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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1160
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2081-2088
Copyright © 2005 by The Endocrine Society

Mifepristone Is an Effective Oral Alternative for the Prevention of Premature Luteinizing Hormone Surges and/or Premature Luteinization in Women Undergoing Controlled Ovarian Hyperstimulation for in Vitro Fertilization

Ernesto L. Escudero, Peter J. Boerrigter, Herjan J. T. Coelingh Bennink, Roberto Epifanio, José A. Horcajadas, Francois Olivennes, Antonio Pellicer and Carlos Simón

Pantarhei Bioscience B.V., Institute for Clinical Concept Research (P.J.B., H.J.T.C.B.), 3700 AL Zeist, The Netherlands; Foundation Instituto Valenciano de Infertilidad (E.L.E., R.E., J.A.H., A.P., C.S.) and Departments of Pediatrics and Obstetrics and Gynecology (J.A.H., A.P., C.S.), Valencia University School of Medicine, 46015 Valencia, Spain; and Unitéd de Medicine de la Reproduction, Hopital Cochin (F.O.), 75014 Paris, France

Address all correspondence and requests for reprints to: Dr. Herjan J. T. Coelingh Bennink, Pantarhei Bioscience B.V., Institute for Clinical Concept Research, P.O. Box 464, 3700 AL Zeist, The Netherlands. E-mail: hcb{at}pantarheibio.com.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The present clinical study was conducted to investigate the effectiveness of a daily dose of 40 mg mifepristone in preventing premature LH surges in women undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization and to study the effect of this antiprogestin cotreatment on endometrial receptivity. This was a prospective, open-label, randomized, exploratory study in 15 healthy volunteer oocyte donors who were randomly allocated to the experimental COH group, including mifepristone (group 1), or the control group, using a long protocol with GnRH agonists (group 2), in a ratio of 2:1, i.e. 10 and five subjects, respectively. In group 1, human chorionic gonadotropin (hCG) was randomly administered (group 1A) or was withheld (group 1B) at the end of stimulation, so that two subgroups of five subjects each were formed, differing in the final oocyte maturation trigger. In all patients receiving mifepristone, 50 mg progesterone were administered im at the time of hCG administration to counteract residual antiprogestogenic activity of mifepristone. Serum estradiol, progesterone (P), LH, and FSH levels were monitored in each patient on d 3 and 6 and every 48 h thereafter. Endometrial biopsies were taken 2 and 7 d after hCG or P administration. Endometrial tissue was processed and evaluated in a blinded fashion for endometrial dating and quantitative PCR of at least four genes known to be up-regulated in receptive endometrium. The total FSH dose and duration of treatment in the two arms of the study were similar. The mean LH levels on d 6 of stimulation and the day of hCG/P treatment in the mifepristone group were 0.8 ± 0.7 and 0.5 ± 0.6 mIU/ml, and those in control subjects were 2.4 ± 3.8 and 2.0 ± 1.7 mIU/ml, respectively. No LH surges were observed in any subject treated with mifepristone. Serum P levels on the day of hCG/P were below the cut-off level (1.2 ng/ml) in all subjects of the mifepristone group (range, <0.5 to 1.05 ng/ml). The mean numbers of cumulus-oocyte complexes retrieved were 11.6 ± 6.6 and 19.6 ± 11.8 in the subgroup treated with mifepristone and hCG and in the control group, respectively. The mean percentages of metaphase II, metaphase I, and germinal vesicle stage oocytes were 86.2, 6.9, and 3.4% in the mifepristone group and 68.4, 6.1, and 11.2% in the control group. In the mifepristone group that did not receive hCG and received P only at the end of stimulation, an endogenous LH surge was not observed nor were oocytes obtained. Histological evaluation of endometrial samples in patients treated with mifepristone and hCG (group 1A) confirmed normal development, whereas in patients treated with mifepristone only (group 1B), there was a complete arrest of the endometrial maturation. The expression patterns of glycodelin, IGF-binding protein-7, glutathione peroxidase-3, and solute carrier family 1 member 1 show a striking absence of up-regulation in patients treated with mifepristone (groups 1A and 1B) compared with controls (group 2). The results of this exploratory study provide evidence that mifepristone is effective for the prevention of premature LH surges and/or premature luteinization in women undergoing COH for in vitro fertilization. However, endometrial receptivity status requires additional evaluation after decreasing RU-486 doses before this strategy can be considered as a new alternative to GnRH agonist/antagonist treatment.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE CURRENT STANDARD of controlled ovarian hyperstimulation (COH) for assisted reproductive technologies, e.g. in vitro fertilization (IVF), includes the administration of FSH to induce multiple follicular growth and cotreatment with a GnRH agonist/antagonists to suppress endogenous gonadotropins. The positive pituitary feedback of rapidly rising estrogen levels produced by the cohort of growing follicles resulting from FSH stimulation gives rise to a high risk of an early LH peak associated with premature luteinization and early ovulation, leading to cycle cancellation (1). GnRH agonists have been found to be effective and have been widely used to prevent premature LH surges (2). In recent years, GnRH antagonists have been shown to be an effective alternative to GnRH agonists and to enable simplification of COH protocols. GnRH antagonists do not require a desensitization period, because they act immediately and can be started during the late follicular phase (3). However, GnRH analog cotreatment is associated with significant drawbacks: GnRH agonists administered as injections or multiple nasal puffs have a long desensitization period along with side effects (hot flushes, headache, bleeding, and vaginal dryness); GnRH antagonists administered as sc injections may produce unwanted LH surges (4). Despite this cotreatment, premature luteinization is reported to occur in 2–35% of the agonist cycles [depending on the progesterone (P) cut-off value used] and in 38% of antagonist cycles (5, 6). New methods with improved efficacy, safety profile, and user convenience are therefore much welcomed.

