Investigation of the 12-month efficacy and safety of low-dose mifepristone in the treatment of painful adenomyosis : Reproductive and Developmental Medicine

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Special Issue: Adenomyosis and Endometriosis

Investigation of the 12-month efficacy and safety of low-dose mifepristone in the treatment of painful adenomyosis

Chen, Shu-Yi; Zhao, Meng-Dan; Sun, Wen-Ting; Zhu, Li-Bo; Zhang, Xin-Mei*

Author Information
Reproductive and Developmental Medicine: September 2022 - Volume 6 - Issue 3 - p 152-161
doi: 10.1097/RD9.0000000000000031
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Abstract

Introduction

Adenomyosis is a benign chronic inflammatory gynecological disorder characterized by the abnormal location of the endometrial glands and stroma within the uterine myometrium, resulting in dysmenorrhea, hypermenorrhea, and infertility[1]. The incidence of adenomyosis is increasing; however, the etiology and pathogenesis of this disease remain unclear. Currently, traditional radical hysterectomy is generally unaccepted by women with adenomyosis, and conservative surgical resection of adenomyotic lesions often results in residual lesions that increase postoperative recurrence rates and the risk of uterine rupture during pregnancy[2–5]. Moreover, women with adenomyosis still need to be continually treated with drugs after conservative surgery[3]. Although uterine artery embolization (UAE) and high-intensity focused ultrasound (HIFU) can also be used to treat adenomyosis, the efficacy and safety of these treatments are still being debated[6–9]. Therefore, medication is the mainstream treatment in the lifelong management plan for adenomyosis[10].

The current effective drugs for adenomyosis are gonadotropin-releasing hormone agonists (GnRHas), levonorgestrel-releasing intrauterine devices (LNG-IUDs; e.g., Mirena), oral contraceptives (OCs), and dienogest (DNG)[3,11–14]. GnRHas can significantly reduce uterine volume and relieve pain symptoms, but they are expensive and can only be used for a short duration due to side effects causing low estrogen levels[12,13]. LNG-IUDs can relieve pain symptoms and be used for a long time; however, their side effects of prolonged irregular vaginal bleeding and unexpected expulsion of the device often make them unbearable to women[11]. Similar to LNG-IUDs, DNG can also be used for a long duration, but it is expensive and can cause irregular vaginal bleeding[13,14]. Additionally, the efficacy and long-term use of DNG still need to be further investigated. Although OCs can be used to treat adenomyosis, most Chinese women are reluctant to accept them. Therefore, in view of the current status of the available drugs used for treating adenomyosis, it is necessary to find better drugs that are inexpensive, have fewer side effects, and are suitable for long-term use.

Mifepristone (RU486) is a progesterone receptor modulator, a synthetic steroid compound, which mainly works by blocking progesterone[15]. Mifepristone plays an anti-tumor role, primarily by inhibiting the proliferation and invasion of cancer cells and promoting cancer cell apoptosis[16]. A series of short-term clinical investigations using different doses has revealed that mifepristone is effective in treating uterine leiomyoma and endometriosis, both in terms of reducing the size of leiomyoma and endometriotic lesions and improving the clinical symptoms[17–21]. In vivo and in vitro studies have shown that mifepristone can inhibit endometrial cell proliferation, reduce angiogenesis and oxidative stress, and promote cell apoptosis[22–25]. In experimentally induced animal studies, mifepristone inhibits the growth of adenomyotic lesions and prevents the occurrence of adenomyosis[26]. Short-term clinical use of mifepristone has demonstrated that it is well tolerated and its side effects are usually mild[27]. In addition, the cost of mifepristone for long-term treatment is very low. Adenomyosis is a disease that requires long-term management; hence, an evaluation of the efficacy and safety of low-dose mifepristone in the long-term treatment of adenomyosis is imperative.

Materials and methods

Patients

The Ethics Committee of the Women’s Hospital, Zhejiang University School of Medicine approved this study and did not require written informed consent from the subjects (No. IRB-20210192-R and 20180135). The retrospective cohort study was conducted between December 2015 and December 2019. Another group of women with adenomyosis were included in the immunohistochemical study as a control group; they had never received conservative treatment, except for occasional painkillers, and underwent either hysterectomy or adenomyomectomy and diagnostic curettage due to severe dysmenorrhea during the same research period.

