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Pii: s0015-0282(01)01834-9

FERTILITY AND STERILITY
Copyright 2001 American Society for Reproductive Medicine Printed on acid-free paper in U.S.A.
Effect of a combined oral contraceptive
containing 3 mg of drospirenone and 30

g of ethinyl estradiol on the human
endometrium
Frank Lu¨dicke, M.D.,a Elisabeth Johannisson, M.D.,a Frans M. Helmerhorst, M.D.,bAldo Campana, M.D.,a Jean-Michel Foidart, M.D.,c and Renate Heithecker, Ph.D.d University Hospital Geneva, Geneva, Switzerland; Leiden University Medical Center, Leiden, The Netherlands;Universite´ de Lie`ge CHR Citadelle, Lie`ge, Belgium; and Schering AG, Berlin, Germany Objective: To provide an in-depth assessment of the effects of the combined oral contraceptive containing
30 ␮g of ethinyl estradiol and 3 mg of drospirenone (Yasmin, Schering AG, Berlin) on the endometrium by
means of endometrial morphometry in comparison to an untreated cycle.
Design: The open, multicenter study consisted of one untreated precycle and 13 treatment cycles.
Setting: Four gynecologic clinics in Belgium, The Netherlands, and Switzerland were involved.
Patient(s): Forty women with a history of regular menstrual cycles.
Intervention(s): Before the commencement of the trial, 3 months without any hormonal intake was oblig-
atory. The first endometrial sample was done in the untreated precycle, adjusted to the day of LH peak plus
5 to 6 days. During the medication phase, endometrial samples were taken at cycle 3, 6 and 13.
Main Outcome Measure(s): Primary outcome measure of the study was the morphologic assessment of the
endometrium with measures such as glandular diameter, glandular epithelial height, and number of vacuolated
cells per 1,000 glandular cells. Furthermore, the endometrial thickness was measured by ultrasound.
Result(s): After 13 cycles of medication use the endometrium had an atrophic appearance in 63% of the
subjects. The size of the glands, the glandular epithelial height, and the number of glands per square millimeter were already significantly reduced after 3 months’ use. Histological and ultrasonographical evaluation of the endometrium indicated a suppression of the proliferative activity of the endometrium.
Conclusion(s): The combination of 30 ␮g of ethinyl estradiol with 3 mg of drospirenone induces changes of
the endometrium that are comparable with other combined oral contraceptives and exhibits a marked antiproliferative effect on the endometrium. The medication was proven to be an effective oral contraceptive and revealed good cycle control characteristics. (Fertil Steril௡ 2001;76:102–7. 2001 by American Society for Hospital Geneva,Switzerland, 30 bld de la Key Words: Combined oral contraceptive, drospirenone, endometrium
Cluse, 1211 Geneva 14,Switzerland.
a Department of Obstetricsand Gynecology, University erts antimineralocorticoid effects in humans (3, chemically a 17-␣-spirolactone derivative, was 4). DRSP given in a dosage of 2 mg inhibits characterized in receptor-binding studies and in ovulation in normally cycling women and has transactivation assays as a progestogen with shown to increase sodium excretion when com- Reproductive Medicine,Leiden University Medical antimineralocorticoid and antiandrogenic ac- pared with placebo. This mild natriuretic effect tivity and was found to be devoid of any an- of DRSP is expected to be of potential benefit drogenic, estrogenic, glucocorticoid, and anti- in terms of prevention of sodium retention and Gyne´cologie-Obste´triquede l’Universite´ de Lie`ge glucocorticoid activity (1). These in vitro rise in blood pressure in susceptible women findings were in accordance with the pharma- cological profile found by in vivo studies in medication (5, 6). The antiandrogenic activity Fertility Control & HormoneTherapy, Schering AG.
various animal models (2). These characteris- of DRSP is due to a functional blockade of the tics place DRSP in close resemblance to the androgen receptor, and the potency in vivo in 0015-0282/01/$20.00PII S0015-0282(01)01834-9 natural hormone progesterone, which also ex- the rat was found to be one-third that of cypro- terone acetate (2). Both the antimineralocorticoid and anti-androgenic effects of DRSP made it a promising candidate for further drug development as progestogenic agent for oral Baseline characteristics of age, height, weight, age at contraception and hormone replacement therapy.
