Hum. Reprod. Advance Access originally published online on March 5, 2007
Human Reproduction 2007 22(6):1696-1704; doi:10.1093/humrep/dem026
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The effects of the selective progesterone receptor modulator asoprisnil on the morphology of uterine tissues after 3 months treatment in patients with symptomatic uterine leiomyomata
1 Department of Pathology, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh, Scotland, UK 2 Centre for Reproductive Biology, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, Scotland, UK 3 DOHaD (Developmental Origins of Health and Disease) Division, University of Southampton, Princess Anne Hospital, Southampton, UK 4 TAP Pharmaceutical Products Inc., Lake Forest, Illinois, IL, USA
5 To whom correspondence should be addressed at: Department of Pathology, University of Edinburgh, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh EH16 4SA, Scotland, UK. Tel.: +44 131 242 7120; E-mail: a.williams{at}ed.ac.uk
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Abstract |
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BACKGROUND: Asoprisnil is a selective progesterone receptor modulator with mixed progesterone agonist/antagonist activity which controls uterine bleeding via an endometrial effect. This study examined full-thickness endometrial, leiomyoma and myometrial morphology in hysterectomy specimens from patients with uterine leiomyomata, after treatment with asoprisnil for 3 months.
METHODS: In this double-blind, randomized, placebo-controlled study, 33 subjects with uterine leiomyomata were randomized to receive asoprisnil 10, 25 mg or placebo for an average of 95 days prior to hysterectomy. Samples of endometrium, myometrium and leiomyoma tissue were subjected to systematic morphological assessment with quantification of mitotic activity.
RESULTS: In patients treated with 10 or 25 mg asoprisnil, a unique pattern called ‘non-physiologic secretory effect’ was evident in endometrium, recognizable through partially developed secretory glandular appearances and stromal changes. Endometrial thickness was decreased, and there were low levels of mitotic activity in endometrial glands and stroma. Unusual thick-walled muscular arterioles and prominent aggregations of thin-walled vessels were present in endometrial stroma, but not in myometrium or non-endometrial vascular beds. Mitotic activity was decreased in leiomyomata.
CONCLUSIONS: Asoprisnil induces unique morphological changes and is associated with low levels of glandular and stromal proliferation in endometrium, and in leiomyomata. These changes are likely to contribute to the amenorrhoea experienced after exposure to the medication.
Key words: asoprisnil/cell-proliferation/endometrium/histopathology/progesterone receptor
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Introduction |
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Asoprisnil is a member of a developing class of selective progesterone receptor modulators (SPRMs) being studied in women with symptomatic uterine leiomyomata. SPRMs are progesterone receptor (PR) ligands that exert clinically relevant tissue-selective progesterone agonist, antagonist or partial or mixed agonist/antagonist effects on various progesterone target tissues in an in vivo situation depending on the biological action studied (Chwalisz et al., 2005b
). Asoprisnil, a steroidal 11
-benzaldoxime-substituted SPRM, exhibits mixed progesterone agonist/antagonist activities in animals (DeManno et al., 2003; Schubert et al., 2005), and high degree of uterine selectivity in women (Chwalisz et al., 2005a). In contrast to pure progesterone antagonists that inhibit ovulation and do not show tissue-selective effects, asoprisnil suppresses uterine bleeding in the presence of follicular phase estrogen concentrations irrespective of the effects on ovulation. It controls uterine bleeding in a dose-dependent manner by an unknown mechanism and appears to target endometrium specifically (Chwalisz et al., 2005a). In addition, asoprisnil exerts endometrial antiproliferative effects in non-human primates (Chwalisz et al., 2005b) and reduces leiomyoma volume in humans (Chwalisz et al., 2003). These effects provide the rationale for its use in the treatment of uterine leiomyomata, in which abnormal uterine bleeding is a common symptom.
Uterine leiomyomata (fibroids or myomas) are common benign smooth muscle neoplasms originating in myometrium. They occur in at least 50% of women of reproductive age, and although many are asymptomatic, they commonly lead to troublesome symptoms of heavy menstrual bleeding, as well as pressure symptoms and infertility. Symptoms attributable to leiomyomata are the primary indication for 200 000–300 000 hysterectomies performed each year in the USA among premenopausal women (Schwartz, 2001). At present, there is no entirely satisfactory medical treatment for management of women with symptomatic leiomyomata.
