This clinical trial research was done in Maternity Hospital Minia University during the period from March 2016 till April 2017.
Patients who are undergoing infertility investigation in Minia university hospital were recruited for the study if they fulfil the following eligibility criteria
- Female age is < 35 years
- Duration of subfertility <3 years
- Unexplained infertility (confirmed with semen analysis, basic hormonal profile, pelvic ultrasound and diagnostic laparoscopy and dye test)
- No history of previous ART
Patients who fulfil the previous criteria were enrolled for the study after obtaining written informed consent and were booked for diagnostic laparoscopy as part of their infertility work-up to coincide with their luteal phase preferably between day 17-24 of their cycles after performing a sensitive urine pregnancy test on the day of the laparoscopy to exclude luteal phase pregnancy.At laparoscopy after confirming unexplained infertility, patients were randomised to two groups
had gentle endometrial curettage (endometrial injury) by the use of sharp curette on the anterior and posterior wall for 1 minute and specimen was sent for histopathological investigation.
Laparoscopy procedure was continued as per routine procedure without additional endometrial curettage.
Patients in both groups were followed up in the outpatient clinic for 3 months and were contacted if not attending clinic (contact details of patients were taken in advance to avoid loss of follow-up) to assess if they have got pregnancy or not.
At the first follow-up visit to assess patients in group A who did not get pregnant were have a repeat endometrial scratch using a pipelle endometrial sample in the clinic which was also sent for histopathology. Patients were counselled for intrauterine insemination with ovulation induction whilst patient in group B have counselling for IUI and ovulation induction without any further intervention. Both groups were followed up for further 3 months to assess patients who achieved pregnancy whether naturally or after IUI
Specimens sent for histopathology were assessed by immunohistochemistry for the population of uterine natural killer cells(uNK) by the use of CD56 marker as well as by routine H&E section to assess for decidual reaction.
Specimens sent for histopathology were assessed by a pathologist who was blind to the different random groups but informed by the study design. The specimens were fixed for 24h in 10% formaldehyde, processed and paraffin embedded. Multiple 5μm sections were cut from each case and stained by hematoxylin and eosin stains. Examination by light Olympus microscope CX41 (Olympus, Tokyo, Japan) to assess the efficiency and diagnosis of the cases as well as to assess for decidual reaction. Photos were taken for selected sections were taken using an Olympus microscopic camera (C5050Z, Olympus, JP).
Multiple 5μm sections were cut from all studied cases for immunohistochemical analysis of the uterine natural killer cells population (uNK) using CD56 marker. Sections were all subjected to xylene for deparaffinization followed by descending grades of alcohol for rehydration. Blocking of endogenous peroxidase activity was done using 3% H2O2 and washing in phosphate buffer solution (PBS). Antigen unmasking was done for all slides by citrate buffer pH 6.0 in microwave for 30min. After cooling down to the room temperature, the slides were incubated with the primary antibody cd56 (Santa Cruz Biotechnology, ready-to-use) overnight in a humidity chamber at 4°C. Secondary biotinylated antibody (Lab Vision) was applied after rinsing with PBS for 30min. Incubation for 30min was applied with streptavidin-biotin (Lab Vision) followed by DAB treatment of the slides for 5 min to obtain the brown color of the positive cells. Finally, the slides were washed with water and counter stained with Mayer’s hematoxylin. Dehydration, clearing, mounting of the slides was done followed by covering with slips. Positive and negative control slides were processed with the cases in each run.
Sections were examined under Olympus light microscope to count the number of positive cells in at least nine 400x high power fields and at three different sites per slide if sufficient area was available (14)
The number required for recruitment for the study was 136 ( 68 patients in each arm) patients assuming a difference in cumulative pregnancy rate between the two arms of 20% and for type 1 error of 0.05 and power of 80% and assuming a drop-out rate of 10%
The outcome measures:
Primary cumulative pregnancy rate over 6 months of follow-up
Secondary: Time to pregnancy (in days) , complication secondary to scratch.(bleeding ,infection)
The trial was registered prospectively in Pan African clinical site Registry with unique registration number PACTR201604001405465