Endometriosis, an estrogen-dependent inflammatory disease
affecting 10-25% of women, is associated with significant
reductions in fertility and is one of the most common
benign gynecological diseases. Retrograde menstruation
with subsequent adhesion formation, invasion, and neovascularization
are believed to give rise to the occurrence
of endometriosis lesions. The most common locations for
endometrial implants are the ovaries, fossa ovarica, uterosacral
ligaments, and posterior cul-de-sac (
Although different medications are used to control endometriosis,
their adverse effects following long-term use
and recurrence of disease after discontinuation of therapy
limit their applications. Additionally, these medications
are not useful for endometriosis-associated infertility
Gonadotropin releasing hormone agonists (GnRHa)
such as diphereline, as standard medications for the treatment
of endometriosis, are able to induce inactivation and
degeneration of endometrial implants via suppression of
hypothalamic-pituitary-gonadal axis and ovarian estrogen
It is known that in women with endometriosis, the growth
of endometrial cells within the peritoneal cavity is induced by
inflammatory mechanisms (
Using herbal medicine has always played a significant
role in Iranian culture and civilization and some of these
herbs have been recommended for treatment of infertility-
related diseases. Licorice (
We hypothesized that licorice or celecoxib might be good candidates for treatment of endometriosis as an inflammatory condition. In the present study, we compared the effects of licorice, celecoxib and diphereline on the growth of endometrial implants in rats.
In this experimental study, 48 mature female Sprague- Dawley rats (almost 8 weeks old, weighting 220 ± 20 g) were purchased from the Center of Comparative and Experimental Medicine at Shiraz University of Medical Sciences (SUMS), Shiraz, Iran. The animals were kept on 12 hours light: 12 hours dark cycles at a controlled temperature with free access to water and food. The animal experiments were performed according to the principles of the care and use of laboratory animals established by the National Institutes of Health, Bethesda, MD, USA, and approved by the Institutional Animal Ethics Committee at SUMS (No. 92-01-01-6869). These animal experiments were performed in the animal house of Shiraz University of Medical Sciences. To select the rats with normal estrous cycle, daily vaginal smears were taken and evaluated by a light microscope. Rats with three normal estrous cycles were used in the next steps.
Licorice roots were purchased from herbal stores in
Endometriosis was induced surgically using the method
described by Vernon and Wilson with little modifications
Briefly, all the female rats were anesthetized using ketamine hydrochloride 10% (100 mg/kg, Alfasan, Netherlands) and xylazine 2% (10 mg/kg, Alfasan, Netherlands). Then, rats’ abdomen was opened through a 4 cm midline incision starting from 1 cm below the xiphoid. The left horn of uterus was ligated at both the uterotubal and cervical junction ends and removed. A longitudinal cut was made through the uterine horn. By a punch biopsy, 4 round pieces of the distal part of uterine tissue were excised (4×4×1 mm) and placed in warm sterile saline 0.9%. Two implants were sutured with proline 5-0, one on the left and the other on the right side of the peritoneal cavity on the areas of well-visible vasculature with endometrial surface facing the peritoneum. Finally, the abdominal muscles, fascia and skin were sutured. Then, chlortetracycline (Cyclo Spray, Eurovet, UK) was sprayed on the incisions site and animals were allowed to recover from anesthesia. Two rats died due to hemorrhage at this stage. Six weeks after the first surgery, a second look laparotomy was performed and the viability of endometrial implants was confirmed by observation of good vascular supply and pinkish colored tissue in contrast to necrosis and fibrosis seen in two rejected cases as showed in Figure 1.
The flow diagram of the study.
At this stage, 44 female rats were divided into 4 groups (11 rats in each group). The control group was treated by 0.5 ml of saline 0.9%/day, the second group by licorice root extract (3000 mg/kg/day) and the third group took celecoxib (Damloran Razak Pharmaceutical Co., Iran, 50 mg/kg, twice a day, dissolved in 0.5 ml of saline 0.9%) for the next 6 weeks. All the treatments of these three groups were administered by oral gavage. The fourth group received a single IM injection of diphereline S.R. 11.25 mg (3 mg/kg, Ipsen, France). Six weeks after the treatments the rats were sacrificed and endometrial implants were evaluated as shown in Figure 2.
Endometrial implants in different times and groups. A. The first laparotomy: auto-transplant of endometrial implants on the peritoneum, B. The second look surgery: shows the adhesion bands and endometrial implants six weeks after induction of endometriosis, C. The third surgery: necropsy of a rat in the control group showing growth of implanted lesions of endometriosis, and D. The third surgery: necropsy of a rat in diphereline group showing regression of the implants.