The present randomized double-blind placebo-controlled study was therefore designed to allow detailed histological assessment of full-thickness endometrium, myometrium and leiomyomata in samples from hysterectomy specimens of patients with symptomatic leiomyomata who had been administered 10 or 25 mg asoprisnil daily for 12 weeks prior to surgery. These doses were selected as previous studies (Chwalisz et al., 2005a, b) have shown dose-dependent reversible suppression of menstruation at doses 
10 mg, and dose-dependent reduction of leiomyomata and uterine volume.
The focus of this study was on the endometrial effects. The endometrium seems to be the most sensitive target for SPRMs, since the effects of asoprisnil on uterine bleeding and endometrial morphology were evident at doses that did not markedly inhibit luteinization indicative of ovulation and ovarian estrogen secretion. In addition, compounds with partial progesterone antagonist effects have the potential to induce ‘unopposed’ estrogen effects on the endometrium and create, therefore, concerns about endometrial safety. Finally, early studies with asoprisnil revealed that this compound induces unique changes in the endometrium, characterized as ‘non-physiologic secretory effects’ that have not been described with any known pharmacological agent before (Chwalisz et al., 2005a). It was clear in this and other unpublished studies that hitherto undescribed changes were present in the endometria of asoprisnil-treated patients, and conventional assessment using the criteria of Noyes et al. (1950) was not appropriate. The evaluation of full thickness, highly oriented endometrial specimens obtained during hysterectomy provided a unique opportunity to study the effects of asoprisnil in all layers of the endometrium, including the basalis, which is not accessible by Pipelle endometrial biopsies.
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Women studied
The study group was composed of premenopausal women volunteers from four centres (Edinburgh, Southampton, Glasgow and Liverpool) in good general health with a menstrual cycle between 17 and 42 days, and symptoms related to overall fibroid size, pressure and/or heavy uterine bleeding (per protocol), who were scheduled for hysterectomy. Each patient had at least one intramural non-pedunculated, submucosal or subserosal fibroid with a diameter of at least 2 cm or multiple small fibroids with uterine volume
200 cm3 on ultrasonography. Other inclusion criteria included age over 18 years; negative pregnancy test; a washout period of 2–12 months for hormonal therapies; serum FSH of <30 mIU ml–1 at commencement; agreement to use double barrier method of contraception (condom/diaphragm/sponge plus spermicide) throughout the study until hysterectomy, unless surgically sterile by bilateral tubal ligation or vasectomy of partner and normal Papanicolaou (PAP) test. Subjects were not permitted to enter the study without a normal endometrial biopsy report based on an adequate specimen taken within 3 months of commencement. Screening data were collected in the clinic by study nurses or gynaecologists. All subjects voluntarily signed a full informed consent form. The study was performed according to the ethical principles of the Declaration of Helsinki (1989 revision) and the protocol was approved by the Institutional Review Board (Multicentre Research Ethics Committee).
Study design
This was a Phase 2 multicentre randomized double-blind placebo-controlled study of asoprisnil administered for 12 weeks. Dose selection was based upon data from Phase I and Phase II studies (publications in preparation) in which asoprisnil at doses of 5, 10, and 25 mg has been shown to be safe in subjects treated for 12 weeks. Asoprisnil 10 and 25 mg doses and the 12-week duration of treatment were chosen because they were effective for treatment of excessive bleeding, in reducing leiomyoma and uterine volumes and in decreasing mass effect symptoms, such as pelvic pressure and bloating, over a 3-month treatment period.