Mifepristone was the first developed and best characterized antiprogestin with a 19-nor steroid structure, a high affinity to the P receptor effectively competing for P binding and good oral bioavailability (7, 8). The rationale for the potential usage of mifepristone is that this compound possesses both central inhibiting and antiprogestogenic properties that justify an evaluation of its use in COH protocols. It has been known for a long time that continuous low-dose treatment with mifepristone, i.e. 2–10 mg daily, is capable of inhibiting ovulation and is associated with disrupted folliculogenesis or follicular arrest (9, 10). Most likely, mifepristone inhibits the positive feedback of estrogens at the level of the pituitary (11, 12). In contrast, COH is associated with advanced endometrial histology and relatively high P levels in the late follicular phase occurring in a relatively large proportion of IVF cycles despite GnRH analog treatment, which is associated with impaired implantation and lower pregnancy rates (5, 13, 14, 15). Low dosages of mifepristone have been shown to delay endometrial maturation (16). A P antagonistic effect during the follicular phase can be hypothesized to counteract any premature P activity during COH, overcoming the histological advancement demonstrated. The functional genomics of human endometrial receptivity have been recently developed (17, 18, 19, 20). Genome-wide analysis with DNA microarray technology demonstrates that receptivity is an active process involving hundreds of up- and down-regulated genes (17, 18, 19, 20). Therefore, we are in the position to investigate the impact of this cotreatment on endometrial receptivity by comparing the expression of a defined cluster of window of implantation genes in patients treated with this new strategy vs. controls.

The present clinical study was conducted primarily to investigate the effectiveness of a daily dose of 40 mg mifepristone in preventing premature LH surges in women undergoing COH for IVF and to further analyze the effect on the endometrium of a protocol of mifepristone including COH. We also investigated whether P alone was able to induce an endogenous LH surge and/or initiate final oocyte maturation, because there is still controversy about the role of P in the mechanism of ovulation (21, 22).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study design and subjects

The study was designed as an open-label, randomized, controlled, exploratory study in healthy females qualifying as oocyte donors. The primary objective of the study was to explore the potential effectiveness of mifepristone in a COH protocol to prevent premature LH surges and the effect on endometrial receptivity compared with that of a standard long agonist protocol. Secondary objectives were to investigate the potential use of P in lieu of human chorionic gonadotropin (hCG) in inducing a spontaneous LH surge and/or final oocyte maturation at the end of stimulation.

Mifepristone and FSH were administered daily from the beginning of the COH and concomitantly with an im injection of 50 mg P to reverse residual antiprogestogenic activity of mifepristone on the day at least two preovulatory follicles were observed by ultrasound (21). Specific end points were to evaluate the endocrinological profile, the number and distribution of follicles, the number and quality of oocytes retrieved, the comparative expression of the genes involved in endometrial receptivity, and the safety for the subjects treated.