menarche and intracyclic bleeding before the start of the The progestogenic activity of DRSP was evaluated in a variety of animal models, and the results obtained suggest a relative progestational potency of the same order as that ofnorethisterone acetate (2). The borderline ovulation inhibi- tion dose in women was found to be 2 mg and the combi- nation of 30 ␮g of ethinyl estradiol (EE) and 3 mg of DRSP did enter into the clinical development of the COC (7). In addition to ovulation inhibition, the contraceptive effect by oral contraceptives are attained by induced changes of the endometrium hampering ovum implantation. The adminis- Note: Data given as mean Ϯ standard deviation.
tration of a COC produces variable histological pictures, but Lu¨dicke. Combined oral contraceptive and human endometrium. Fertil Steril 2001. in general, results in an underdevelopment of endometrialstroma and glands, whereas the effect on the endometrialglands are often more pronounced. The endometrium be- gave her written consent before enrollment. The study was comes scanty and contains only few glands with empty and performed with the regulations and recommendation of the narrow-lined lumina (8). With time of COC intake, the Declaration of Helsinki (Hong Kong 1989).
height of the epithelium decreases, the glands become atro- Episodes of bleeding and any adverse events were re- phic, pseudodezidualisation of the stroma can be detected, and, moreover, the development of the spiral arteries does Trial Plan
not take place to the same extent as in a normal menstrual The women who volunteered for the study were assigned randomly to two independent groups. In group A, the treat- These induced changes in the endometrium are consid- ment effects were investigated after 3 and 13 treatment ered to avoid hyperplasia and to minimize the long-term risk cycles. In group B, the treatment effects were investigated for endometrial cancer and could also lead to less menstrual after 6 and 13 treatment cycles. Both groups received the bleeding. In the study reported here, the degree to which the same medication, thus the only difference between the two DRSP-containing oral contraceptive affects the endome- assessment groups was the timing of the endometrial biop- trium in healthy women was investigated; it was aimed at assessing the effects of the combined oral contraceptive The study consisted of one untreated precycle and 13 containing 30 ␮g of EE and 3 mg of DRSP on the endome- treatment cycles. Before the commencement of the trial, a trium by means of endometrial morphometry in comparison minimum of 3 months without any hormonal intake was to an untreated cycle. Evaluation of the effect of the study obligatory. The first endometrial sample in the untreated medication on the endometrium characterizes the progesta- precycle was performed adjusted to the day of LH peak plus tional potency of DRSP, and the results were compared to 5 to 6 days. A home monitoring kit to detect the LH peak in already existing data on endometrial changes with other urine (OvuQuick, Quidel, San Diego, CA) was used for this combined oral contraceptives. Finally, the gathered data purpose. Baseline characteristics for all 40 subjects are sum- adds valuable information to the safety evaluation of this Test Material
MATERIALS AND METHODS
The study medication consisted of 21 film-coated tablets of 30 ␮g of ethinyl estradiol and 3 mg of drospirenone per Subjects
blister pack. One tablet was taken daily for 21 days, followed Forty apparently healthy women with a history of regular menstrual cycles volunteered for the study. None of them Endometrial Samples and Morphometric
had used steroidal contraceptives or an intrauterine device Analyses
during the previous 3 months, and none had had an abortion The endometrial sample was performed with a Pipelle de within the last 6 months or delivery within 1 year before cornier device without dilatation of the cervix and without anesthesia. The endometrial strips (10 –15 mm) were imme- The medical ethics committee of each of the study sites diately fixed, embedded in paraffin, sectioned, and stained (Geneva, Switzerland; Leiden, The Netherlands; Lie`ge and with hematoxylin and eosin. In addition to a general histo- Nijmegen, Belgium) approved the study, and each woman logic assessment of the endometrial biopsies, numeric data FERTILITY & STERILITY
difference was seen concerning the histologic assessment between VCA and ITT population. No endometrial pathol-ogy was detected in the ITT nor in the VCA population. For Morphological indices applied for the trial.
the morphometric parameters, the VCA is reported only.
Disposition of Study Subjects
After having given informed consent, 40 women were assigned to two different groups according to a list reflecting the different time points of the intermediate biopsy, 19 subjects to group A (intermediate biopsy cycle 3) and 21 subjects to group B (intermediate biopsy cycle 6). Two a Score 0 –3; 0 ϭ none, 1 ϭ slight, 2 ϭ moderate, and 3 ϭ marked.
women initially assigned to group A were erroneously Lu¨dicke. Combined oral contraceptive and human endometrium. Fertil Steril 2001. switched to group B before the their first intermediate bi-opsy, so that they were evaluated in group B; thus, a total of40 subjects were included in the ITT analysis: 17 were was generated on morphometric parameters of the endome- evaluated in group A and 23 in group B. Intermenstrual trium so as to obtain quantitative information about the bleeding characteristics and adverse events during the course endometrial morphology. The indices examined are shown of the study were evaluated by the ITT analysis.