At baseline, screening procedures (performed within 45 days of study commencement) included pelvic and breast examination, complete physical examination, PAP test (ThinPrep® Pap Test, Cytyc Corp), ECG, transvaginal ultrasound, endometrial biopsy (Unimar Pipelle® Endometrial Suction Curette, Medscand), contraception counselling and clinical laboratory investigations: chemistry, haematology, urinalysis, endocrine panel, lipid profile, coagulation screen and serum and urine pregnancy tests. After successful enrollment based on inclusion and exclusion criteria, women were sequentially assigned subject numbers in ascending numerical order that encoded the assignment of the woman, via a randomization schedule, to one of the three treatment arms of the study. Subjects were randomized to one of three parallel dose groups in a 1:1:1 ratio to receive daily doses of asoprisnil 10, 25 mg or placebo. Subjects and all study personnel were blinded to treatment groups. Asoprisnil or placebo tablets were supplied in blister cards of identical appearance, supplied to the site packaged in sealed kits. Drug was self-administered as an oral dose taken once daily. Treatment was initiated no later than the fifth day of the subject's menstrual cycle, and continued for 12 weeks, when subjects were to undergo hysterectomy within 24 h of the final dose of drug. The subjects returned all used and unused study medication blister cards at 8-week and final visits so that verification of medication compliance could be monitored. The effects of asoprisnil on clinical and safety parameters will be reported separately.
Hysterectomy and processing of uterine samples
After removal of the uterus, the unfixed specimen was placed on ice immediately and taken without delay to the local pathology laboratory, where the study pathologist or deputy was on hand to open and sample the specimen. The specimen was oriented and a probe inserted through the external os of the cervix to define the position of the cavity. The uterus was opened using a long-bladed knife along the plane of the probe. The opened specimen was then placed in an adequate volume of 10% buffered formaldehyde and allowed to fix overnight. For large specimens, parallel parasagittal slices 2 cm in thickness were made to permit adequate fixation overnight. The following day, the pathologist sampled the specimen for routine diagnostic reporting, and took additional blocks for study assessment. The additional blocks consisted of three blocks from the uterine fundus, mid-corpus and isthmus, respectively, ideally to include full thickness from endometrium, myometrium and serosal surface, but in any case including full thickness of endometrium with underlying myometrium. Endometrial samples were taken from areas away from leiomyomata wherever possible. Leiomyomata were separately sampled. All specimens were processed by routine methods to paraffin wax, and 3 µm haematoxylin–eosin sections were prepared by microtomy and mounted on glass slides. Histological sections of endometrium were considered unsuitable for assessment if a leiomyoma was present within 10 mm of the endometrium.
Histological assessment of endometrium
During Phase I studies with asoprisnil, it became clear that SPRMs including asoprisnil induce unique changes in the endometrium that cannot be assessed by currently used criteria of endometrial dating (i.e. the Noyes criteria) (Noyes et al., 1950), because of differential effects on glands and stroma. As a result, TAP Pharmaceutical Products Inc., Lake Forest, IL, Diagnostic Cytology Laboratories, Indianapolis, IN, USA, and a group of expert gynaecological pathologists developed a list of diagnostic criteria that allows for classification of changes induced by SPRMs (‘Dictionary of Endometrial Biopsy Diagnoses for Clinical Trials with SPRMs’—data on file). This system supplements the conventional descriptive criteria of the normal menstrual cycle as described by Noyes, and in Blaustein's Pathology of the Female Genital Tract (Mutter and Ferenczy, 2002), but includes additional categories that are considered characteristic of the effects of SPRMs.
Haematoxylin–eosin stained sections of full-thickness endometrium and underlying myometrium from fundus, mid-corpus and isthmus and samples of fibroids were evaluated by a gynaecological pathologist blinded to treatment groups. The maximum single endometrial thickness on each section was measured using an eyepiece micrometer. Overall endometrial appearances were classified according to the classification system described earlier. Individual histological characteristics of endometrial glands, stroma and vessels were analysed by assigning a descriptive subcategory, as shown in Tables 1 and 2. Myometrium was assessed for any histological changes or abnormalities, and for the presence or absence of adenomyosis. Leiomyomata were assessed for histological type (usual, cellular, epithelioid, symplastic, myxoid, leiomyoma with heterologous elements, other) and for degenerative effects (no degenerative changes, hyaline degeneration, cystic degeneration, myxoid or mucoid degeneration, coagulative necrosis, calcification, fatty degeneration).
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Non-endometrial tissues, when submitted with the hysterectomy specimens, were also examined histologically. These comprised samples of ovaries, Fallopian tubes and cervix. As these were the only non-uterine tissue samples available, it was of particular interest to assess the vascular beds to determine whether the changes in endometrial vessels were specific to that site, or could be seen in other tissues.