Inclusion and exclusion criteria

The inclusion criteria for patients with adenomyosis were as follows: (1) presence of severe dysmenorrhea, with or without menorrhagia (hypermenorrhea); (2) age 20 to 50 years, with a regular menstrual cycle (21–35 days) and/or a basal level of follicle-stimulating hormone <25 IU/L; (3) a diagnosis of pure adenomyosis, verified using ultrasonography and magnetic resonance imaging, affecting both the internal and external area of the uterus, with extensive small scattered lesions distributed in the anterior and posterior myometrial layers consistent with subtype IV (indeterminate) diffuse adenomyosis proposed by Kishi et al.[28]; (4) mifepristone (Xianju Pharmaceutical Co., Ltd, Taizhou, China) was administered without contraindications; and (5) sex hormone treatment was not received in the 6 months before using mifepristone, except for LNG-IUD. The exclusion criteria for patients were as follows: (1) current incidence or history of neoplasm (e.g., breast cancer); (2) presence of endometrial-related abnormalities, such as endometrial polyps; (3) any unexplained cause of pelvic pain; (4) incomplete data and drop-out after less than 1 month of treatment; and (5) previous treatment with UAE or HIFU.

Evaluation of therapeutic efficacy and safety

Pain severity was scored using a standardized questionnaire with a Visual Analogue Scale (VAS). The scale ranged from zero to ten; the extreme left side of the scale denoted “no pain,” and the extreme right side indicated “maximum pain.” A score of more than 6 was considered severe pain[29]. The treatment was judged effective in improving dysmenorrhea when the VAS score decreased by more than 2 points[1,30]. In this study, menorrhagia was defined as requiring the use of more than 5 menstrual pad products per day[31]. We defined “ineffective LNG-IUD” as no change or a worsening in either the pain score or amount of menstrual loss after using LNG-IUDs in the adenomyosis patients. Six months after LNG-IUD insertion was considered sufficient time for stabilization, and all patients in our study had utilized the device for more than 6 months.

Uterine size was measured using transvaginal ultrasound (uterine volume = length × width × depth × 0.52). The thickness (in cm) of the endometrium (2 layers) was recorded using ultrasound. The level of serum CA125 was determined using ELISA (HM10776; Bio-Swamp, Shanghai, China)[1]. Hemoglobin, sex hormone profiles, blood lipids, and liver and kidney function were routinely measured in accordance with the manufacturers’ instructions (LH750, Beckman-Coulter, Fullerton, USA; Cobas e602, Roche, Mannheim, Germany; and ARCHITECT C16000, Abbott, Chicago, USA), and adverse reactions caused by mifepristone, such as nausea and vomiting, were closely observed.

Follow up

Mifepristone was continuously administered at a dose of 5 mg per day, beginning from day 1 to 5 in patients with regular menstrual cycles and from any time in women with LNG-IUD. The follow-up results of most patients were derived from their hospitalization and outpatient visit records, and a few patients were followed up by telephone to collect missing data. The evaluation data of most patients were collected every 3 months until the end of the medication, while some women were evaluated 3 to 6 months after they stopped the treatment.

Endometrial biopsy

The endometrial sections from biopsies were stained with hematoxylin and eosin and were evaluated by 2 experienced pathologists from our hospital. If the findings were inconsistent, the specimen was judged by a third pathologist for final diagnosis. All the specimen information was blinded to the pathologists. The endometrial diagnosis criteria were set according to a panel of pathological experts specializing in endometrium, especially that influenced by mifepristone. The key histopathologic characteristics induced by mifepristone were as follows: (1) the presence of cystically dilated glands; (2) abnormal vascular changes, especially thick-walled vessels; (3) glands exhibited combinations of mitosis, inactivity, secretory change, and apoptosis; and (4) compacted and non-decidualized stroma, occasionally showing pseudodecidual changes[32,33].

Immunohistochemistry

Samples of eutopic and ectopic endometrial lesions were obtained from women with adenomyosis who had undergone either adenomyomectomy and diagnostic curettage or hysterectomy. The sample collection and immunohistochemical staining were performed as previously described by Wang et al.[24] The serial sections were immunohistochemically stained against mouse anti-human nerve growth factor (NGF) primary antibody (dilution 1:150, ab6199; Abcam, Cambridge, USA), mouse anti-human cyclooxygenase-2 (COX-2) (dilution 1:600, ab15191; Abcam), and Ki-67 (dilution 1:200; Gene Company, Shanghai, China).

