This study was designed as a retrospective cohort study. We enrolled all symptomatic patients aged 20 to 45 years in whom endometriosis was confirmed by physical examination, history taking, and imaging techniques who were referred to two referral governmental hospitals between March 2017 to March 2019. The approval of the local ethics committee was obtained (Code: IR.SUMS.REC.1398.1395), and all the patients gave informed consent to the protocol.
The inclusion criteria were as follows: women with a definitive diagnosis of endometriosis based on pathology report, complete demographic and follow-up information even with a phone call, those whose VAS score in any of pain symptoms of endometriosis (dysmenorrhea or dysparonea) was moderate to severe before surgery , with data about pain response to progesterone-based therapies after surgical treatment, unwillingness to conceive until at least two years after surgery, tendency to continue treatment, even though they knew they would not have a routine and monthly mensturation period and those who have had regular follow-ups after surgery.
Exclusion criteria were as follows: incomplete demographic and follow-up information, those with diseases such as GI or urinary tract diseases, as well as those with PID disease, taking hormonal or infertility drugs up to six months before surgery and having previous endometriosis surgery, FSH>10 before operation ,or age >45 at the time of operation, those who stopped taking the drug after the operation for any reason or used it irregularly and existence of concomitant malignancy and unavailability of pathology slides.
Patients' records were used to collect data. Demographic information including patients' age, BMI, pain symptoms, the stage of endometriosis disease according to ASRM (American society for reproductive medicine) classification, affected area, type of hormone therapy, duration of treatment and response to treatment were recorded in a checklist.
During operation after all adhesions were lysed and excised by sharp dissection to fully mobilize the ovaries and ovarian cystectomy, all areas of superficial active endometriosis involving the other ovary or the pelvic peritoneum were fulgurated. Deep infiltrative endometriosis lesions located in the utero-sacral, retrocervical, rectovaginal area, Douglas pouch, rectum and bladder were separated and resected from the surrounding normal tissue with preserving important structures such as ureter, uterine vessels and pelvic nerves.
Patients were aware of the two methods of treatments before their operation, and they had been informed that none has been proven to be superior yet. All of them were on stage three or four of diseases according to the ASRM classification.
Medical treatment was started on the day of discharge for patients based on the patient's preference, which included 30 mg daily medroxyprogesterone or contraceptive pills with 0.03 mg ethinyl estradiol and 15 ug desogestrel. All patients were followed for at least 12 months after surgery (8 to 25 months).
The sample size was determined by NCSS software, 80% power and the first type error of 5% and using similar articles (sensitivity = 93%).
After patients selection based on inclusion and exclusion criteria and extracting the required data from patients' records, Pathological slides were examined and stained immunohistochemically for PR and ESR.
First, the samples in paraffin was cut to a size of 5 micrometers and placed on a slide [17, 18]. The slides are deparaffined and hydrated by a series of washes with xylene and ethanol. After rinsing in distilled water for 5 minutes, the slides are immersed in 0.01M sodium citrate buffer for 15 minutes and then cooled for 45 minutes.
Then the slides are rinsed in PBS 1% (Tween 20) PBST for 5 minutes and cut with a hydrophobic pen. Endogenous peroxidase is quenched for 5 minutes with 3% hydrogen peroxide and then rinsed with PBST for 5 minutes. Non-specific binding with 5% natural goat serum in PBST is blocked for 1 hour at room temperature.
The primary used antibodies (PR H-190) (sc _7208; 1: 800) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The slides were incubated with the initial antibody at 4 ° C overnight. Normal goat IgG (Biotechnology Santa Cruz) was used as a negative control.
Natural endometrium on day 14 was also considered as a positive control. Goat antirabbit biotinylated secondary antibody was used for PR (Vector Laboratories, Burlingame, CA) and for 1 hour at room temperature.
Slides were washed in 1% PBS and incubated at ABC Elite (Vector Laboratories) for 30 minutes at room temperature and then washed again in 1% PBS and incubated with diaminobenzidine (Vector Laboratories) for 41 seconds.
Then exposed to hematoxylin as a counterstain for 30 seconds. Finally, the slides were rinsed with ethanol and xylene for 5 minutes and washed and mounted by Permount (Thermo Fisher Scientific, Waltham, MA) [6, 19, 20].
H score for immunohistochemical staining was determined based on the percentage of receptor staining for each slide. Each slide was scored separately by two pathologists unaware of the subject, and H scores were averaged. The H score was calculated using the modified H score, which is expressed as: negative (score 0), weak positive (score 1), positive (score 2) and strongly positive (score 3) [7, 19].
Finally, the collected data were entered in SPSS software version 20. For the final analysis, patients were divided into two groups based on response to treatment and the data of the two groups were compared. Qualitative data were compared between the two groups using chi-square test and if necessary with Fisher's exact test, and quantitative data were compared using t-test. Kramer-Phi and Spearman tests were used to examine the correlation. P value less than 0.05 was considered statistically significant.