Assessment of mitotic activity
Glandular and stromal mitoses per unit area of endometrium were quantified separately by counting the number of unequivocal mitotic figures present per 10 high-power microscope fields of endometrium (Olympus BX51,x40 objective, field diameter 0.55 mm). Counting was commenced in the area identified by prior microscopic assessment to show the greatest density of mitoses, and a minimum of 10 non-overlapping fields randomly selected for counting thereafter. The same method was used to assess mitotic activity in leiomyomata, but uninvolved myometrial tissue was not assessed in this way as it shows no mitotic activity.
Data analysis and statistical methods
For the overall diagnostic category of endometrium as well as categorical assessments of morphological features in glands and stroma, number and percentage of patients in each category were summarized for each treatment group and each of the three locations (uterine fundus, mid-corpus and isthmus), with no statistical inference performed. For endometrial thickness measured by eyepiece micrometer, mean and standard deviation were calculated for each treatment group and each of the three locations. Although not prespecified, the thickness was also compared, for each location separately, between the placebo group and the combined asoprisnil group, using t-tests. The multiplicity resulting from the three locations was adjusted for using Hochberg's multiple comparison procedure.
No sample size calculation was performed for this mechanism of action study. Although a power analysis was carried out for resistance index (primary efficacy endpoint), and a uterine blood flow parameter assessed with colour Doppler imaging in this study, none was performed for morphological characteristics as no statistical inference was to be performed on these characteristics.
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Demographic information
There were 10, 12 and 11 subjects, respectively, who received placebo, asoprisnil 10 and 25 mg. Twenty-nine subjects were Caucasian and four were black. Placebo and asoprisnil groups (10 and 25 mg) were well matched with respect to age (mean 41.8, 45.1 and 44.6 years, respectively), weight (mean 73.4, 73.8 and 75.9 kg) and height (165.3, 164.3 and 165.6 cm). The treatment period for this study was between July 2003 and March 2005. Drug compliance was satisfactory in all groups. On average, the subjects took the study drug for 95 days. All hysterectomies were performed within 2 days of the last dose of study drug.
Histological evaluation of endometrium
Baseline endometrial biopsies taken prior to commencement of medication all showed normal histological appearances, classified as inactive (10%), proliferative (28%), menstrual (7%) or secretory—cycling/physiologic (55%).
In 29 subjects, endometrial samples from fundus, mid-corpus and isthmus were taken, and were available for histological assessment. Table 3 shows the overall diagnostic category assigned to endometrium from three locations (fundus, mid-corpus and isthmus) in specimens from subjects taking placebo, 10 or 25 mg asoprisnil. In fundus, mid-corpus and isthmus (this order will be used throughout the text), respectively, normal secretory appearances (cycling/physiologic) were seen in five of seven, five of seven and five of seven samples from patients taking placebo, whereas specimens from patients taking 10 or 25 mg asoprisnil showed normal secretory appearances in 1 of 10, 2 of 10 and 0 of 10 and in 1 of 11, 1 of 11 and 0 of 10, respectively. In contrast, a non-physiologic secretory effect was found in 7 of 10, 5 of 10 and 5 of 10 samples from patients taking 10 mg asoprisnil, and 8 of 11, 8 of 11 and 5 of 10 samples from patients taking 25 mg. The diagnosis of non-physiologic secretory effect was assigned in one of seven, zero of seven and one of seven samples from patients receiving placebo.