Seven women discontinued the trial prematurely, in six Ultrasound Examination of the Endometrium
women, the endometrial biopsy at baseline or in treatment At each study visit, a transvaginal sonographical exami- cycle 13 was not available or yielded not sufficient material, nation of the endometrium was done with the use of high and one woman was excluded from the analysis because she frequency (5–7 MHz) transducers. Endometrial thickness switched accidentally to a different COC; therefore, 26 cases was measured as the distance between both endometrial/ entered into the valid case analysis, 13 subjects in each group. Some of the biopsies during treatment resulted in toofew material for morphometric evaluation, thus, at cycle Statistical Analysis
three, 11 biopsies, and at cycle six, 10 biopsies, were evalu- The primary target variables were the individual changes from baseline (precycle) to the 3rd, 6th, and 13th treatmentcycle of the diameter of glands, the height of the glandular Intermenstrual Bleeding
epithelium, and the number of vacuolated glandular cells.
Bleeding that occurred outside the tablet-free interval was All other morphological indices variables were considered described as intermenstrual bleeding, presenting either as secondary target variables (Table 2).
scanty bleeding (spotting) or with the strength of a menstrualperiod (breakthrough bleeding). Thirteen women reported The two-sided 95% confidence intervals were computed spotting in the first treatment cycle. The incidence of spot- under the assumption of normal deviates for all variables ting declined continuously afterwards. Nineteen subjects re- except to the score variables pseudostratification and prede- ported intermenstrual bleeding at least once from the second to the 13th cycle. One woman reported an episode of break- Intention-to-Treat and Valid Case Analyses
through bleeding during the treatment period. No correlation Two types of analyses were done: a valid-case (VCA) between episodes of intermenstrual bleeding and the mor- analysis for the primary outcome measures and an intention- phometric assessment of the endometrium was detected.
to-treat (ITT) analysis on all variables in addition to the VCA.
Adverse Events
A subject was reckoned as a valid case if she had no Three women discontinued the trial because of the fol- protocol deviation that might have affected the primary lowing adverse events: acne, headache, and weight gain. The outcome measures of the trial, meaning the morphologic most frequently mentioned complaints during drug intake assessment of the endometrium. According to the study were headache (eight cases), breast tension (five cases), and protocol, the following criteria had to be fulfilled: at least one biopsy at baseline and at the end of the study had to beexistent, not more than four missed pills in each medication Morphometry
cycle were allowed, and the months without hormonal med- According to the collected tissue samples at baseline, all ication before the trial had to be adhered to. In contrary, the 26 women showed a histologically secretory endometrium, only prerequisite for a subject to be included into the ITT which contained glands in which the epithelial cells exhib- analysis was that she had taken at least one pill. No notable ited sub- or supranuclear vacuolization or showed apocrine- Lu¨dicke et al.
A combined oral contraceptive and human endometrium Indices of endometrial morphology before (pre-cycle) and during administration of 3 mg of DRSP and 30 ␮g of EE.
Note: Data given as mean Ϯ SD; (mean absolute change from pre-cycle Ϯ SD); [Confidence interval for mean change from pre-cycle].
Lu¨dicke. Combined oral contraceptive and human endometrium. Fertil Steril 2001. type secretion into the gland lumina. During treatment, an lated cells. At the baseline biopsy in one case, “slight pre- increasing atrophy of the endometrium was found, as char- decidualization” was scored. During treatment, two histolo- acterized by small glands with a single layer of inactive gies were reported with signs of slight predecidualization, epithelium, without mitotic figures or secretory activity, and two cases with moderate predecidualization, and one case no evidence of decidual changes. The endometrium appeared with marked predecidualization. Except in one case, no to be atrophic in 41% and 44% of women having used the pseudostratification was seen under treatment.
medication for 3 and 6 cycles, respectively. After 13 cycles Finally, the ultrasonographic measurements of the endo- of COC use, atrophy was described in 63% of the cases.
metrium correlated well with the results of the morphometric Table 3 summarizes the different morphometric indices measurements and indicated a suppression of the endome- and their change from baseline during the study period. A trium. The full sonographic reduction of endometrial thick- distinct suppression of proliferative activity of the functional ness was reached after six cycles (Table 4).