We separated the stroma and epithelial parts and calculated the expression of NGF and COX-2 in each sample. NGF and COX-2 staining was evaluated using semiquantitative analysis with an immunoreactivity (IR) score[34]. Five random fields were observed and averaged for each specimen. The percentage of immunoreactive cells was determined by counting 100 cells at high magnification (200×). The IR score for each sample was assigned according to the percentage of positive cells (0 for 0%, 1 for ≤10%, 2 for 11%–50%, 3 for 51%–80%, and 4 for >80%) multiplied by an intensity factor (0, negative; 1, weak; 2, moderate; and 3, strong). For Ki-67, only nuclear staining was considered a positive result. The percentage of positive cells was evaluated by counting 100 endometrial epithelial and stromal cells at 400× magnification and averaged over 10 areas to obtain an overall Ki-67 score for each case. For Ki-67 staining, we scored cases as follows: 0 for <5% nuclei stained, 1 for 6%–25% of nuclei stained, 2 for 26%–50%, 3 for 51%–75%, and 4 for 76%–100%[35].

Statistical analysis

SPSS program version 22.0 was utilized to perform the statistical analyses. Data were expressed as mean ± standard derivation unless otherwise specified. The Student’s t test and Mann-Whitney U test were used to compare parametric data between 2 groups. Variables within the same group before and after mifepristone treatment were compared using the Friedman test and one-way repeated analysis of variance analysis. We performed an analysis of covariance on the change from the baseline to that after therapy, with the baseline data as a covariate between the 2 groups. Fisher’s exact test was performed for categorical variables. The Kruskal-Wallis test was used to compare variables and immunostaining scores among 3 groups. P <0.05, P <0.01, and P <0.001 were considered statistically significant, highly significant, and extremely significant, respectively, in all cases.

Results

Comparison of clinical features of 3 groups of patients before treatment

As shown in Fig. 1, all 66 patients across the 3 groups had severe dysmenorrhea before treatment, including the 2 mifepristone groups: group A (n = 45) was administered 5 mg mifepristone daily, and group B (n = 13) was administered 5 mg mifepristone daily with previously ineffective LNG-IUD. Another 8 women with adenomyosis who directly underwent either hysterectomy or adenomyomectomy immunohistochemical and diagnostic curettage were included in the immunohistochemical study (group C, n = 8).

F1
Fig. 1.:
Flowchart of the study protocol. LNG-IUD: levonorgestrel-releasing intrauterine device; D&G: dilatation and curettage; GnRHa: Gonadotropin-releasing hormone agonist.

In group A, when followed up at 12 months, 4 of the 45 adenomyosis patients had chosen to have a hysterectomy, and 6 of the 45 patients had chosen to undergo adenomyomectomy because they experienced thickened endometrium and irregular bleeding. In group B, when followed up at 12 months, 2 of the 13 patients had an adenomyomectomy, and 1 of the 13 patients had terminated treatment as a result of wanting to conceive. Among all groups, 36 of 45, 3 of 13, and 5 of 8 women had the symptom of menorrhagia in groups A, B, and C, respectively. There were no significant differences between the mifepristone group (group A) and mifepristone with LNG-IUD group (group B) in age, uterine size, VAS score, serum CA125 levels, and endometrial thickness at baseline (P >0.05) (Table 1). However, the hemoglobin levels were significantly lower, and the menstrual volume was greater in group A than those in group B (P <0.01).