The study was conducted in accordance with the last revision of the Declaration of Helsinki and the International Conference on Harmonization guideline for Good Clinical Practice. Written informed consent was obtained from all subjects, and the study was approved by the internal institutional review board.

Sixteen subjects who qualified as oocyte donors were enrolled as volunteers; they had a body mass index between 19 and 29 kg/m2, a normal karyotype and were between 21 and 35 yr of age. Excluded were subjects with polycystic ovary syndrome, as documented by ultrasound and endocrinological evidence, and/or endometriosis; hormone levels outside the normal range [FSH, ≥10 mIU/ml; estradiol (E2), ≥60 pg/ml; LH, ≥10 mIU/ml; prolactin, ≥24.2 ng/ml; TSH, ≥4.7 µIU/ml; T4, ≥13 µg/dl); the presence of a follicular cyst with concomitant E2 levels greater than 60 pg/ml at the start of stimulation; a low response in a previous COH cycle(s); gross uterine or ovarian alterations, as documented by ultrasound; positive blood tests for hepatitis B, hepatitis C, and/or HIV; and/or contraindications of any drug used. Fifteen subjects completed the study, and one subject in the control group was replaced due to an insufficient ovarian response.

A mifepristone dose of 40 mg was chosen for this concept testing study for practical reasons, because it had to be manufactured from standard tablets available in 200-mg strength only.

Treatment

Subjects qualifying for the study were randomly allocated to the experimental COH group including mifepristone (group 1) or the control group (group 2) in a ratio of 2:1, i.e. 10 and five subjects, respectively. In group 1, hCG was randomly administered (group 1A) or was withheld (group 1B) at the end of stimulation, so that two subgroups of five subjects each were formed, differing in the final oocyte maturation trigger.

In all subjects in group 1 (COH plus RU486), controlled ovarian hyperstimulation started on menstrual cycle d 2 with 150 IU recombinant FSH (Gonal-F, Serono S.A., Madrid, Spain), sc, for 2 d. On stimulation d 3, the serum E2 level was assessed, and the FSH dose was adjusted according to a step-up or step-down protocol (if E2 was >200 pg/ml: 100 IU FSH/d for 3 d; if E2 was 100–200: 150 IU FSH/d for 3 d; if E2 was 50–100: 200 IU FSH/d for 3 d).

From stimulation d 6 onward, E2 levels and follicle size were monitored every 48 h. Mifepristone was administered as a daily dose of 40 mg, orally, concomitantly with FSH, starting from d 2 of the menstrual cycle. When at least two follicles were 18.5 mm or larger, 50 mg P was administered im to counteract any residual antiprogestogenic activity of mifepristone in all subjects. In subgroup 1A, 6500 IU recombinant hCG (Ovitrelle, Serono S.A.) was also administered at this time to trigger oocyte maturation, whereas in subgroup 1B, hCG was not administered.

In group 2, GnRH agonist treatment with 800 mg nafarelin (Synarel, Seid, Barcelona, Spain) was started on d 21 of the preceding cycle as two nasal puffs twice daily. On menstrual cycle d 2, the nafarelin dose was reduced to 400 mg as one nasal puff twice daily to be continued until the day of hCG administration. On the same day, stimulation with recombinant FSH was started, and the administration of FSH and hCG as well as monitoring followed the protocol described for subgroups 1A and 1B.

Oocyte retrieval was scheduled in all patients 36 h after the administration of P and/or hCG. Oocytes from the subjects in the mifepristone group were not used for fertilization/donation for safety reasons. In the luteal phase, all subjects received P supplementation intravaginally with 400 mg micronized P (Utrogestan, Seid).

Endometrium processing and blood sampling

Endometrial biopsy using a Pipelle device was scheduled in all subjects on d 2 and 7 after P and/or hCG administration. A portion of each specimen was included in formalin for histological dating, according to the criteria of Noyes et al. (23), and immunohistochemistry. Immunohistochemistry was performed on endometrial sections using an LSAB peroxidase kit (DakoCytomation, Carpinteria, CA) with appropriate primary and secondary antibodies. A second portion of the sample was placed in RNALater (Sigma, St. Louis, MO) and immediately frozen at –80 C to quantify the expression pattern of selected genes involved in endometrial receptivity (20), including glycodelin (PP14), IGF-binding protein-7 (IGF-BP-7), glutathione peroxidase-3, and solute carrier family 1 member 1, by quantitative fluorescent RT-PCR (QF-PCR).