layer of the endometrium was reflected by the reduced sizeof the glandular diameter compared with baseline duringtreatment. This effect was already seen after three cycles of DISCUSSION
COC intake and was even more pronounced and statisticallysignificant at treatment cycle 6 and 13. Equally, a reduction Throughout the normal menstrual cycle, the morphologic of the number of glands and the gland’s epithelial height was variation of the endometrium has been documented, and found to be reduced when compared with baseline. A sig- some of the major features of these changes that are also nificant decrease in epithelial height was already observed at represented in the morphometric indices applied for this 3 months of medication and remained in the same range at study are schematically shown in Figure 1.
treatment cycle 6 and 13 (Table 3). The number of vacuo- The number of glands per square millimeter is approxi- lated cells per 1,000 glandular cells was suppressed to zero mately 20 and does not change significantly during the under treatment from a mean baseline value of 376 vacuo- normal menstrual cycle, whereas the glandular diameter Endometrial thickness (mm) as assessed by ultrasound—mean values and absolute changes.
Lu¨dicke. Combined oral contraceptive and human endometrium. Fertil Steril 2001. FERTILITY & STERILITY
Selected endometrial morphologic changes during the menstrual cycle.
Lu¨dicke. Combined oral contraceptive and human endometrium. Fertil Steril 2001. shows a linear increase from day LH-1 to day LHϩ11/ϩ12.
intake of combined contraceptive steroids in the endome- From day LHϩ6, glycoprotein-rich cytoplasmatic products trium. Its glandular component is a sensible indicator for are expelled into the glandular lumen, and the peak of measuring the effect of contraceptive steroids. The progestin intraglandular secretions coincides with the time of implan- component in the COC inhibits proliferation of the endome- tation of the free blastocyst (day 7 after ovulation) if fertil- trium, which is in line with our findings in which signs of ization takes place (10). The glandular epithelial cells are characterized by rapid growth and increasing epithelial pseudostratification, and epithelial height were found to be height from LH-11 through LHϩ2, and the first reliable clearly surpressed. When given at a dose of 30 ␮g of EE, in histologic alteration that is considered specific to ovulation is combination with 3 mg of DRSP, atrophic appearance of the basal vacuoles that start to appear in the glandular epithelium endometrium was found in 44% of the women at six treat- on day LH 0, and their number increases steadily up to day ment cycles and in 63% of the women at 13 treatment cycles.
LHϩ4 (11). The preovulatory endometrium is characterized These results are similar to what has been reported for other by proliferation of glandular cells, stromal cells, and vascu- COCs, such as the combination of 75 ␮g of gestodene with lar endothelial cells, and the maximum number of endome- 20 ␮g of EE in which the rate of atrophy was about 50% at trial cells engaged in DNA synthesis is seen between cycle 6 months of treatment (14). The reduction of the number of day 8 and 10 and corresponds to maximal mitotic activity, glands as seen in our study and others is a phenoma that peak plasma estradiol levels, and maximum concentration of cannot be observed in the menstrual cycle without COC estrogen receptors (10). The size of the glands does not keep medication and is apparently progestin-dose dependent. It pace with this glandular growth, and the tall columnar epi- has been shown that the release of 50 ␮g of norethisterone thelial cells finally pile up against each other with their per 24 hours via a vaginal ring did not reduce the number of nuclei at different levels, giving rise to pseudostratification glands, whereas the release of 200 ␮g/24 hours diminished (12). Predecidualization starts to take place around day significantly their number after 10 weeks of application (15).
LHϩ8 and is characterized by the conversion of stromal In view of the fact that the induced endometrial changes are cells into plump epithelial-like cells with enlarged nuclei and limited to the functionalis layer of the endometrium but do increased cytoplasm. The cells have metabolic functions not affect the basalis layer, this effect is known to be revers- related to pregnancy or, if conception has not occurred, to the ible after withdrawal of the contraceptive steroids (8).
menstrual breakdown of the endometrium by phagocytotic Although histology and morphometric measurements al- digestion of extracellular collagen matrix (13).
low for the assessment of the impact of contraceptive ste- Distinct histological changes can be observed during the roids on the endometrium when compared with the normal Lu¨dicke et al.
A combined oral contraceptive and human endometrium cycle, give insights in the progestogenic potency of certain Schellschmid, Schering AG, Clinical Development, Fertility Control and progestins, and provide common grounds for the comparison Hormone Therapy, Berlin, Germany. We also thank Christoph Gerlinger forthe statistical analysis, Schering AG, Biometry, Fertility Control and Hor- of endometrial effects of different COCs, they do not predict intermenstrual bleeding. Like earlier reports, the presentstudy did not reveal any correlation between the number ofdays of intermenstrual bleeding and the morphometric as- References
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2. Muhn P, Krattenmacher R, Beier S, Elger W, Schillinger E. Dro- Cycle control with both 30 ␮g of EE/3 mg of DRSP and 30 spirenone: a novel progestogen with antimineralocorticoid and antian- ␮g of EE/150 ␮g of desogestrel (Marvelon) was evaluated in drogenic activity. Pharmacological characterization in animal models.