Table 1 - Patient demographics and clinical characteristics before treatment.
Parameters Group A (n = 45) Group B (n = 13) Group C (n = 8) P
Age (year) 41.78 ± 5.15 40.92 ± 4.82 42.88 ± 4.49 0.581
Parity 1.13 ± 0.59 0.77 ± 0.60 1.38 ± 0.52 0.057
Gravity 2.62 ± 1.32 3.15 ± 2.23 3.00 ± 1.41 0.573
Abortion 1.49 ± 1.04 2.38 ± 1.85 2.13 ± 1.36 0.180
Body mass index 22.56 ± 2.61 22.08 ± 2.03 23.37 ± 3.23 0.541
VAS score 7.80 ± 2.26 7.15 ± 2.44 7.63 ± 1.30 0.387
Menorrhagia (pad) 7.07 ± 3.30 3.54 ± 3.15 5.88 ± 3.00 0.001**
Uterine size (cm3) 235.20 ± 122.64 175.61 ± 101.10 426.94 ± 318.44 0.115
Serum CA125 (U/mL) 169.55 ± 151.88 128.32 ± 204.75 210.60 ± 129.36 0.056
Hemoglobin (g/L) 103.71 ± 22.89 125.69 ± 20.36 107.38 ± 16.93 0.003**
Endometrial thickness (cm) 0.22 ± 0.09 0.20 ± 0.03 0.25 ± 0.06 0.757
Estradiol (pmol/L) 335.71 ± 237.20 282.30 ± 152.97 308.69 ± 210.38 0.649
Differences between group A and group B are analyzed by Student’s t test or Mann-Whitney U test.
**P <0.01. VAS: visual analogue scale.

Efficacy of 2 groups after mifepristone treatment

As shown in Table 2, all 45 patients in group A and all 13 patients in group B participated in the 6-month follow-up; however, only 30 and 10 patients from the 2 respective groups completed the investigation at the 12-month time point. In group A, the VAS scores of patients receiving mifepristone were markedly decreased after 6 or 12 months compared with the baseline (P <0.001). Furthermore, at 6 or 12 months during treatment, hemoglobin levels were remarkably different compared with the baseline (128.11 ± 11.93, n = 45; 136.10 ± 10.35, n = 45; vs. 103.71 ± 22.89, n = 30; P <0.001). After 6 months, the levels of serum CA125 in group A were lower than baseline (P = 0.001); however, serum CA125 levels were not significantly different between the baseline and level at 12 months after taking mifepristone (P >0.05). The uterine volume at 12 months was significantly reduced compared with baseline in group A (P <0.05). The effective rates of improvement in dysmenorrhea in group A patients were 100% (45/45) and 100% (30/30) after treatment at 6 and 12 months, respectively.

Table 2 - Change of efficacy and safety variables after mifepristone treatment.
Items Mifepristone (n = 45) Mifepristone + LNG-IUD (n = 13) #
VAS score change
 At 6 months -6.26 ± 1.96*** -5.69 ± 2.56*** 0.449
 At 12 months -6.01 ± 1.96*** -5.11 ± 1.97* 0.255
Menorrhagia change (pad)
 At 6 months -7.07 ± 3.30*** -3.31 ± 3.22*** 0.001
 At 12 months -6.20 ± 3.67*** -1.50 ± 1.65 0.306
Serum CA125 change (U/mL)
 At 6 months -47.12 ± 141.73** -23.84 ± 95.24 0.837
 At 12 months -14.10 ± 106.72 -15.66 ± 83.48 0.867
Uterine size change (cm3)
 At 6 months -25.07 ± 90.48 -15.98 ± 71.28 0.805
 At 12 months -36.74 ± 99.84* -26.98 ± 94.91 0.451
HB change (g/L)
 At 6 months 24.40 ± 15.87*** 8.31 ± 13.46* 0.265
 At 12 months 35.27 ± 21.21*** 5.60 ± 5.56** 0.037
ET change (cm) (2 layers)
 At 6 months 0.06 ± 0.15 0.10 ± 0.18 0.672
 At 12 months 0.31 ± 0.21** 0.11 ± 0.12 0.003
E2 change (pmol/L)
 At 6 months 40.29 ± 245.20 -12.74 ± 142.74 0.591
 At 12 months -235.17 ± 278.59** -175.77 ± 159.37** 0.642
Efficacy, n/n (%)
 At 6 months 45/45 (100%) 13/13 (100%) -
 At 12 months 30/30 (100%) 10/10 (100%) -
Amenorrhea, n/n (%)
 At 6 months 45/45 (100%) 13/13 (100%) -
 At 12 months 17/30 (56.67%) 3/10 (30.00%) 0.273
There were 30 and 12 patients who completed the investigation at 12 months respectively in 2 groups. Variables in 1 group at 3-time points analyzed by Friedman test and one-way repeated analysis of variance, variables between 2 groups analyzed by analysis of covariance and Mann-Whitney U test, categorical variables analyzed by Fisher’s exact test. VAS: visual analogue scale; HB: hemoglobin; ET: endometrial thickness; E2: estradiol; LNG-IUD: levonorgestrel-releasing intrauterine device.
*P <0.05,
**P <0.01,
***P <0.001.
#Difference between 2 groups.