Blood samples were obtained from all subjects in each visit. Serum LH, P, and E2 were measured on stimulation d 3 and every 48 h from stimulation d 6 onward. Samples were tested with a microparticle enzyme immunoassay Axsym System (Abbott Cientifica S.A., Madrid, Spain). The serum E2 kit had a sensitivity of 28 pg/ml and intra- and interobserver coefficients of variation of 6.6 and 7.7%, respectively. The serum LH kit had a sensitivity of 0.5 IU/liter, with intra- and interobserver coefficients of variation of 4.6 and 1.9%, respectively. The serum P kit had a sensitivity of 0.2 ng/ml, with intra- and interobserver coefficients of variation of 6.6 and 7.7%, respectively.

RNA isolation

Total RNA was extracted using the TRIzol method according to the protocol recommended by the manufacturer (Invitrogen Life Technologies, Inc., Gaithersburg, MD). Briefly, homogenized biopsies (1 ml TRIzol reagent/75 mg tissue) were incubated at room temperature for 5 min. After addition of chloroform (0.2 vol TRIzol), samples were incubated for 2.5 min at room temperature; thereafter, they were centrifuged for 15 min at 12,000 x g (4 C). The aqueous phase was precipitated with an equal volume of 2-propanol, stored on ice for 5 min, and centrifuged for 30 min at 12,000 x g (4 C). The pellet was washed with 75% ethanol and dissolved in diethylpyrocarbonate-treated H2O. Approximately 1–2 µg total RNA was obtained per milligram endometrial tissue. RNA quality was checked by agarose gels. To 1 µg total RNA, 0.5 µg oligo(deoxythymidine)12–18 primer (Invitrogen Life Technologies, Inc., Carlsbad, CA) and 0.5 µg pd(N)6 (Amersham Biosciences, Arlington Heights, IL) were added. The mixture was heated at 70 C for 10 min, then quickly chilled on ice for 2 min. cDNA was synthesized in a total volume of 20 µl containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM deoxy-NTPs, and 200 U SuperScript II ribonuclease H– reverse transcriptase (Invitrogen Life Technologies, Inc.). After incubation for 1 h at 37 C, the cDNA was diluted to a concentration equivalent to 100 ng/µl.

Real-time QF-PCR

The experiments for real-time QF-PCR were performed with the LightCycler System (Idaho Technology, Inc., Salt Lake City, UT). Primers were designed using the Primer Express software provided with the aforementioned system to accomplish the manufacturer universal conditions. SYBR Green I double-stranded DNA binding dye was used as the assay chemistry. Total RNA (1 µg) was reverse transcribed using an Advantage RT-for-PCR kit (Clontech, Palo Alto, CA). We employed 200–300 ng cDNAs for each sample analyzed. cDNA from placenta tissue was used to obtain the standard curve. All real-time QF-PCR assays were carried out according to the manufacturer’s universal thermal cycling parameters. The primer-cDNA sequences are listed in Table 1Go. Each assay was performed in duplicate. Data were presented as the relative value of the gene expression investigated and normalized to the housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase) in each patient. Melting curves were analyzed to confirm amplification specificity.


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  		TABLE 1. Primer sequence of genes involved in endometrial receptivity and housekeeping gene investigated

 
Definitions and statistics

A premature LH surge was defined as an LH rise of 10 mIU/ml or more; premature luteinization was defined as a measurement of serum P of 1 ng/ml or more. Given the exploratory character of the study, only descriptive statistics were used. For subjects with LH levels below the lower detection limit of 0.5 mIU/ml, a value of 0.25 mIU/ml was used for descriptive statistic calculations.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
One cycle cancellation occurred in a subject in the control group (subject 12) due to a poor ovarian response, and the subject was replaced so as to end up with the intended five completers in the control group according to the protocol. Table 2Go presents the subject characteristics and clinical features of COH. Overall, the 10 subjects in group 1 and the six subjects in group 2 did not differ in mean age, weight, height, or body mass index. The total FSH dose used to arrive at the present criteria for administering hCG and/or P was 1560 ± 288 IU in the mifepristone group and 1370 ± 524 IU in the control group. The duration of treatment was 10.0 ± 1.3 d in the mifepristone group and 9.4 ± 1.7 d in group 2.