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two randomized, open-label studies of 13 and 26 treatment 3. Oparil S, Ehrlich EN, Lindheimer MD. Effect of progesterone on renal cycles, respectively. Taken together, data were generated for sodium handling in man: relation to aldosterone excretion and plasmarenin activity. Clin Sci Mol Med 1975;49:139 – 47.
29,376 cycles in the DRSP group and 14,850 cycles in the 4. Kuhl H. Comparative pharmacology of newer progestogens. Drugs Marvelon group. Cycle control with both preparations was 5. Oelkers W, Berger V, Bolik A, Bahr V, Hazard B, Beier S, et al. Dihy- shown to be very good. A total of 8% in the DRSP group and drospirorenone, a new progestogen with antimineralocorticoid activity: 7% in the Marvelon group had had intermenstrual bleeding effects on ovulation, electrolyte excretion, and the renin-aldosterone sys-tem in normal women. J Clin Endocrinol Metab 1991;73:837– 42.
during the 6 months before the start of the study, a reported 6. Oelkers W, Foidart JM, Dombrovicz N, Welter A, Heithecker R.
incidence that was similar to the rate of occurrence during Effects of a new oral contraceptive containing an antimineralocorticoidprogestogen, drospirenone, on the renin-aldosterone system, body weight, blood pressure, glucose tolerance, and lipid metabolism. J ClinEndocrinol Metab 1995;80:1816 –21.
Depending upon dosage and type of progestin, prede- 7. Rosenbaum P, Schmidt W, Helmerhorst FM, Wuttke W, Rossmanith cidual changes of the stroma take place and strong prede- W, Freundl F, et al. Inhibition of ovulation by a novel progestogen(drospirenone) alone or in combination with ethinyl estradiol. Eur J cidual reaction was found in the stroma using high doses of Contracept Reprod Health Care 2000;5:16 –24.
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reaction was found (19), whereas 35 ␮g of EE in combina- 9. Klebe U, Moltz L, Pickartz H. Effects of cyproterone acetate and ethinyl estradiol on endometrial histology. Arch Gynecol 1983;234: tion with norgestimate (0.25 mg) yielded no decidual re- sponse, nor did the combination of 50 ␮g of EE and 2 mg of 10. Ferenczy A. Anatomy and histology of the uterine corpus. In: Kurman RJ, ed. Blaustein’s pathology of the female genital tract. Heidelberg, cyproterone acetate (CPA) (9). In this study, the overall rate of decidual reaction under treatment was found to be 19% 11. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J what is in the range that has been reported for the combina- 12. Johannisson E. Endometrial morphology during the normal cycle and tion of 20 ␮g of EE with 75 ␮g of gestodene. Taking under the influence of contraceptive steroids. In: d’Arcangues C, FraserIS, Newton JR, Odlind V, eds. Contraception and mechanisms of together the key information that might summarize the pro- endometrial bleeding. Cambridge, UK: Cambridge University Press, gestogenic potency of a progestin on the endometrium-like 13. Lawn AM, Wilson EW, Finn CA. The ultrastructure of human decidual antiproliferative activity, atrophic changes, and predecidual- and predecidual cells. J Reprod Fertil 1971;26:85–90.
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sion of endometrial activity, and the histological markers for 16. Foidart JM, Wuttke W, Bouw GM, Gerlinger C, Heithecker R. A comparative investigation of contraceptive reliability, cycle control and antiproliferative activity and induced atrophic changes com- tolerance of two monophasic oral contraceptives containing either dro- pare well to other modern low-dose combined contracep- spirenone or desogestrel. Eur J Contracept Reprod Health Care 2000;5:124 –34.
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18. Wienke EC Jr, Cavazos F, Hall DG, Lucas FV. Ultrastructural effects of norethynodral and mestranol on human endometrial stroma cell.
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19. Landgren BM, Johannisson E, Masironi B, Diczfalusy E. Pharmacoki- netic and pharmacodynamic effects of small doses of norethisterone Acknowledgments: The authors would like to acknowledge the contribution released from vaginal rings continuously during 90 days. Contraception of Christiane Klipping, Dinox, b.v., Nijmegen, The Netherlands and Ilka FERTILITY & STERILITY

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