Molecular Human Reproduction, Vol. 6, No. 8, 726-734,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Modulation of endometrial transformation in gonadotrophin-stimulated and unstimulated pseudo-pregnant rabbits: studies with the progesterone receptor antagonist, onapristone
1 Department of Anatomy and Reproductive Biology, School of Medicine, RWTH University of Aachen, 52057 Aachen, and 2 Research Laboratories, Schering AG, 13342 Berlin, Germany
Abstract
Advanced endometrial transformation often occurs in IVF and embryo transfer therapy after ovarian stimulation with gonadotrophins. One reason is probably the early rise in peripheral progesterone concentration after ovulation induction. Consequently, we studied in a rabbit model, whether the post-ovulatory application of the progesterone receptor antagonist, onapristone, could prevent such an advancement of endometrial transformation after stimulation with different gonadotrophin preparations. The inhibitory effect of onapristone on the endometrium is dependent upon the strength of ovarian stimulation. In unstimulated animals or animals treated with recombinant LH (nine corpora lutea/animal in both groups), secretory differentiation and proliferation of the endometrium was strongly inhibited by onapristone. After weak ovarian stimulation with a 3:1 mixture of FSH and LH (22 corpora lutea/animal), secretory differentiation was strongly inhibited, while proliferation was enhanced. After strong stimulation with either a 1:1 mixture of FSH and LH, or human menopausal gonadotrophin (HMG; >40 corpora lutea/animal), only limited inhibitory effects of onapristone on secretory transformation or proliferation could be detected. In conclusion, these graded effects of onapristone after stimulation with gonadotrophins, resemble the basic observations from which a therapeutic strategy emerges, to modulate the advanced endometrial transformation which occurs in many IVF patients after ovarian stimulation.
assisted reproduction/endometrium/luteal phase/ovarian stimulation/progesterone receptor antagonists
Introduction
Ovarian stimulation with gonadotrophins is commonly used in human IVF and embryo transfer. However, post-ovulatory endometrial transformation is often advanced after ovarian stimulation (Benadiva and Metzger, 1984
; Garcia et al., 1984; Kolb et al., 1997; Fanchin et al., 1999; Nikas et al., 1999). As a consequence, the precisely synchronized development of endometrium and embryo is disrupted and the embryos do not meet the implantation window of the endometrium. Hence, implantation rates are low if ovarian stimulation is used and embryos are retransferred 48 h after IVF.
Advanced endometrial development is thought to be triggered by the premature progesterone serum elevation, which occurs in 30% of stimulated IVF cycles prior to administration of human chorionic gonadotrophin (HCG), and the advanced post-ovulatory rise in progesterone serum concentrations (Schoolcraft et al., 1991; Silverberg et al., 1991; Develioglu et al., 1999; Fanchin et al., 1999). However, other investigators have not been able to confirm these observations (Bustillo et al., 1995; Ubaldi et al., 1996; Lass et al., 1998) and have proposed that other factors should be considered (Noci et al., 1997). In this context, it has been reported that the expression of progesterone receptors was decreased in the luteal phase after ovarian stimulation (Molina et al., 1989; Seppala and Tiitinen, 1995). Until now, scientific results have not been available to explain the causal relationship of this phenomenon.
The aim of this study was to evaluate the contribution of progesterone and progesterone–receptor interactions to the advancement of endometrial transformation induced by ovarian stimulation. In addition, we assessed whether the advanced endometrial development could be slowed by intervention in this regulatory hormonal system. Due to the well-known effects of progesterone receptor antagonists in causing a delay of endometrial transformation when applied after ovulation in the rabbit (Beier, 1986; Beier et al., 1987, 1994; Hegele-Hartung et al., 1992), we chose to administer the progesterone receptor antagonist, onapristone. This intervention aims to provide a strategy to modulate advanced endometrial development in human IVF.