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Overall description of endometrial morphology associated with asoprisnil
Endometrium of patients treated with asoprisnil showed a combination of morphological features affecting glands, stroma and vessels. Although none of these features was on its own specific for asoprisnil, the occurrence of several features together allowed designation of the endometrium as showing the ‘non-physiologic secretory effect’. In this pattern, the endometrial glands showed some features associated with secretory differentiation, often with serpentine or tortuous profiles, composed of columnar epithelial cells with infrequent or absent mitotic activity (see Fig. 1A as an example). However, glands rarely showed any evidence of active secretion, as cytoplasmic vacuolation was absent in most cases. There was a tendency for cystic dilatation of glands, but no abnormal folding patterns or increase in gland-stroma ratio above normal (Fig. 1B). The stroma showed a tendency for increased cellularity without evidence of decidual change. One of the most consistent changes with asoprisnil was the effect on vessels, of which there were two characteristic appearances. First, thick-walled muscularized vessels, similar to those seen in the stroma of the common benign endometrial polyp, were commonly found in the stroma of asoprisnil-treated patients (Fig. 1C), but were not identified in any subjects receiving placebo. Secondly, aggregates of thin-walled vessels were seen at all levels of the endometrium, from within stratum basalis to just beneath surface epithelium (Fig. 1D). These were not specific to asoprisnil, as they were occasionally observed in placebo-treated patients, but they were much more common among asoprisnil-treated patients. In asoprisnil-treated patients, endometrium was commonly thin (Fig. 1D) and frequently it was not possible to distinguish stratum basalis from the stratum functionalis. The morphological features associated with asoprisnil were similar in the thin endometrium of both basalis and functionalis.
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Individual features of endometrial glands
With asoprisnil, cytoplasmic secretory vacuolation was relatively infrequent (up to 30%) in patients taking 10 mg, and absent from glands in all locations in all patients taking 25 mg. With asoprisnil, gland architecture varied from simple tubules with minimal undulation to a tortuous appearance resembling that of the mid- or late secretory phase. Simple tubular gland architecture with minimal undulation was more commonly seen in endometrium from the isthmus in patients taking 25 mg (40%) than in fundus or mid-corpus (9%). Asoprisnil 25 mg led to some degree of cystic gland dilatation in all uterine locations but especially in the isthmus (80% compared to 29% with placebo). No differences in the frequency of other glandular architectural abnormalities, such as the abnormal folding patterns seen in the disordered proliferative pattern (Mutter and Ferenczy, 2002
) were identified between asoprisnil-treated patients and controls. The predominant epithelial cell type seen in glands in all locations was comparable between asoprisnil and placebo, consisting of tall columnar cells showing a degree of nuclear stratification that did not vary significantly between treatment groups. Epithelial cell nuclei showed no cytological atypia in any patient. Although a systematic assessment was not performed, histologically there was no apparent difference in numbers of apoptotic glandular cells between asoprisnil-treated patients and controls.
Mitotic activity in endometrial glands and stroma
Mitotic activity for endometrial glands is shown in Table 4 and for stroma in Table 5. As five out of seven, five out of seven and five out of seven samples from placebo-treated patients showed normal secretory endometrial appearances in which mitoses are very infrequent or absent, and only one out of seven, one out of seven and one out of seven specimens showed normal proliferative appearances (Table 3), mitotic activity cannot properly be compared between asoprisnil-treated patients and the placebo group. However, it is notable that in the group of patients treated with 25 mg asoprisnil, no mitotic activity in endometrial glands was identified in any specimen except for one sample of fundic endometrium in one patient. At least 70% of specimens from patients taking 10 mg asoprisnil showed one mitosis or less per 10 high-power fields (HPF) in endometrial glands. In endometrial stroma, patients who received 25 mg asoprisnil showed absence of mitotic activity in 8 out of 11, 9 out of 11 and 9 out of 9 assessable slides, and with 10 mg, 1 mitosis or less per 10 HPF was seen in 7 out of 10, 10 out of 10 and 8 out of 10 assessable slides.
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Stromal changes
Stromal decidual change was seen around vessels in a specimen from fundus only in one patient taking 25 mg asoprisnil, but in the remaining assessable samples from patients taking either 10 or 25 mg asoprisnil there was no decidual change identified. In contrast, decidual change was present in endometrial stroma in three of seven, three of seven and three of seven samples from patients taking placebo. With asoprisnil, there was an increased frequency of stromal compactness in all uterine locations, compared with placebo. Stroma was assessed as compact in zero of seven, zero of seven and one of seven placebo samples, compared to 6 of 10, 3 of 10 and 3 of 10 samples from patients taking 10 mg asoprisnil and 6 of 11, 5 of 11 and 7 of 10 on 25 mg.
