This prospective randomized double-blind mono- center study approved by the local ethics committee of the Faculty of Medicine, Minia University hospital under institutional review board number (623-4/2020), and registered on clinical trial (NCT04414020), involved 252 adult patients of both sexes, ASA I-II, who underwent elective craniotomy. Exclusion criteria included candidate refusal to participate in the study, emergency craniotomy, any risk for developing venous thromb-embolism (previous deep venous thrombosis, systemic lupus or cerebrovasculer stroke), tumor recurrence, population with stented coronary arteries, morbid obesity and giving relevant history of ovarian or endometrial neoplasia.
An informed written consent obtained from all participants who were randomly and equally allocated into two groups through web-based randomizer (https://www.randomizer.org). Computerized randomization was performed using variable block sizes. Opaque sealed envelopes were used for allocation concealment. Boxes with patient numbers containing unlabeled coded vials were provided for all patients. Saline prepared for injection was in equi-volume with progesterone. Serum progesterone was checked twice, first immediately before initiating first dose and the second one was taken at one week after therapy. Double blind fashion disputed (Patient allocation was concealed from patient and the histopathologist). Data acquisition was tasked by two anesthetic lecturers who were not informed about study design. Candidates were assigned in two equal groups, Control group received intramuscular saline (2ml- NaCL0.9%) as a placebo for five days daily before and after surgery, while progesterone group (PR group) received intramuscular progesterone (1mg/ kg) for five days daily before and after craniotomy. Saline prepared for injection was in equi-volume with progesterone.
Both injectate, progesterone and placebo are similar regarding color, duration of therapy and route of injection. Serum progesterone was checked twice, first immediately before initiating first dose and the second one was taken at surgery morning. In both groups, general anesthesia and endotracheal intubation was accomplished with Propofol (2mg/kg) and Fentanyl (2 mcg/kg) and neuromuscular blockade by cis-atracurium (0.5 mg/kg). Anesthesia was maintained with sevoflurane (2-4 %). Intra-operative monitoring included 5 lead elecrtocardiogram, invasive blood pressure, temperature probe, oxygen saturation, exhaled CO2 (end-tidal capnography), train of four and bispectral index. Tube of polyvinyl chloride size 7 secured at the angle of the mouth with a stabilizer. Fixation decided to be on mouth angle opposite to surgical site. Full sensory and motor neurological examination were executed on 3 months visit post-operative.
2.1.Surgical biopsy
Tumors were excised following standard surgical techniques regarding positioning, skin incision, craniotomy, dural opening, dissection, hemostasis and closure. Biopsy obtained from multiple points at the tumor-brain interface by cotton tipped swab under cover of surgical microscope.
2.2. Histopathology examination
2.2.1. Case selection and tissue sample preparation
Paraffin blocks with clinico-pathological data of the patients were collected. H&E slides were prepared to detect histopathological changes as congestion, cytoplsmic edema, necrosis and inflammation.
2.2.2. Immunohistochemistry
Serial sections were cut 4μm thick on positively charged slides. The slides were de-paraffinized with xylene, rehydrated through graded ethyl alcohol. Then, they were immersed in 3% hydrogen peroxide for 30 min and rinsed in phosphate buffer solution (PBS). Citrate buffer (pH 6.0) was used for antigen retrieval by the microwave for 10 minutes. Sections were left to cool at room temperature then washed in PBS. Afterwards, rabbit polyclonal anti-PR anti-body (ready to use, Abcam) was incubated overnight at 4°c in humidity chamber. Slides were then rinsed with PBS before applying secondary antibody for 30 min. After wash in PBS, streptavidin–biotin complex was added for another30 min. Brownish color developed by using diamino-benzoate (DAB), then the slides were washed in distilled water. Lastly, they were stained with hematoxylin, dehydrated, cleared by xylene and covered slipped.
2.2.3. Evaluation of immunostaining.
According to Allred scoring system, The intensity score (IS) of nuclear staining evaluated as follows: 0= no positive cells, 1= mild positivity, 2= moderate positivity and 3= strong positivity. The proportion of staining power evaluated according to the percentage of positive cells: 0= negative, 1=1%, 2=2‑35%, 3=36‑65% and 4=66‑100%. PR immune-expression score was calculated as the algebric summation of the scores for the intensity of staining and the extent of staining power. Final scores were then classified into low (< 3) and high (≥ 3) (0).
2.3. Statistical analysis
2.3.1.Sample Size Calculation:
Before the study runover, the number of patients required in each group was determined after a power calculation according to data obtained by a Pilot study performed on ten consented candidates, five in each group. In that study, the percentage of cellular damage in Progesterone group was 20% ( one case with cytoplasmic oedem), while in Placebo control group was 80%( four cases with cytoplasmic degeneration). A sample size of 126 patients in each group was determined to provide 99% power for Fisher’s exact test at the level of 0.05 significance using G Power 3.19.2 software.
Figure 1 reflecting sample size calculation at power 99%.
Exact - Proportions: Inequality, two independent groups (Fisher's exact test)
Options: Exact distribution
Analysis: A priori: Compute required sample size
Input: Tail(s) = Two
Proportion p1 = 0.2
Proportion p2 = 0.8
α err prob = 0.05
Power (1-β err prob) = 0.99
Allocation ratio N2/N1 = 1
Output: Sample size group 1 = 126
Sample size group 2 = 126
Total sample size = 252.
Statistical analysis
- The collected data were coded, tabulated, and statistically analyzed using SPSS program (Statistical Package for Social Sciences) software version 20.
-Descriptive statistics were done for Parametric quantitative data by mean, standard deviation and minimum & maximum of the range, while they were done for categorical data by number and percentage.
-Analyses within each group were done for parametric quantitative data using paired sample t test, and for qualitative data using Wilcoxon signed rank test. Analyses were done for qualitative data using Fisher Exact test.
-The level of significance was taken at (P value < 0.05).