In the present study we investigated ovarian stimulation in the rabbit model. We used different gonadotrophin stimulation protocols for ovarian stimulation. The potency of each stimulation protocol in inducing follicle maturation and supporting luteal progesterone production was monitored. Unstimulated pseudo-pregnant animals formed the control group. In all stimulation groups, the effects of the application of onapristone on endometrial transformation was assessed on days 4 and 5 of pseudo-pregnancy. Onapristone was administered s.c. at a dose of 20 mg/kg body weight on day 2 of pseudo-pregnancy (day 2 post-HCG). As parameters for endometrial transformation we used endometrial morphology, epithelial cell proliferation (Ki 67 immunohistochemistry), apoptosis (TUNEL method) and the expression and distribution of mRNA for uteroglobin and aminopeptidase N (APN) as marker molecules for the secretory transformation of the endometrium (Classen-Linke et al., 1987; Krusche and Beier, 1994; Krusche, 1999).
Materials and methods
Animals
We used 74 sexually mature nulliparous New Zealand White rabbits with a body weight of 3.2–4.0 kg. The rabbits were housed in individual cages in controlled temperature and lighting conditions (12 h light:12 h dark). Food and water were provided ad libitum. All experiments on animals were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction and were run with the permission of the District Government, Cologne, Germany (AC 9a 26/92 and AC 39 15/95).
Hormones
Five different gonadotrophin preparations were used for stimulation: (i) FSH-P (purified FSH, Rigaux®; Schering-Plough Sante Animale, La Grindoliere, Segre, Belgium). Animals received a s.c. injection of 0.3 Armour Units FSH, which was calculated by the standardized FSH content of this pituitary gonadotrophin preparation, dissolved in 0.5 ml 0.9% NaCl; (ii) human menopausal gonadotrophin (HMG, Pergonal®; Serono Pharma, Unterschleißheim, Germany); one dose contained 4.25 IU FSH and 5.35 IU LH in 0.5 ml 0.9% NaCl; (iii) a 1:1 mixture of 5 IU recombinant human (rh) FSH (Gonal F®) and 5 IU rhLH (both kindly supplied by Serono Pharma) dissolved in 0.5 ml 0.9% NaCl; (iv) a 3:1 mixture of 5 IU rhFSH (Gonal F®) and 1.3 IU rhLH (both Serono Pharma) dissolved in 0.5 ml 0.9% NaCl; and (v) 5 IU rhLH (Serono Pharma) dissolved in in 0.5 ml 0.9% NaCl.
Ovulation was induced with i.v. injection of 75 IU HCG (Primogonyl-1000; Schering AG, Pharma, Berlin, Germany) in 0.5 ml 0.9% NaCl.
The progesterone receptor antagonist, onapristone (11ß-(4-N,N-dimethylamino-phenyl)-17
-hydroxy-17ß-(3-hydroxy-propyl)-13-methyl-4,9-gonadiene-3-one), kindly provided by Schering AG, was solubilized in an vehicle of benzylbenzoate and ricinus oil (2:3) and administered s.c. The antagonist was given in a dose of 20 mg/kg body weight. Control animals received vehicle only.
Experimental design
Gonadotrophin-stimulation was carried out for 3 days (day 3, day 2 and day 1 before-HCG) by s.c. application of the above preparations in order to induce multiple follicular growth. Each gonadotrophin preparation was injected s.c. twice daily (once in the morning and once in the evening). Pseudo-pregnancy was induced with an i.v. injection of 75 IU HCG. The day of HCG injection was designated as day 0 of pseudo-pregnancy (day 0 post-HCG).
Two days after ovulation induction (day 2 post-HCG), onapristone was injected once s.c. Control animals received vehicle only. The animals were killed on days 4 and 5 post-HCG in groups I and II. In group III animals were killed on day 5 post-HCG. The detailed treatment schedule is shown in Figure 1.
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Uteri were removed and one piece was snap-frozen in liquid nitrogen for in-situ hybridization. Another piece of the uterus was fixed in 4% formaldehyde in phosphate-buffered saline (PBS) and embedded nuclease-free in paraffin for Ki 67 immunohistochemistry and assessment of apoptosis by the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) method. The remaining piece of uterus was dissected, the endometrium was scraped out and processed for RNA isolation. The ovaries were also removed and the number of corpora lutea determined.
Peripheral hormone concentration
Blood samples were taken from the central ear artery on day 0 (before HCG-application), and days 1, 2, 3 and 5 post-HCG in experimental group I and II. The serum concentrations of progesterone and testosterone were measured using an enzyme immunoassay (EIA; IBL, Hamburg, Germany), according to the manufacturer's protocol. The intra- and inter-assay coefficients of variation were 9–12 and 15–19% respectively. Prolactin was measured by a radioimmunoassay (RIA). The inter- and and intra-assay coefficients of variation were 12.9 and 11% respectively.