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  		TABLE 2. Subject characteristics and clinical features of controlled ovarian hyperstimulation

 
Endocrinology

Premature LH surges were never observed in any subject treated with mifepristone (groups 1A and 1B). Moreover, in the subjects assigned to the mifepristone group who did not receive hCG (group 1B), no oocyte was obtained.

One subject in the control group (subject 13) had a serum LH level of 10 mIU/ml with a concomitant serum P level of 1.3 ng/ml on d 6 of stimulation (Fig. 1Go). All serum LH levels in the subjects treated with mifepristone were 2 mIU/ml or less, even below the detection limit of 0.5 mIU/ml in four of 10 subjects on d 6 of stimulation and in seven of 10 subjects on the day of hCG/P administration. Two subjects in the control group had a serum LH level below the detection limit on d 6 of stimulation, and none of the controls had a serum LH below detection on the day of hCG treatment. The mean LH levels of subjects treated with mifepristone on d 6 of stimulation and on the day of hCG/P administration were 0.8 ± 0.7 and 0.5 ± 0.6 mIU/ml, respectively, and those of subjects in the control group were 2.4 ± 3.8 and 2.0 ± 1.7 mIU/ml, respectively (Fig. 1Go).



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  		FIG. 1. Serum LH levels on stimulation d 6 and on the day of hCG administration in subjects undergoing COH with concomitant mifepristone (40 mg, orally) and GnRH agonist in a long protocol (control group). {square}, Undetectable LH values, i.e. values under the lower limit of detection for LH of 0.5 mIU/ml. *, Missing value.

 
Individual serum E2 levels of all subjects on d 6 of stimulation and the day of hCG/P treatment are presented in Fig. 2Go. Serum E2 levels in the subjects treated with mifepristone ranged from 580-2300 pg/ml on the day of hCG/P administration, whereas those of the subjects in the control group varied between 1486 and 7316 pg/ml; the mean serum E2 levels were 1171 ± 597 and 3727 ± 2434 pg/ml in the mifepristone group and the control group, respectively.



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  		FIG. 2. Serum E2 levels on stimulation d 6 and on the day of hCG administration in subjects undergoing COH with concomitant mifepristone (40 mg, orally) and GnRH agonist in a long protocol (control group). *, Missing value.

 
Serum P levels on the day of hCG/P treatment (Fig. 3Go) were below the critical cut-off level (1.2 ng/ml) for the definition of premature luteinization in all subjects treated with mifepristone (range, <0.5–1.05) and in only three of the five subjects in the control group (subject 14, 1.34 ng/ml; subject 13, 6.1 ng/ml).



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  		FIG. 3. Serum P levels on the day of hCG administration in subjects undergoing COH with concomitant mifepristone (40 mg, orally) and GnRH agonist in a long protocol (control group). *, Missing value.

 
Follicles and oocytes

Figure 4Go represents a graphical overview of the number of follicles observed in the two treatment groups separated by size categories (10–16 and >16 mm in diameter). On the day of hCG administration, the mean number of mature follicles (>16 mm) was 6.4 ± 5.2 in subgroup 1A and 7.6 ± 6.4 in the control group, respectively. The mean number of cumulus oocyte complexes retrieved was 11.6 ± 6.6 in subgroup 1A and 19.6 ± 11.8 in the control group, respectively. An intact zona pellucida was observed in 96.6 and 85.7% of the oocytes in the two groups, respectively. The mean percentages of metaphase II, metaphase I, and germinal vesicle stage oocytes were, respectively, 86.2, 6.9, and 3.4% in the mifepristone plus hCG group and 68.4, 6.1, and 11.2% in the control group. Oocytes obtained from group 1 were not used for fertilization or for reproductive purposes because of the experimental nature of the study.



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  		FIG. 4. The mean number of follicles in two categories, 10–16 mm in diameter and more than 16 mm in diameter, on stimulation d 6 and 8 and on the day of hCG administration in subjects undergoing COH with concomitant mifepristone (40 mg, orally; {blacksquare}) and GnRH agonist in a long protocol (control group; {square}).

 
Endometrial hematoxylin-eosin dating and QF-PCR

Endometrial samples obtained on LH+2 and LH+7 (2 d and 7 d after hCG administration) in patients treated with mifepristone plus hCG (group 1A) confirmed development as expected in a natural cycle. Endometrium from patients treated with mifepristone only (group 1B) showed a complete arrest of endometrial maturation regardless of P administration, whereas the endometria from group 2 (control) resulted in advanced histological dating (Table 3Go).