In group B, the VAS scores were significantly decreased after 6 (n = 13) or 12 months (n = 10) of mifepristone treatment (P <0.001 and P = 0.029, respectively). A significant increase in hemoglobin levels was also found after 6 and 12 months of treatment compared with baseline levels (P < 0.05 and P = 0.002, respectively). In group B, there was no significant difference in the levels of serum CA125 and uterine size among the baseline, 6 months, and 12 months treatment data (P >0.05). The rated effectiveness of the treatment in relieving pain was 100% (13/13) and 100% (10/10) at 6 and 12 months, respectively.

After 6 or 12 months of mifepristone treatment, there was no significant difference between groups A and B (P >0.05) regarding the percentage reductions in VAS score, uterine volume, and serum CA125 levels. The percentage reduction in menstrual volume in group A was significantly higher than that in group B at 6 months (P <0.01). Moreover, the percentage increase in hemoglobin level after 12 months was significantly higher in group A than that in group B (41.66 ± 34.16, n = 30 vs. 4.74 ± 5.59, n = 10; P = 0.037).

Evaluation of side effects during mifepristone treatment

As shown in Table 2, after mifepristone treatment, the 6-month amenorrhea rates in groups A and B were both 100% (45/45 and 13/13, respectively). The amenorrhea rates at 12 months were 56.67% (17/30) and 30% (3/10) in groups A and B, respectively. In addition, there was no significant difference in amenorrhea or irregular bleeding rates between the 2 groups (P >0.05).

In group A, the endometrium was significantly thicker after 12 months (n = 30) of treatment than that before treatment (P <0.01). In group B, endometrial thickness did not change significantly after 6 (n = 13) or 12 (n = 10) months of treatment. After 12 months of treatment, the percentage increase in endometrial thickness in group A was significantly greater than that in group B (176.28 ± 134.27, n = 30 vs. 70.77 ± 83.45, n = 10; P = 0.003).

The levels of plasma E2 in group A patients treated with mifepristone for 12 months were significantly decreased compared with those at 6 months or before treatment (P = 0.001 and P = 0.002, respectively). For group B, with long-term (12 months) use of 5 mg mifepristone daily with LNG-IUD, the decline in E2 levels was statistically significant at 12 months compared with the baseline (P <0.01). Regarding the changes in E2 levels before and after mifepristone treatment, there was no significant difference between groups A and B.

After 6 months of treatment, 2 patients from group A showed a slight increase in hepatic alanine aminotransferase (91 U/L and 44 U/L, respectively); whereas no patients in group B had abnormal liver function. The transaminase rapidly decreased to normal levels when treated with liver medication. After 6 months of mifepristone treatment, 2 patients in group A and 1 patient in group B had mild hypercholesterolemia. However, all patients with slightly elevated blood cholesterol had levels less than 6.2 mmol/L, and their blood cholesterol returned to normal after being controlled by a low-fat diet. The liver function and blood lipid results were normal for all patients at the 12-month treatment point. To understand whether mifepristone affected the adrenal function during treatment, cortisol levels were examined in 9 patients after 12 months of treatment. The results were all within the normal range. In addition, none of the patients in either treatment group complained of serious gastrointestinal reactions, hot flushes, breast pain, neuropsychiatric symptoms, or other side effects.

Efficacy and side effect evaluation after discontinuation of treatment

Patients with severe adenomyosis in Group A who received mifepristone for 12 months (n = 12) entered the extension study and completed a 6-month follow-up after drug withdrawal (Table 3). The mean VAS score for dysmenorrhea was 7.75 ± 1.54 at baseline and significantly decreased to 1.08 ± 1.08 at 12 months of treatment but remarkably increased back to 7.25 ± 1.48 at the 6-month post-treatment mark. Bleeding was significantly increased after stopping mifepristone. There was no significant difference between the hemoglobin levels after discontinuation and the baseline (P > 0.05).