Probe preparation
To detect uteroglobin mRNA in Northern blotting and in in-situ hybridization experiments we used sense and antisense oligonucleotide probes (24mers), which corresponded to the codons of the amino acids 49–56 of uteroglobin protein:
sense: CCC CAG ACG ACC AGA GAG AAC ATC
antisense: GAT GTT CTC TCT GGT CGT CTG GGG
The oligonucleotides were labelled at their 3' end with the enzyme terminal deoxynucleotidyl transferase with digoxigenin dUTP (Roche GmbH, Mannheim, Germany) and dATP at a ratio of 1:10. Control hybridizations were performed with an oligonucleotide for 18S rRNA (Krusche and Beier, 1994) tailed with digoxigenin dUTP.
The Northern hybridization of APN mRNA (the plasmid KS7 was kindly provided by Ove Noren, Copenhagen, Denmark) was carried out with random primed probes. The probes were labelled with the DIG DNA Labeling Kit (Roche GmbH) according to the manufacturer's protocol.
Northern blotting
Total endometrial RNA was isolated using a previously published method (Chomczynski and Sacchi, 1987). 10 µg of RNA were loaded to a 1.2% agarose gel, containing 0.66 mol/l formaldehyde and 1x MSE-buffer (20 mmol/l morpholinopropane sulphonic acid, 5 mmol/l sodium acetate, 0.5 mmol/l EDTA pH 8). The gel was run for 3 h at 80 V. The RNA was transferred onto a neutral nylon membrane (Qiagen, Hilden, Germany) by capillary blotting with 10x SSC buffer (20x SSC = 3 mol/l sodium chloride, 0.3 mol/l sodium citrate, pH 7) and fixed by UV cross-linking. Thereafter the membrane was successively hybridized with the probes for uteroglobin mRNA and 18S rRNA. Further conditions for the Northern hybridization of uteroglobin mRNA have been described elsewhere (Krusche and Beier, 1994).
The hybridization with the random primed labelled probes for APN mRNA was carried out at 42°C in DIG-Easy-Hyb hybridization solution (Roche GmbH) overnight. 16 ng of probe/ml hybridization solution were used. The stingency washes were performed with 0.1x SSPE/0.1% SDS (20x SSPE = 3.6 mol/l NaCl, 0.2 mol/l NaH2PO4, 20 mmol/l EDTA, pH 7.4) at 40°C for 30 min.
The specific hybridized probe was detected by an anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche GmbH) and the chemiluminescence substrate CSPD (Serva, Heidelberg, Germany). The endometrial RNA of each individual animal was studied twice on independent Northern blots.
In-situ hybridization
The detection of the uteroglobin mRNA was performed as described previously (Krusche and Beier, 1994) with minor modifications. We used 12 µm thick cryostat sections, which were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) in PBS. The sections were incubated in 2x SSC at 70°C for 30 min. After washing in PBS the sections were acetylated (0.1 mol/l triethanolamine, 0.9% NaCl, pH 8 with 1 ml acetic anhydride/500 ml solution). Prehybridization and hybridization buffer were supplemented with 100 µg poly A/ml. The hybridization was carried out overnight with 0.75 µg uteroglobin antisense oligonucleotide/µl hybridization buffer at 37°C. Stringency washes were performed three times for 30 min at 48°C with 1x SSC. The immunological detection of the digoxigenin-labelled hybrids was carried out according to our previous report (Krusche and Beier, 1994). In each experiment, a negative control using a uteroglobin sense probe for hybridization was included. Two cryostat sections of each animal of all experimental groups were investigated.