Endometrial thickness
Table 6 shows the mean single layer thickness of the endometrium, as measured by eyepiece micrometer on histological sections, for specimens from patients taking placebo, 10 or 25 mg asoprisnil. There was no statistically significant difference in endometrial thickness between placebo and combined asoprisnil groups, although there was a trend for decreased thickness with asoprisnil treatment.
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Vessels
Aggregates or leashes of thin-walled vessels were seen with increased frequency in endometrial stroma of asoprisnil-treated patients compared with controls (Table 7). Samples from patients on placebo showed the presence of at least one aggregation of thin-walled vessels in 2 of 7, 1 of 7 and 0 of 7, whereas these were present in 3 of 10, 5 of 10 and 2 of 10 samples from patients on 10 mg asoprisnil and in 5 of 11, 5 of 11 and 4 of 10 on 25 mg.
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Thick-walled vessels, similar to those seen in the stroma of endometrial polyps, were seen with increased frequency compared with controls in all uterine locations in patients treated with asoprisnil. Such vessels were not seen in any samples from placebo subjects, whereas they were found in 2 of 10, 3 of 10 and 1 of 10 samples from patients on 10 mg asoprisnil and in 5 of 11, 5 of 11 and 3 of 10 on 25 mg.
Although there are some similarities, the changes associated with asoprisnil differ from those associated with endometrial polyps. In the latter, thick-walled vessels occur in the stroma of a rounded, projecting polypoid mass in which gland crowding may be observed, and the stroma frequently has a collagenous appearance. After 12 weeks treatment with asoprisnil, gland crowding is not seen, the stroma does not appear prominently collagenized, and the endometrium remains flat and often thinned.
Non-endometrial vascular beds
There were 12 ovaries from eight patients, 15 Fallopian tubes from 10 patients and 10 cervices available for assessment of vascular beds in non-endometrial tissues. No specific histological abnormalities were observed.
Myometrium
Myometrium samples were obtained from all 33 patients and morphological assessments were performed. Adenomyosis was infrequent (present in one control subject, one taking 10 mg and four taking 25 mg asoprisnil). No further abnormalities or changes were identified in these samples.
Leiomyomata
Histological evaluation of the largest leiomyoma of each hysterectomy specimen showed 29 of 30 to be of the usual histological type, and one to be of cellular type. Degenerative changes (hyaline or myxoid/mucoid changes, or coagulative necrosis) were seen in 4 of 9 leiomyomata from the placebo group, 8 of 12 in the 10 mg and 7 of 9 in the 25 mg asoprisnil group. In the placebo group, mitotic activity was not identifiable in one of eight leiomyomata, and in the range 1–2 mitoses per 10 HPF in the remaining seven of eight assessable leiomyomata (Table 8). In the 10 mg asoprisnil group, 8 of 12 showed no identifiable mitoses and 4 of 12 showed 1–2 mitoses per 10 HPF. In the 25 mg asoprisnil group, six of nine showed no mitoses, two cases showed 1–2 mitoses per 10 HPF and one case showed 3–5 mitoses per 10 HPF.
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Discussion |
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Asoprisnil has a variety of clinical effects that have so far not been described with any other pharmaceutical agent. It controls uterine bleeding in a dose-dependent manner without causing suppression of estradiol from follicular phase levels and irrespective of the effects on luteinization (Chwalisz et al., 2005a
). In patients with leiomyomata, it suppresses heavy uterine bleeding, reduces leiomyoma and uterine volumes and has beneficial effects on pressure-related symptoms (Chwalisz et al., 2003). These distinctive clinical effects are associated with equally distinctive morphological effects on endometrium, and the existing compendium of pathological diagnoses does not include appropriate categories to describe the effects of asoprisnil on endometrium. Early clinical studies (Chwalisz et al., 2005a) have examined the asoprisnil effect in endometrial biopsies taken by Pipelle aspiration after treatment for 28 days. The present study, however, has allowed for the first time, a detailed morphological investigation of samples of full thickness endometrium and underlying myometrium, mostly well oriented and undisrupted by the sampling procedure after a 3-month asoprisnil treatment. Most of the subjects recruited were suffering symptoms of heavy uterine bleeding associated with fibroids, and the endometrium cannot, therefore, be considered functionally normal. Although efforts were made to sample endometrium at least 10 mm away from the nearest fibroid, it is known that vascular changes occur in the endometrium of these patients, and results must be interpreted with caution. However, the baseline endometrial biopsies taken at screening were within normal limits histologically.