The length, width and height of each implant were carefully measured using collis rulers by one researcher who was blinded to the treatment arms. The area of the endometrial implants in four groups was measured by multiplying length by width), and the volume was calculated by ellipsoid volume formulation (π/6×length×width×height).
All of the endometrial implants were fixed in formalin,
placed in paraffin, cut into 5 µm sections, stained with
hematoxylin-eosin and evaluated by the same pathologist.
Photographs were taken by a digital camera (Sony,
Japan). To classify the persistence of epithelial cells in
grafts, the scoring system applied by Keenan et al. (
For statistical analysis, the software SPSS 15 (SPSS Inc., Chicago, USA) was employed. To compare the mean area and the mean volume, ANOVA followed by Tukey HSD test was performed. To compare the histopathologic scoring, Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction were applied. A P<0.05 was considered significant.
The mean area, volume and pathologic scores of implants in control, licorice, celecoxib and diphereline groups
|Groups||Area (cm2)||Volume (cm3)||Pathologic score||Hemosiderin-laden macrophages|
|Control||42.94 ± 11.76||125.90 ± 11.69||2.5 ± 0.70||51.00 ± 9.90c|
|Licorice||27.57 ± 17.84a||90.86 ± 19.32a||1.90 ± 1.04||1.20 ± 1.07d|
|Celecoxib||39.87 ± 13.57||121.03 ± 7.08||2.44 ± 0.88||41.80 ± 6.4|
|Diphereline||8.60 ± 2.53b||11.00 ± 2.56b||0.54 ± 0.68b||1.2 ± 1.00|
Data was shown as mean ± SD. P<0.05 were considered statistically significant. a; Statistically significant differences between licorice and the control group, b; Statistically significant differences between diphereline and the control group, c; Statistically significant differences between control and other groups, and d; Statistically significant differences between Licorice, diphereline and celecoxib group.
Two rats died during laparotomy due to hemorrhage
and in two other rats the implants did not grow. The
remaining 44 rats were divided into 4 groups and
treated. In licorice group, the mean area and volume
values of endometrial implants were significantly
lower than those of the control group (P=0.042 and
P<0.001, respectively) (
Specimens of the treated groups stained by hematoxylin-eosin.
The percentage of HLMs in endometrial implants of
rats in celecoxib, licorice and diphereline group was significantly
lower than that of the control group (P=0.004,
P=0.000 and P=0.000, respectively). Also, the percentage
of HLMs was significantly lower in licorice and diphereline
group compared to celecoxib group (P<0.001 and P<0.001,
respectively). The percentage of HLMs was not different
between licorice and diphereline group (P=1.000,
Hemosiderin-laden macrophages (HLMs) in the specimens of different
We compared the effects of licorice, celecoxib, and
diphereline in a rat model of endometriosis induced by
auto-transplantation of endometrium on the peritoneal
surface as a well-established method (
Previous studies showed that glycyrrhetinic acid as a
constituent of licorice extract, inhibits thrombin-induced
platelet aggregation (
COX-2 overexpression has been detected in both eutopic
and ectopic endometrium, and also in peritoneal
macrophages derived from women with endometriosis
Histhopathologic slides prepared from endometriosis showed higher counts of HLMs that serve as an indirect evidence for diagnosis of endometriosis. As shown in our study, endometrial implants had normal growth with intact endometrial lining and more scattered foci of HLMs after treatment with celecoxib or normal saline. However, after taking licorice or diphereline, growth of endometrial implants were highly limited with lower HLMs. These findings are in favor of the potential therapeutic effect of licorice in suppression of endometrial implants growth.
In this study, celecoxib did not significantly reduce
the growth of endometrial implants that was against our
primary hypothesis. There are studies that showed that
celecoxib, dexketoprofen trometamol or rofecoxib were
able to cause regression and atrophy of endometriosis lesions
Since endometriosis is a chronic disease and needs
long-term treatment, complications of prolonged use of
licorice such as hypokalemia, hypernatremia, edema, hypertension
and cardiac complaints should be kept in mind
before human application. It should be considered that
the maximum permitted dosage of licorice root is 5 to
15gr/day and the duration of treatment should not exceed
6 weeks in humans (
As a limitation of our study, we did not evaluate inflammatory markers such as white blood cells counts, nor interleukin-6 (IL-6), vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-a) levels in the peripheral blood and peritoneal fluid before and after interventions to assess the anti-inflammatory properties of licorice.
We believe that licorice might have the potency to be used as a novel and excellent alternative in the management of endometriosis after in-depth investigations in animals and humans.
Licorice decreased the growth and histopathologic grades of auto-transplanted endometrial implants. However, celecoxib had no significant effect and diphereline had the highest potency in reduction of the endometrial growth.