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  		TABLE 3. Comparison of chronological vs. histological dating in endometrial samples from patients in each treatment group

 
The expression patterns of four genes strongly up-regulated in endometrial receptivity (20) (PP14, IGF-BP-7, glutathione peroxidase 3, and solute carrier family 1 member 1) were quantified by QF-PCR in endometrial samples from all groups on LH+7. The results show a striking absence of gene up-regulation in those endometria from patients treated with mifepristone (groups 1A and 1B) compared with controls (group 2) and with our previous report (Fig. 5Go).



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  		FIG. 5. Quantitative PCR of a cluster of endometrial receptivity genes up-regulated during the window of implantation in natural (20 ) and IVF (group 2) cycles (results are shown as the average). This up-regulation is lacking in all patients treated with mifepristone (1A1, 1A2, 1B1, 1B2, and 1B3).

 
Safety

One subject in the control group (subject 2) developed moderate ovarian hyperstimulation syndrome, but recovered completely after appropriate treatment. No other side effects occurred during the study.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The results of this exploratory study provide evidence that mifepristone is effective as an oral treatment for the prevention of premature LH surges and/or premature luteinization in women undergoing COH for IVF. Nevertheless, at the indicated doses even with P supplementation on the day of hCG treatment, there is an adverse impact on endometrial receptivity at the gene level that requires additional modifications.

Mifepristone has been primarily investigated as an abortifacient and contraceptive drug in the past. The results of those efforts have led to the development and regulatory approval of the drug in combination with prostaglandins for the termination of early pregnancy. For this purpose, dosages as high as 600 mg must be administered to be optimally effective (24). The fear of potential misuse of antiprogestins and difficulties in finding generally acceptable dosing regimens have obstructed additional development of those compounds for contraceptive purposes.

Few efforts have been made to investigate antiprogestins as a cotreatment in women undergoing COH for IVF to suppress premature LH surges and to prevent premature luteinization, which are associated with a poor outcome of IVF cycles. One clinical study was performed in healthy women with male factor infertility undergoing intrauterine donor insemination with the aim to investigate the mechanism of disruption of folliculogenesis caused by mifepristone (25). Eleven women with ovulatory cycles were treated with 150 IU recombinant FSH on d 2, 4, 6, and 8 and daily thereafter until the administration of hCG during one cycle and were cotreated with a daily dose of 50 mg mifepristone from d 2 until hCG injection during a subsequent cycle. Treatment resulted in the development of a single preovulatory follicle, which was delayed in the mifepristone treatment cycle. The researchers suggested that mifepristone might have a potential application for the prevention of LH surges in COH. The current study for the first time shows that low-dose mifepristone cotreatment is effective in preventing premature LH surges in women undergoing COH for IVF. The results show that the FSH dosages employed in standard COH protocols suffice to overcome mifepristone-induced growth retardation of the follicles, which had already been established in a monkey study (26). In the study reported here, a dose of 40 mg mifepristone was used to prove the concept, which resulted in a profound and sustained suppression of endogenous LH, with the majority of subjects having undetectable LH levels on the day of hCG, whereas none of the subjects in the control group had undetectable serum LH levels. This implies that the dose employed in this study resulted in oversuppression of the hypothalamic-pituitary unit, which was associated with reduced E2 production and lower oocyte yields, as also shown in other studies (27, 28). Because the occurrence of an LH surge is not always observed when follicles reach 18 mm, a larger study will be necessary to confirm these data. Also, in the present study there is a lack of post-hCG LH measurements, which will be interesting to perform in additional investigations.

One subject in the control group exhibited premature luteinization with a high preovulatory serum P level, and one subject had a borderline P value just beyond the cut-off value established for the research center. In contrast, none of the subjects in the mifepristone group showed any sign of premature P rise. Especially in subjects with a premature increase in P, an antiprogestin may be beneficial to block an increased P action thought to impair IVF outcome.