The majority of asoprisnil-treated subjects showed endometrial morphology that was consistently recognizable as showing a ‘non-physiologic secretory effect’, a category that was assigned in only one placebo-treated subject. It was present to a similar extent in all three uterine locations sampled (fundus, mid-corpus and isthmus), and was seen in a higher proportion of subjects on 25 mg of asoprisnil. This appearance is unfamiliar to histopathologists, as it is not seen in normal cycling endometrium or in any described condition, and is believed to reflect the partial agonist/antagonist effect of asoprisnil described in animal studies (DeManno et al., 2003). The histological features defining the effect are endometrial glands showing tortuosity similar to the architecture of the secretory phase, but with a rarity of secretory activity or cytoplasmic vacuolation. Glands frequently showed cystic dilatation, however, and although nuclear stratification was present, there was a paucity of mitotic activity. Appearances of cystic glandular dilatation are frequently associated in the minds of histopathologists with simple hyperplasia of endometrium, but the non-physiologic secretory effect differed in that glandular crowding was absent, cystic change was often focal involving one or two glands in a field of non-dilated glands and mitotic activity was very sparse or absent. Gland dilatation is a feature commonly seen in the inactive, non-secretory endometrium of the perimenopausal and post-menopausal woman, and is not considered indicative of glandular secretory activity. It may in fact represent glands in which the opening to the surface has become occluded, perhaps, as a consequence of the lack of secretory activity.
In this 3-month study, endometrial thickness was not increased in the asoprisnil group compared with placebo, and indeed the trend was for mean endometrial thickness to be less than that of placebo. With asoprisnil, the endometrial stroma showed increasing compactness without decidual change, but the most characteristic stromal effect involved vessels. Aggregations of thin-walled vessels were seen in endometrial stroma more frequently in asoprisnil-treated patients than in placebo-treated patients. Additionally, vessels with thickened muscularized walls were seen with greater frequency in asoprisnil patients than in controls.
The mechanism of amenorrhoea induced by asoprisnil is not known, but it is probable that the drug exerts a specific effect on endometrial vessels through action on the PR. This may involve perivascular cells that express PR, and are believed to be pivotal in control of menstruation (Kelly et al., 2002), or other stroma-mediated events. The perivascular cells are intimately apposed to the endothelial cells of spiral arterioles, and are believed to respond to falling progesterone concentrations by initiating vasoconstriction–vasodilatation cycles with resulting hypoxia leading to endometrial sloughing. Although no specific histological changes were seen in perivascular cells in this study, the results of detailed immunohistochemical studies are not yet available.
The thick-walled aggregates of vessels resemble those seen in the stroma of endometrial polyps, and it is likely that in asoprisnil-treated subjects these evolve from the thinner-walled aggregates as time goes on. The process appears specific to endometrium, as no similar changes were identified in the limited range of non-endometrial tissues examined in this study. In the endometrium, it is possible that there is an active process of angiogenesis stimulated by hypoxia through an action on the perivascular cells. However, the development of vascular aggregates may be analogous to the stromal vessels of endometrial polyps, where it is believed that focal areas of non-shedding endometrium persist for several cycles to form the polyp. In both situations, therefore, endometrium is not shed at menstruation, and this may be the mechanism whereby such morphologically abnormal vessels develop. However, although some similarities with stromal changes of endometrial polyps were observed, with asoprisnil, the endometrium remained flat and often thin, without gland crowding and without prominent stromal collagen. It is notable that there was a low incidence of polyp formation in the asoprisnil-treated patients in this study, with only one small polyp found in one subject taking 10 mg.