Table 3 - Clinical characteristics of patients with severe adenomyosis in group A by evaluation periods (n = 12).
Evaluation periods VAS score Serum CA125 (U/mL) Uterine volume (cm3) Hemoglobin (g/L) Endometrial thickness (cm)
Baseline 7.75 ± 1.54 217.63 ± 196.43 287.30 ± 192.99 103.58 ± 26.87 0.23 ± 0.05
At 12 months’ treatment 1.08 ± 1.08** 197.15 ± 144.39 242.22 ± 135.64 137.00 ± 12.05** 0.56 ± 0.32**
6 months off treatment 7.25 ± 1.48 300.62 ± 258.08* 374.10 ± 237.26* 110.33 ± 18.79 0.27 ± 0.07
Differences between 3 periods were analyzed by Friedman test and one-way repeated analysis of variance.
*P <0.05
**P <0.01.
VAS: visual analogue scale.

After stopping the mifepristone treatment, the levels of serum CA125 were significantly increased compared with those both before and during treatment (P = 0.013 and P = 0.024, respectively). Similar to the serum CA125 levels, a significant increase in uterine volume was observed from 287.30 ± 1.92.99 cm3 at baseline and 242.22 ± 135.64 cm3 at 12 months to 374.10 ± 237.26 cm3 at 6 months after mifepristone withdrawal. The endometrial thickness after discontinuation of therapy was not significantly different from that before mifepristone administration.

Endometrial assessments during and after treatment

We obtained 35 endometrial samples from 31 adenomyosis patients in group A, including 26 collected during the mifepristone treatment and 9 collected after the termination of treatment for 3 to 6 months (Fig. 2, B and C). We found that the timing of endometrial biopsy was as follows: (1) there was endometrial thickness >0.5 cm (1 layer), (2) there had been episodes of vaginal bleeding lasting more than 10 days, (3) there had been irregular bleeding, or (4) there had been bleeding leading to moderate anemia.

F2
Fig. 2.:
Sections showing proliferation markers in different eutopic and ectopic endometrium with or without mifepristone treatment and mifepristone discontinuance in adenomyosis patients. (A-C) Sections stained with hematoxylin and eosin (50×). (A) Proliferative control. (B) Cystic dilatation of endometrial glands and densely packed stroma in a disordered status after 9 months of treatment with 5 mg/d mifepristone. (C) Physiologically proliferative endometrium after 3 months of stopping 5 mg/d mifepristone. (D) Changes of endometrial thickness (cm) by 3 evaluation periods (n = 12). (E–L) Immunohistochemical staining for determining Ki-67 expression. The image represents high magnification (400×). The percentage of positive cells was averaged over 10 areas to obtain an overall Ki-67 labeling index for each case. Data are expressed as mean ± SEM. Statistical difference was analyzed using Kruskal-Wallis test. *P <0.05, **P <0.01. Scale bars = 500 μm. Eu: eutopic; Ec: ectopic.

In general, the endometrial histology results after 6 to 12 months of therapy showed a variety of disordered changes which were different from normal-appearing cycling endometrium. These findings, such as thickening of the endometrium, pseudo-decidualized compact stroma, confluent structures of glands, abnormal vessel morphology, asymmetry of stromal and epithelial growths, and cystically dilated glands mixed with mitotic and secretory effects, were characteristic features corresponding to the spectrum of progesterone receptor modulator-associated endometrial changes (PAECs)[36]. No signs of hyperplasia, atypical hyperplasia, or malignancy were discovered in any of the 26 samples. After discontinuation of mifepristone treatment for 3 to 6 months, the pathology of all 9 endometrial tissue samples presented a proliferative pattern.

Immunohistochemical analysis of proliferation and pain markers

We collected endometrial and adenomyotic lesion samples from 23 adenomyosis patients who underwent surgery, comprising mifepristone-treated patients (group A, n = 8), patients after 3–6 months of mifepristone discontinuance (group A, n = 7), and control patients (group C, n = 8) (Fig. 1). We discovered that all the control and discontinuance specimens were collected during the follicular and early secretory phases of the menstrual cycle, depending on routine hematoxylin and eosin staining, probably due to our choice of operation time.

Additionally, these 23 patients were all symptomatic with severe dysmenorrhea (VAS > 6), and there were no significant differences regarding demographics and clinical data at baseline among the 3 groups (P >0.05). The expression of Ki-67 is shown in Fig. 2E–L. Ki-67 expression on the eutopic endometrium was significantly increased in the mifepristone group compared with that in the control and discontinuance groups (P <0.05).