Ki 67 immunohistochemistry
Paraffin sections, 5 µm thick, were dewaxed, rehydrated in a decreasing alcohol series and transferred to distilled water. Thereafter the sections were incubated for 4x5 min in citrate buffer (10 mmol/l; pH 6) in a microwave oven (600 W). The sections were washed in PBS and then treated with 0.3% H2O2 in methanol for 30 min. The sections were rinsed in PBS/0.1% bovine serum albumin (BSA) (fraction V; Serva) and blocked with 5% normal goat serum in PBS for 10 min. The anti-Ki 67-antibody (MIB-I; Dianova, Hamburg, Germany) was diluted 1:40 in 5% normal goat serum in PBS and incubated for 1 h. The sections were washed twice in PBS/0.1% BSA and were covered with the biotin-conjugated goat anti-mouse antibody (Dianova) diluted 1:300 for 1 h. After two washes with PBS/0.1% BSA, the sections were incubated with steptavidin–peroxidase conjugate diluted 1:300 in PBS for 30 min. After two washes with PBS and one in distilled water, the reaction was detected with aminoethyl carbazole (AEC)/H2O2. In two individual sections of each animal 500 luminal epithelial cells and 500 glandular epithelial cells were counted to determine the percentage of proliferating (Ki 67-positive) cells.
TUNEL
Paraffin sections, 5 µm thick, were mounted on nuclease-free, APES (3-aminopropyltriethoxysilane; Sigma, Deisenhofen, Germany)-coated slides, deparaffinized, rehydrated and incubated in PBS. The endogenous peroxidase was inhibited by treatment with 0.3% H2O2 in methanol for 30 min. After two washes in PBS the sections were incubated with proteinase K (2 µg/ml) (Roche GmbH; 10 mmol/l Tris, 5 mmol/l EDTA pH 8) for 15 min at 37°C. After extensive washing with PBS the sections were incubated with 1x terminal deoxynucleotidyl transferase (TdT) preincubation buffer (MBI Fermentas, St Leon-Rot, Germany) for 30 min at 37°C. The labelling mixture contained 35 pmol/µl digoxigenin–dUTP (Roche GmbH), 350 pmol dATP/µl and 0.3 IU terminal transferase/µl (MBI Fermentas) in 1x TdT incubation buffer. The incubation was carried out at 37°C for 1 h. Thereafter the sections were washed twice in 2x SSC buffer for 10 min, rinsed with POD-buffer (0.1 mol/l Tris, 0.15 mol/l NaCl pH 7.5) and blocked with 1% blocking reagent (Roche GmbH) in peroxidase (POD) buffer. The sections were incubated with anti-digoxigenin–POD antibody (Roche GmbH) diluted 1:300 in 1% blocking solution. After two washes with PBS/0.1% Tween 20 for 15 min and one wash in distilled water the reaction was detected with AEC/H2O2. Sections that were incubated with a labelling mixture without terminal transferase served as negative controls. As positive controls, we used additional sections that were incubated with DNAse I prior to the labelling reaction. Two individual sections from each animal were TUNEL-labelled. To determine the number of apoptotic cells in the endometrial epithelium, 400 luminal and 400 glandular epithelial cells were counted in each section.
Statistical analysis
For testing three or more different parameters against each other, the Kruskal–Wallis test was applied: (i) the effects of different ovarian stimulation protocols on the number of corpora lutea and progesterone serum concentration; and (ii) the effects of different treatments in group III on endometrial proliferation. Post-hoc Mann–Whitney U-tests were applied to test whether means differed significantly from each other (pairwise comparisons). P < 0.05 was considered to be statistically significant. The multistage Bonferroni correction was used to reveal unacceptably high levels of type I error (Shaffer, 1995).
To test two parameters against each other, Mann–Whitney U-tests were applied to determine whether hormone serum concentrations in FSH-P-stimulated and unstimulated animals differed significantly from each other on the days indicated. P < 0.05 was considered to be statistically significant.
Results
Group I: unstimulated pseudo-pregnant animals
The administration of onapristone to unstimulated animals on day 2 post-HCG caused an inhibition of endometrial transformation, as indicated by differences in progesterone-dependent epithelial cell proliferation and differentiation of secretory active epithelium. The expression of uteroglobin mRNA and APN mRNA was strongly inhibited by onapristone on day 5 post-HCG (Figures 2 and 3A). In onapristone-treated animals, endometrial glands were short and did not show ramifications (Figure 3B). In contrast, the endometrial glands of vehicle-treated control animals were well developed (Figure 3B).
