This is a case-control study that measure the P450 Aromatase in patient’s menstrual blood with endometriosis by using laparoscopic or laparotomy surgery, confirmed with histopathology at Department of Obstetrics and Gynecology, Dr. Hasan Sadikin Hospital (RSHS), Bandung, Public Hospital in Sumedang and Ujung Berung, also from Dr. Slamet Hospital, Garut. Sample size was based on a 95% confidence level, by setting the sensitivity of the P450 aromatase examination results in estimating the endometriosis event by 80% and choosing a precision of 15% from the sample size formula and considering the mild endometriosis incidence rate according to the literature is 30%, then the number of n are: 92, plus 10%, so that the minimum sample reaches 100 patients. Patient selection was carried out by team (obstetrics-gynecology specialists and residents); patient will undergo diagnostic laparoscopic or definitive surgery both laparoscopic and laparotomy. Patients who underwent Pomeroy sterilization had their menstrual blood collected prior the surgery, in the first days of menstruation out a lot (day I-II) to P450 aromatase immunocytochemistry.
Inclusion criteria: histopathological results of endometriosis; non-endometriosis included as control group. The exclusion criteria: pregnant women, on hormone treatment, at least three months, except GnRH after nine months of the last administration; other gynecological diseases (infections, malignancies), patients who have received hormone therapy before menstrual blood sampling and surgery, whose data were incomplete and samples are less representative were also excluded. In the implementation, there were 63 (42.28%) cases of endometriosis, while 86 (57.72%) were classified as non-endometriosis. All representative sample evaluation: 37 endometriosis vs 33 control groups.
1 ml menstrual blood was collected, one per subject on the 1-2 day of the cycle and into a sterile container; were then centrifuged 15 min. Immunocytochemistry smears were made on the object's glass. They were soaked in xylol and ethanol I, II and III, as well as in alcohol 70, 80 and 90% for 5 min each, then washed with distilled water. The antigen unmasking retrieval procedure was done for 3x 5 min, then cooled at room temperature 15 min; washed with phosphate buffer saline (PBS). Then incubated with 0.3% H2O2 in methanol 10 min; washed with PBS. The smears were added the blocking reagents and were incubated 5-10 min. Then given primary antibodies, incubated 60 min; washed PBS 3x. Secondary antibodies were then added, incubated 10 min; washed PBS 3x. Later on, streptavidin were used and were incubated 10 min; washed PBS 3x. Chromogen was dropped and incubated 10 min; washed with running water 5 min. The smears were Incubated in mayer hematoxylin for 2 min; washed with running water after. The last steps of the procedure were to dehydrate alcohol 70, 80, 90% for 3 min and soak the smears into xylol for 3 min, then mounting.
Assessment of Immunocytochemistry
Histopathological endometriosis criteria: found endometrial gland epithelial cells and endometrial stromal in the tissue examined. The criteria for viable cells in this study were: there were cells or groups of stromal cells in their entirety in menstrual blood smear preparations. Generally, most cells will soon die when exposed to sunlight. Menstrual blood contains many proteolytic enzymes that are released from lysosomes which break due to a decrease in steroid hormones before menstruation, so that the menstrual blood cell component is easier to experience lysis. Lymphocyte cells around the group of stromal cells that are also stained with dark brown are used as positive controls.
Data normality were assessed. Comparison of continuous variables were done using unpaired t-test if normally distributed; otherwise, Mann-Whitney U test was used. Categorical variables were compared using Chi-square test. To determine the cut-off value of aromatase P450 expression in detecting endometriosis, ROC curve was used. p value < 0.05 were considered significant.