The levels of NGF and COX-2 are shown in Fig. 3. COX-2 and NGF levels were significantly different between the mifepristone group and no-therapy or discontinuance groups (P <0.05), and the expression of COX-2 and NGF was distinctly decreased on the eutopic and ectopic endometrium in the mifepristone treatment group (Fig. 3).

F3
Fig. 3.:
Immunohistochemical staining for protein expression of NGF (A–H) and COX-2 (I–P) in different eutopic and ectopic endometrium with or without mifepristone treatment and mifepristone discontinuance in adenomyosis patients. The image represents a semiquantitative analysis of the immunostaining for 5 fields performed for each case at high magnification (200×). Data are expressed as mean ± SEM. Statistical difference was analyzed using Kruskal-Wallis test. *P <0.05, **P <0.01. Scale bars = 500 μm. NFG: nerve growth factor; COX-2: cyclooxygenase-2; Eu: eutopic; Ec: ectopic.

Discussion

In recent years, adenomyosis clinical studies have found that mifepristone can play a role in inhibiting ovulation and directly affects the progesterone receptor, which prevents the proliferation and differentiation of ectopic endometrium, reducing its growth potential and making it shrink[37,38]. Further experimental studies have found that mifepristone can inhibit ectopic endometrial angiogenesis and simultaneously promote ectopic endometrial apoptosis[20,39]. These findings provide direct laboratory data on the clinical efficacy of mifepristone in the treatment of adenomyosis.

The results suggested that various indicators were significantly improved in patients with adenomyosis after 6 and 12 months of low-dose mifepristone treatment. Low-dose mifepristone treatment could effectively control the clinical symptoms of adenomyosis in patients with dysmenorrhea and hypermenorrhea, which was consistent with the results reported in recent years in the literature for large doses of mifepristone[40]. Moreover, the uterine volume was smaller in patients after 12 months of treatment than that before dosing, and their levels of serum CA125 were significantly decreased after 6 months of treatment, indicating that a low dose of mifepristone has some effect on the severity of adenomyosis after 12-month treatment regimes. After taking mifepristone for 12 months, eutopic endometrial thickening with an increased Ki-67 index and irregular uterine bleeding contributed to an increase in the serum CA125 level. There were fewer patients in group B who underwent the treatment of 5 mg mifepristone daily with LNG-IUD; hence, this group did not present with an apparent decrease in uterine volume or serum CA125 levels, but their symptoms improved similarly to patients in group A. Consequently, the results suggest that a 5 mg/day mifepristone treatment plan can be applied to control the symptoms of dysmenorrhea and menorrhagia for all patients, especially those with ineffective Mirena.

Although low-dose mifepristone effectively relieves the dysmenorrhea symptom of adenomyosis, the mechanism of this treatment in improving dysmenorrhea is unclear. Inflammation and innervation may be the key causative factors in adenomyosis-associated pain[41,42]. NGF is a multifunctional factor for neurotrophic activity, and NGF expression correlates with the severity of adenomyosis[42]. COX-2 is induced by various stimuli, such as inflammatory transmitters, growth factors, cytokines, and carcinogens, and is involved in inflammation and the proliferation and differentiation of cancer cells[43]. COX-2 expression is not only correlated with the levels of IL-6 and IL-8 but also with the degree of dysmenorrhea in patients with adenomyosis[44]. The COX-2 selective inhibitor, celecoxib, markedly reduces the severity of adenomyosis in mice[45]. In the present study, we investigated immunohistochemical alterations in the expression of neurotrophic and inflammatory factors. We found that the expression of NGF and COX-2 decreased significantly during mifepristone treatment and gradually increased after its discontinuance, suggesting that NGF- and COX-2-related factors may be involved in the occurrence and development of adenomyosis-associated pain. There may be a positive feedback loop between these factors. Peng et al. reported that endometriosis-related sexual pain may be mediated by NGF signaling through increased nerve bundle density and may be associated with COX-2/PGE2 stimulation[46].The involvement of these molecules in specific signaling pathways associated with adenomyosis pain should be further explored.