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In unstimulated pseudo-pregnant animals, onapristone treatment caused an inhibition of proliferation, as indicated by Ki 67 staining (Figure 3C
) and induced apoptosis in ~20–30% of the epithelial cells (Figure 3E). In control animals endometrial epithelial cells proliferated (Figure 3D) and <1% of endometrial epithelial cells were apoptotic (Figure 3F).
Group II: FSH-P-stimulated pseudo-pregnant animals
In rabbits, stimulated with the pituitary gonadotrophin preparation FSH-P (a strong ovarian stimulant), the progesterone-dependent uteroglobin mRNA and APN mRNA expression continued (Figure 4) on day 5 post-HCG after onapristone administration on day 2 post-HCG. The morphological appearance of the endometrium was only marginally influenced: the size of the endometrial glands was slightly reduced (Figure 5 A and B), and there was a stronger hybridization signal in the surface epithelium of the onapristone-treated animals (Figure 5A).
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Proliferation was not inhibited after onapristone-treatment on day 4 post-HCG, as Ki 67 staining was similar in onapristone-treated and control animals (Figure 5C and D
). In both groups, <1% endometrial epithelial cells were apoptotic (Figure 5E and F).
Assessment of post-ovulatory peripheral hormone concentration in FSH-P-stimulated and unstimulated animals
Table I shows the serum concentrations of progesterone, testosterone and prolactin. The progesterone serum concentration in stimulated animals from day 2 post-HCG to day 5 post-HCG was significantly higher than in unstimulated animals (Mann–Whitney U-test: P 
0.004). On day 0 post-HCG there was no significant difference between the testosterone serum concentration of stimulated and unstimulated animals, whereas on day 2 post-HCG the testosterone serum concentration was significantly lower in stimulated animals (Mann–Whitney U-test: P = 0.017). The serum prolactin concentration on day 0 post-HCG was significantly lower in stimulated than in unstimulated animals (P = 0.005).
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The onapristone serum concentration was determined in three gonadotrophin-stimulated pseudo-pregnant animals 4 and 24 h after onapristone adminstration: 4 h after administration 212.76 ± 49.67 ng onapristone/ml and 24 h after administration 132.76 ± 46.72 ng onapristone/ml blood serum were found.
Group III: ovarian stimulation with four different human gonadotrophin preparations
Figure 6 depicts the effects of gonadotrophin-stimulation on the inhibitory potency of onapristone on endometrial uteroglobin mRNA expression. After stimulation with HMG or FSH/LH (1:1), uteroglobin mRNA expression continued after onapristone adminstration. However, after ovarian stimulation with FSH/LH (3:1) and LH, uteroglobin mRNA expression was suppressed by the onapristone.
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The endometrial morphology and uteroglobin mRNA distribution indicate that in HMG- (Figure 7A and B
) and FSH/LH (1:1)-stimulated animals (Figure 7C and D), onapristone affected gland size and uteroglobin mRNA distribution. In comparison with control animals, the endometrial glands of onapristone-treated animals were shorter and uteroglobin mRNA expression was enhanced in the epithelial surface cells. After onapristone treatment in FSH/LH (3:1)-stimulated animals (Figure 7E), endometrial gland formation was strongly inhibited and uteroglobin mRNA expression was reduced in surface and glandular epithelial cells. In LH-stimulated animals uteroglobin mRNA expression and endometrial gland formation was totally suppressed by onapristone treatment (Figure 7G).
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Figure 8
shows the proliferation of the two endometrial epithelial cell populations (the glandular and the surface epithelium), in gonadotrophin-stimulated, onapristone-treated animals and in gonadotrophin-stimulated control animals, (not treated with onapristone). This experiment showed that after these forms of stimulation, onapristone did not have a negative effect on Ki 67 staining (as seen in Figure 3), but actually increased cell proliferation. There was no significant difference in the epithelial cell proliferation (glandular and surface epithelium) between animals stimulated either with HMG, with FSH-LH (1:1) or with FSH-LH (3:1) after treatment with the onapristone. However, endometrial proliferation was significantly different between HMG-stimulated, onapristone-treated and control animals as well as FSH-LH (1:1)-stimulated, onapristone-treated and control animals. There was no statistically significant difference between FSH-LH (3:1)-stimulated animals and control animals after Bonferroni's correction.