It is well known that preovulatory endometrium exhibits early secretory changes more frequently in women undergoing COH compared with controls even without higher P concentrations (29). Advanced endometrial histology and premature appearance of pinopods have been described as markers of a shifting implantation window (30). Our data in the control group (group 2) confirmed previous results indicating that gonadotropin-stimulated COH cycles resulted in a histological advancement of the endometrium. Interestingly, cotreatment with RU-486 provoked an in-phase development as expected in a natural cycle (group 1A), but if hCG was withheld (group 1B), there was dramatic endometrial development arrest regardless of P administration, probably due to occupancy of the P receptors in the endometrium. Despite the morphological appearance of the endometrium, QF-PCR identified the absence of up-regulation of a cluster of well-characterized genes involved in endometrial receptivity (17, 18, 19, 20) in all patients treated with mifepristone (groups 1A and 1B) compared with the control group. This finding indicates that an additional decrease in mifepristone will be necessary to achieve the endometrial receptivity aspects.

Obviously, a dose-finding study is warranted to establish the appropriate dose of mifepristone for this application. It may be expected that a dose much lower than the 40 mg employed in this study will result in effective suppression of the pituitary. Dosages as low as 2 mg mifepristone/d were associated with complete suppression of ovulation in the murine model (31). This is important from a safety perspective, because the anticipated total dose of mifepristone during stimulation will be far below that required for the induction of abortion (600 mg), practically excluding misuse of the drug.

P plays a key role in the ovulatory process. Experiments have shown that a rise in P precedes the LH surge by several hours in the periovulatory period (32). Also, the administration of exogenous P has been shown to elicit an FSH/LH peak in an estrogen-primed milieu (33). The current study provided an opportunity to address the question of whether P alone is able to induce an endogenous LH peak and/or final oocyte maturation in a COH protocol, an option that would further relieve the burden of COH. From the results, it appears that a 50-mg single dose im injection of P does not result in the retrieval of mature or immature oocytes in this situation. However, it is important to mention that P was administered in the presence of a high concentration of mifepristone; thus, it is difficult to make any solid conclusions about whether P will trigger the LH surge in this experimental design. In any case, in a COH protocol including mifepristone, hCG administration is required to induce final oocyte and endometrial maturation.

Although no side effects were noticed in patients treated with mifepristone, one important issue that was not addressed in this exploratory study was the safety of the oocyte and embryo, because no fertilization of oocytes was allowed in subjects treated with mifepristone. Mifepristone rapidly crosses the blood-follicle barrier of the follicles, and follicular fluid levels are similar to plasma levels (33). In a study including 40 female volunteers requesting laparoscopic sterilization treated with clomiphene and hCG, a dose of 100 mg mifepristone was administered to 20 women 1 h before hCG administration, whereas 20 women served as controls. Oocytes were recovered from follicles larger than 15 mm and fertilized in vitro with donor semen. Neither the cleavage rate of fertilized oocytes, the morphological characteristics of the cleaving embryo (four to eight cells), nor the hormonal concentrations in the follicular fluid were significantly different between groups (34). Mifepristone administration in the envisaged COH protocol would be discontinued at least 3 d before embryo transfer, and P would be administered to reverse any residual antiprogestogenic activity on the day of hCG treatment. Despite the long half-life of mifepristone, embryonic exposure after embryo transfer will be very low. An extensive toxicity and teratogenicity program did not reveal any concern for the regulatory approval of mifepristone; however, embryotoxic effects cannot be completely ruled out. Spontaneous reporting has revealed nine cases of fetal malformations after exposure to mifepristone in combination with gemeprost. Eight of these cases were published (35). Prostaglandins are known to be teratogenic; most likely, it is the prostaglandin cotreatment that accounts for the reported teratogenic effects associated with this treatment (35, 36).

In conclusion, oral administration of mifepristone is able to prevent premature LH surges and/or premature luteinization in women undergoing COH for IVF. Progesterone alone is not effective in inducing an endogenous gonadotropin surge or final oocyte maturation in COH. Endometrial development in patients cotreated with mifepristone is histologically normal, but endometrial receptivity is altered at the gene level. Additional studies are needed to establish the appropriate dose and to investigate the fertilization, implantation, and pregnancy rates with this new type of COH regimen, which will contribute to a more patient-friendly stimulation protocol.


   Footnotes
 
First Published Online October 13, 2004

Abbreviations: COH, Controlled ovarian hyperstimulation; E2, estradiol; hCG, human chorionic gonadotropin; IGF-BP-7, IGF-binding protein-7; IVF, in vitro fertilization; P, progesterone; QF-PCR, quantitative fluorescent RT-PCR.

Received June 17, 2004.

Accepted October 4, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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