Endometrial thickening is the most noteworthy side effect of long-term treatment with low-dose mifepristone. It can cause irregular uterine bleeding during treatment or heavy breakthrough bleeding after stopping the medication; hence, patients experience dissatisfaction with the treatment[47]. Although it is reported that high-dose mifepristone can cause endometrial atrophy, endometrial thickening was found with continuous low-dose mifepristone use in the study due to its weak estrogen-like effects. Whether mifepristone induces growth or atrophy of eutopic and ectopic endometrium depends on the dosage and the length of continuous administration. To avoid abnormal bleeding from endometrium shedding and hypertrophic endometriosis due to a prolonged dosage regime, intermittently combining mifepristone with an oral progestin may be a feasible approach[48]. Six months of continuous oral mifepristone followed by a progestin withdrawal period may be a good strategy.

Mifepristone can induce unusual, specific endometrial appearances described as PAECs[33]. Most researchers believe these changes are not concerning, as the histological endometrium is benign and will rapidly return to normal physiological status with cessation of treatment and resumption of menses[33,49], which is consistent with our Ki-67 results of the eutopic endometrium. Berger et al. reported that the characteristics of PAECs can be explained by altered gene expression that is not involved in the signaling pathways of endometrial cancer[33]. Nevertheless, massive cystic endometrial changes and progressive endometrial thickening similar to endometrial hyperplasia present an urgent need for further diagnosis[50]. Therefore, routine endometrial biopsy to rule out endometrial abnormalities is necessary during the long-term application of mifepristone treatment. We maintain that there is an urgent need to develop standardized and repeatable criteria for diagnosing PAECs, and pathologists need to consider the medication history of a patient when making endometrial diagnoses[32].

Moreover, our observations revealed that the symptoms of dysmenorrhea and hypermenorrhea gradually reappeared after the withdrawal of mifepristone treatment and eventually reached their original pretreatment intensities. Most notably, 6 months after discontinuance, a significant increase was observed in uterine size and serum CA125 levels compared with pretreatment data, which was different from the outcomes seen when using mifepristone for uterine myoma[51]. However, it was unclear whether those results were due to the progression of the disease itself or the adverse effects caused by the cessation of mifepristone. Mifepristone is not only a progesterone receptor antagonist but also a competitive glucocorticoid receptor antagonist and can be used to treat many other diseases, such as Cushing’s syndrome[52]. Nonetheless, symptoms of cortisol withdrawal may occur after the drug treatment is stopped[53]. Therefore, we suppose that mifepristone used for the treatment of adenomyosis could also have similar hormone withdrawal symptoms, such as making the uterus larger than it was before the drug therapy. It is very likely that this symptom is transient and is an indication of the effect of treatment; however, attention should be paid to follow-up monitoring of the disease after the discontinuation of mifepristone.

The present study had some limitations. First, the population size was small, and the research was based on a retrospective, single-center study with various recall biases. Furthermore, some patients dropped out for different reasons during the follow-up period, and the missing data might have affected the results obtained. Lastly, endometrial biopsy results were not evaluated to exclude endometrial lesions before initiating mifepristone treatment. Hence, a large sample size from a prospective randomized study with a longer follow-up time is needed to confirm our results.

Conclusion

In summary, a 5 mg/d mifepristone 12-month regimen provided extremely efficient treatment of adenomyosis with dysmenorrhea and/or menorrhagia, especially for patients with ineffective LNG-IUD, with few side effects. NGF and COX-2 factors may mediate the pathogenesis of adenomyosis with dysmenorrhea. Intermittently combining low-dose mifepristone with an oral progestin may be a feasible approach to reduce endometrial thickness and heavy breakthrough bleeding and ensure endometrial safety. Based on the recurrence of symptoms and progression of the disease after mifepristone discontinuance, when and how to cease the treatment needs to be further explored. Moreover, the efficacy and safety evaluation after medication withdrawal requires a longer follow-up time.

Acknowledgments

None.

Author contributions

Conception and design: Shu-Yi Chen and Xin-Mei Zhang; provision of study materials or patients: all authors; collection and assembly of data: Shu-Yi Chen and Wen-Ting Sun; data analysis and interpretation: all authors; manuscript writing: all authors; final approval of manuscript: all authors.

Funding(s)

This work was supported by grants from the Natural Science Foundation of Zhejiang Province (No. LY19H040011), the National Key R&D Program of China (No. 2017YFC1001202), and the National Nature Science Foundation of China (Nos. 81471433, 81671429, and 82071616).

Conflicts of interest

All authors declare no conflicts of interest.

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Keywords:

Adenomyosis; Mifepristone; Clinical retrospective study; 12-month treatment; Mechanisms

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