3% and <6% for FSH, <5% and <7% for HCG, <5% and <10% for E2 and <3% and <5% for progesterone, respectively.

Outcome measures
Pregnancies progressing beyond the 12th week of gestation were considered to be ongoing. Ongoing implantation rate was calculated by dividing the number of gestational sacs with fetal heartbeat present at 12 weeks of gestation by the number of embryos transferred.

Statistical analysis
Fisher’s exact test was used to analyse nominal variables in the form of frequency tables. Metric variables were analysed by the Mann–Whitney U-test. Values are expressed as median (interquartile range), unless stated otherwise.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Abnormal progesterone levels on day 2 of the cycle were present in 20 out of 410 patients [4.9%; 95% confidence interval (CI) 3.2–7.4%].

Delaying initiation of stimulation resulted in normalization of progesterone values in 16 patients (80%) after a mean period of 1 (1) day. In four patients, progesterone levels remained elevated, despite a drop compared with the initially recorded values. In these patients the cycle was cancelled.

Similar indications for IVF treatment were present in patients with normal and high progesterone levels on day 2 of the cycle (Table I).


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Table I. Indication for IVF treatment in patients with normal progesterone (normal-P) and high progesterone (high-P) levels on day 2 of the cycle
 
Patient and stimulation characteristics are shown Table II. A significantly higher exposure to progesterone and a significantly lower exposure to E2 from day 1–8 of stimulation was present in the high-P compared with the normal-P group (Table II; Figure 1). Moreover, in the high-P group significantly lower E2 levels were present on the day of HCG administration (Table II).


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Table II. Patient characteristics and stimulation data in the normal progesterone (normal-P) and high progesterone (high-P) group
 


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Figure 1. Notched plots of E2 and progesterone levels on days 1, 6 and 8 of stimulation in the normal progesterone (normal-P) and high progesterone (high-P) group. The notches in the notched box plot are a graphic CI about the median of a sample. Differences between the two groups are significant for progesterone on days 1, 6 and 8 of stimulation, and for E2 on days 6 and 8 of stimulation.

 
In the high-P group, all 16 patients in whom progesterone levels were normalized reached oocyte retrieval and had embryo transfer. In the normal-P group, 23 patients did not reach oocyte retrieval due to poor ovarian response, while 31 patients did not have embryo transfer [ovarian hyperstimulation syndrome risk (n = 4), failure of fertilization (n = 11), poor embryo quality (n = 15), no cumulus–oocyte complexes retrieved at ovum pick-up (n = 1)].

A significantly lower ongoing pregnancy rate per started cycle, per oocyte retrieval and per embryo transfer was present in the high-P compared with the normal-P group (Table III).


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Table III. Pregnancy outcome in the normal progesterone (normal-P) and high progesterone (high-P) group
 
Three of the patients in the high-P group have not yet started a new trial; four patients have undergone a further trial under a long agonist scheme starting on day 21 of the menstrual cycle, while three additional patients have performed a frozen–thawed cycle. In the remaining 10 patients, a new trial was initiated with GnRH antagonists (n = 8) or a short agonist protocol (n = 2). In all 10 patients, normal progesterone values (median 0.7, interquartile range 0.5) were recorded on day 2 of the cycle. Two ongoing pregnancies were established in the antagonist group (two out of eight).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study shows that a significantly lower chance of achieving an ongoing pregnancy is present in patients with elevated progesterone levels on day 2 of a GnRH antagonist cycle.

This was a prospective study in which all participants were stimulated with rFSH and GnRH antagonists. Patients with elevated progesterone levels on day 2 of the cycle were always postponed for 1–2 days, and stimulation was started only if progesterone levels returned to normal range.

To our knowledge, no information is available in the literature on the incidence of elevated progesterone levels in healthy patients prior to initiation of stimulation for IVF, although it should be expected that this information would have been available in cycles treated with the short GnRH agonist protocol. In infertile couples, Keck et al. (1999)Go reported the presence of elevated progesterone levels in 11.4% of patients assessed on day 4–5 of the cycle (36/316) patients).

Moreover, little information is available on the association of progesterone levels prior to initiation of stimulation with IVF outcome. In GnRH agonist cycles using the long protocol, Huang et al. (1996)Go showed that elevated progesterone levels after 10 days of analogue treatment were associated with significantly lower E2 levels on day 5 of stimulation and on the day of HCG administration. In these patients, for whom stimulation was started after normalization of progesterone values by extending the period of down-regulation, a significantly higher cancellation rate was also observed.

Limited information is also available on the effect of elevated progesterone levels in the early follicular phase on IVF outcome. Sims et al. (1994)Go, using a short GnRH agonist protocol, showed that following analogue administration elevated progesterone levels during cycle days 2–6 were associated with a higher requirement for gonadotropins, lower peak E2 concentrations and fewer mature oocytes retrieved.

In the current study, the presence of elevated progesterone levels did not identify a subpopulation of women undergoing IVF in terms of age, diagnosis, FSH levels at initiation of stimulation, starting dose of FSH or duration of stimulation (Table I).

No significant differences were observed in the number of follicles present on the day of HCG administration or the number of cumulus–oocyte complexes retrieved, which however, tended to be higher in patients with normal progesterone levels on day 2 of the cycle.

However, it is clear that patients with elevated progesterone levels on day 2 of the cycle had been exposed to a significantly different hormonal environment compared with those with normal progesterone levels.

Progesterone levels, despite normalization, were significantly higher on day 1 of stimulation, as well as on days 6 and 8 of stimulation, in patients with elevated progesterone values on day 2 of the cycle compared with those with normal levels.

This might be attributed to progesterone production by a corpus luteum that did not undergo functional luteolysis completely, and might have retained partially its ability to produce progesterone under stimulation by endogenous and exogenous gonadotropins in the treatment cycle. As follicular development occurred, progesterone was also produced by the developing follicles, which might have led to the similar levels of serum progesterone on the day of HCG administration in the two groups compared.

Moreover, E2 levels were significantly lower in the high-P group on days 6 and 8, and on the day of HCG administration. This might be a reflection of the tendency for an increased number of follicles observed in the normal-P group.

The potential influence of these hormonal differences on pregnancy rates might be exerted at the level of oocyte/embryo and/or at the level of endometrium. However, no evidence is provided by the current study that the difference observed in ongoing pregnancy rates can be attributed to reduced oocyte/embryo quality in patients with elevated progesterone on day 2 of the cycle. Fertilization rates and number of transferred embryos were similar between the two groups, while the quality of the transferred embryos, using established criteria for morphological evaluation, was identical. It is thus possible that the different hormonal environment during the follicular phase between the two groups compared might result in different endometrial receptivity. It is therefore interesting to assess in GnRH antagonist cycles whether high exposure to progesterone levels from initiation of stimulation results in a histologically more advanced endometrium, known to be associated with a lower chance of pregnancy (Kolibianakis et al., 2002Go).

The current study suggests that if elevated progesterone levels are present on day 2 of the cycle in which premature LH inhibition is achieved using GnRH antagonists, the cycle should be cancelled. This might represent a coincidental event that will not reoccur in the next cycle.

In conclusion, the presence of elevated serum progesterone on day 2 of the cycle is associated with a decreased chance of pregnancy in patients treated with rFSH and GnRH antagonists.


    Acknowledgements
 
This work is supported by grants from the Fund for Scientific Research, Flanders, Belgium.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 10, 2004; accepted on March 24, 2004.