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In Figure 9
the number of corpora lutea (as an indicator of the induction of folliculogenesis) and the progesterone serum concentrations on day 5 post-HCG are shown to demonstrate the influence of the four stimulation protocols on the ovary. The most potent induction of folliculogenesis was achieved either by stimulation with FSH-P, or the 1:1 mixture of FSH and LH, or by HMG stimulation (Figure 9A). With the 3:1 mixture of FSH and LH, only weak stimulation of folliculogenesis was achieved; stimulation was significantly lower than with FSH-LH (1:1) (P = 0.001). However, after Bonferroni's correction, it did not differ significantly from the HMG group. Recombinant LH alone did not enhance follicular growth and maturation. There were no significant differences between the number of corpora lutea in FSH-P, HMG and FSH-LH (1:1)-stimulated animals (Figure 9A). However, in HMG-stimulated animals, the progesterone serum concentration was significantly lower than in the FSH-LH (1:1) stimulated ones (Figure 9B). It is also important to recognize that, although the inhibitory effects of onapristone on the endometrium are much greater in the FSH-LH (3:1) group than in the HMG group, the progesterone serum concentration was not significantly different in the HMG and FSH-LH (3:1)-stimulated animals.
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Discussion
Ovarian stimulation with gonadotrophins is commonly used in human IVF and embryo transfer. However, the post-ovulatory endometrial transformation is advanced after ovarian stimulation (Benadiva and Metzger, 1984; Garcia et al., 1984; Kolb et al., 1997; Fanchin et al., 1999; Nikas et al., 1999) and, as a consequence, the precisely synchronized development of the endometrium and the embryo is disrupted. The reasons for this inadequate endometrial development after ovarian stimulation are unclear and remain under discussion.
We studied this problem of advanced endometrial development after gonadotrophin stimulation in the rabbit. The pseudo-pregnancy of the rabbit has proven to be a model system for the human luteal phase (Beier and Kühnel, 1973; Beier, 1982). In this species, ovarian stimulation induces an advancement of secretory transformation (Delbos-Winter et al., 1987) as well as an enhancement of gland formation (Krusche et al., 2000).
FSH-P stimulated and unstimulated animals: effects of onapristone on the endometrium
Whereas in unstimulated animals the post-ovulatory administration of onapristone exerted strong inhibitory effects on endometrial transformation, which comprises epithelial cell proliferation and secretory differentiation, in FSH-P stimulated animals, the same dose only slightly reduced the process of endometrial transformation. Endometrial epithelial cells continued to express uteroglobin mRNA, indicating a retardation of endometrial surface cell differentiation in comparison with FSH-P-stimulated control animals. Glandular size was slightly reduced probably as a consequence of apoptotic cell death in 3–5% of epithelial cells on day 3 post-HCG (data not shown).
This impairment of the antigestagenic potency of onapristone on the endometrium was obviously caused by ovarian stimulation. One consequence of ovarian stimulation was the elevation of the progesterone serum concentration on day 2 post-HCG. However, the elevated progesterone serum concentration could not be the only reason for the reduced antigestagenic potency of the onapristone because, in unstimulated animals, the antagonist acts in a completely inhibitory manner on day 4 post-HCG, when the serum progesterone concentration is as high as in FSH-P-stimulated animals on day 2 post-HCG (Krusche, 1999).
Different gonadotrophin-stimulation protocols and progesterone receptor antagonist effects on the endometrium
We further analysed the effects of four different ovarian stimulation protocols, which were variably potent in the induction of folliculogenesis and progesterone production of the corpora lutea, and on the modulation of the antigestagenic potency of the progesterone receptor antagonist, onapristone. The data reveal a correlation between the outcome of ovarian stimulation and the extent of the antigestagenic effects of onapristone.
Stimulation either with HMG (Pergonal) or the 1:1 mixture of recombinant human FSH and LH [FSH-LH(1:1)] is as potent as FSH-P at inducing folliculogenesis. In animals from these three stimulatory groups, endometrial transformation was only slightly inhibited after onapristone administration. However, the distribution of uteroglobin mRNA expression in the endometrial epithelium indicated a delay of surface epithelium differentiation, which is comparable with unstimulated animals on day 4 post-HCG (Krusche et al., 2000). This is the most important aspect of our study with regard to the major problem of human IVF, i.e. advanced endometrial development, which is claimed to be one reason for the reduced implantation rates. From our data, we deduce that the post-ovulatory application of a progesterone receptor antagonist is able to slow down this advanced endometrial development, in turn achieving better synchronization of the endometrium and the embryo.