No histological changes were found in this 3-month study to give cause for concern about the development of malignant or premalignant changes. Glands were specifically assessed in each sample to identify features of cytological atypia, but none was found. The glandular architectural changes did not suggest complex hyperplasia, and although there was in some cases a superficial resemblance to simple cystic hyperplasia, this was merely due to the presence of dilated glands, whereas the other features of simple hyperplasia were lacking. Moreover, endometrial thickness was not increased; indeed, the trend (which was not statistically significant) was for a decreasing endometrial thickness in asoprisnil-treated patients. It is important that histopathologists are made aware of the potential diagnostic pitfalls of misdiagnosing endometrial polyps and simple hyperplasia, particularly in curettage or aspiration biopsies of endometrium from asoprisnil-treated patients.
There are some morphological similarities between the endometrial effects of asoprisnil in this study, and those of the PR antagonist mifepristone, but there are also significant differences. Cystic glandular dilatation with inactive epithelial appearances is common to both, and there is an antiproliferative effect (Baird et al., 2003; Narvekar et al., 2004). However, no specific vascular changes have been identified in the stroma in mifepristone-treated endometrium, and glands show a less tortuous morphology, generally showing simple tubular appearances or cystic dilatation. This suggests an absence of agonist activity and a pure antagonist effect with mifepristone on PR, whereas with asoprisnil, the gland tortuosity probably reflects a partial agonist effect in addition to antagonist action.
In this study, comparison of mitotic activity in endometrium between treatment and control groups is limited, as five out of seven subjects in the control group showed secretory phase appearances in which mitoses are not expected to be present. However, it is notable that the low level of mitotic activity in the asoprisnil treatment groups is in keeping with the results of studies of asoprisnil in primates (Brenner and Slayden, 2005), and may have parallels in studies of other PR ligands such as mifepristone, in which an antiproliferative effect has also been described (Baird et al., 2003; Narvekar et al., 2004).
Mitotic activity was also decreased in leiomyomata in patients receiving asoprisnil, and in keeping with this, degenerative changes were also more common. This is consistent with the observed diminution in leiomyoma volume in patients receiving asoprisnil (data not shown). However, no histological differences were found in the non-leiomyomatous myometrium between placebo and asoprisnil groups. Asoprisnil does not seem to induce any noticeable changes in the myometrium.
In conclusion, this study has shown that asoprisnil administered as 10 or 25 mg daily doses for 3 months leads to the development of unique histological appearances in the endometrium. As well as consistent effects on gland morphology, thick-walled muscular vessels are seen in the stroma which appear to be specific to endometrial tissue. The mechanism of uterine bleeding suppression is still unknown, but it may involve interaction between asoprisnil, perivascular cells and spiral vessels. This study reports, for the first time, a detailed evaluation of endometrial changes induced with asoprisnil after a 3-month treatment, based on the full-thickness endometrial samples obtained during hysterectomy. Histopathologists examining endometrial biopsy specimens from asoprisnil-treated patients need to be aware of the unique ‘SPRM effects’ induced by asoprisnil (and perhaps other compounds of this class) to avoid misclassifying appearances as simple hyperplasia or polyps, which share some of the features but have significant differences.
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Conflict of interest |
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This work was supported by TAP Pharmaceutical Products Inc., Lake Forest, IL 60045, USA. A.R.W. Williams acts as a Consultant for TAP Pharmaceutical Products Inc. and Schering AG. H.O.D. Critchley has received support for staff costs and consumables for clinical and laboratory based studies on the role of selective progesterone receptor modulators in the management of leiomyomata and heavy menstrual bleeding from TAP Pharmaceutical Products Inc. and Jenapharm GmbH, Jena, Germany (a subsidiary of Schering AG, Berlin, Germany), respectively.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConflict of interestAcknowledgementsReferences |
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The authors are grateful to clinical investigators Professor M.A. Lumsden and Dr D. Hapangama. We thank Dr H. Millward-Saddler, Dr M.M. Khine and Dr D. Millan for providing uterine samples. We also thank Dr Alex Lawrence, Mrs Liz O'Neill, Mrs Joan Kerr and the gynaecology theatre staff in Edinburgh, Southampton, Glasgow and Liverpool for assistance with ultrasound scanning, patient recruitment and specimen collection.
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This work was presented in part at the 2006 Annual Scientific Meeting of the Society for Gynaecologic Investigation, March 2006, Toronto, Canada.
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Submitted on July 3, 2006; resubmitted on November 14, 2006; accepted on December 27, 2006.
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