In FSH-LH (3:1)-stimulated animals, onapristone strongly inhibited secretory transformation, although endometrial epithelial cell proliferation continued. So, as a consequence of low stimulation of folliculogenesis in this treatment group, onapristone exerted nearly complete inhibition of endometrial transformation.
Comparison of these FSH-LH (3:1)-stimulated animals with HMG-stimulated animals showed that the progesterone serum concentrations were similar in both groups, whereas the induction of folliculogenesis and the endometrial effects of onapristone were different. Thus, in our animal model, the predictive marker of the inhibitory effects of the progesterone receptor antagonist was the number of follicles induced and not the progesterone serum concentration. These results are in agreement with previous data (Lass et al., 1998), which showed a strong correlation between advanced endometrial transformation and the number of oocytes retrieved in human IVF cycles.
Mechanism of gonadotrophin action on the endometrium
Prolactin and testosterone are known to affect endometrial transformation and their concentrations were reported to be altered in women undergoing gonadotrophin stimulation (Ben-David and Schenker, 1983; Fliss et al., 1991; KleisSanFransisco et al., 1993; Simón et al., 1995). In our rabbit model, the prolactin serum concentration at day 0 post-HCG and the testosterone serum concentrations at day 2 post-HCG were significantly lower in gonadotrophin-stimulated animals. Consequently, both post-ovulatory ovarian steroidogenesis and pituitary prolactin release are altered, as in the human cycle. However, it has not yet been determined whether these two hormones interfere with the effects of the progesterone receptor antagonist on endometrial transformation.
In human IVF, gonadotrophin stimulation leads to alterations in the pre-ovulatory oestradiol and testosterone output (Martin et al., 1997; Agrawal et al., 1998). Both hormones regulate progesterone receptor expression (Iwai et al., 1995). In this context, it has been reported that progesterone receptor expression is decreased in the luteal phase after gonadotrophin stimulation in human IVF (Molina et al., 1989; Seppala and Tiitinen, 1995). The pre-ovulatory hormonal changes in rabbits have not yet been studied; however, we might speculate that there are similar changes which affect progesterone receptor expression. Furthermore, receptor modifications, such as ubiquitylation (Bebington et al., 2000), have to be taken into consideration in the regulation of endometrial transformation. The reported limited inhibitory effects of onapristone on the endometrium in gonadotrophin-stimulated rabbits might be the consequence of alterations in the progesterone receptor system. This hypothesis is under investigation.
Conclusions
The present study shows that after ovarian stimulation, the progesterone receptor antagonist onapristone retards endometrial transformation, although its effect is limited with stronger forms of ovarian stimulation. Since ovarian stimulation, used in human IVF therapy, is frequently reported to cause an advancement of post-ovulatory endometrial development, we suggest a therapeutic application of progesterone receptor antagonists to slow down such advanced endometrial transformation. Eventually, this modulation of advanced endometrial development may improve implantation rates. With respect to the strength of ovarian stimulation, the dose or even the type of progesterone receptor antagonist, either a pure antagonist or a receptor modulator (mesoprogestin) could be chosen to control post-ovulatory endometrial development in IVF cycles.
Acknowledgments
We thank Sabine Eisner, Elisabeth Brügmann and Marion Battenberg for excellent technical assistance. Thanks are also due to Dr Zurth (Schering AG) for the measurement of onapristone serum concentration and Dr C.Schmitz for statistical analysis. This work was kindly supported by the START Research funding programme of the School of Medicine, RWTH University of Aachen, and by Schering Aktiengesellschaft, Fertility Control and Hormone Therapy Research, Berlin.
Notes
3 To whom correspondence should be addressed at: Department of Anatomy and Reproductive Biology, Wendlingweg 2, 52057 Aachen, Germany. E-mail: ckrusche{at}post.klinikum.rwth-aachen.de 
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Submitted on January 10, 2000; accepted on May 22, 2000.
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