Role of Endometrial Scratch in UnExplained infertility (RESCUE); A Randomized Clinical Trial


 Background Endometrial natural killer (NK) is thought to play a role in implantation and maintenance of a healthy pregnancy. However, their immunological role in unexplained infertility is yet unknown. The aim of the current study was to investigate the role of endometrial injury in treatment of unexplained infertility and to have more insight on the potential underlying mechanisms of its action methodology randomized controlled trial (RCT) done at Minia university hospital, diagnostic laparoscopy was done for both study and control group, study group had gentle scratch and specimens were sent for histopathological examination while control group had Laparoscopy only .Patients in both groups were followed up at the outpatient clinic for 3 months to assess if pregnancy achieved or not. Patients in the study group who had not achieved pregnancy had a repeated scratch using a pipelle sampler which was also sent for histopathology. Patients were counselled for intrauterine insemination (IUI) with ovulation induction while patient in control group had been counselled for IUI and ovulation induction only. Patients in both groups were followed up for further 3 months to assess patients who achieved pregnancy either naturally or after IUI . Results Baseline characteristics were comparable in both groups. Clinical pregnancy rate (CPR) was significantly higher in scratch group compared with the control group (22%; vs. 7.4%, P = 0.001) in the first three months of follow up. After second scratch and IUI cycles cumulative pregnancy rate was significantly higher in the scratch group compared with the control group (36.7% , vs. 16.2%,, P = 0.001). The mean time achieve pregnancy was found to be significantly shorter in the scratch group as compared to the control group (102 days vs.49.5 days, P = 0.01). Conclusion: Endometrial natural killer cells might play a crucial role in endometrial receptivity and therefore embryo implantation. Endometrial scratch could promote the recruitment of endometrial NK cells and give a more favourable results as regard clinical pregnancy in patients with unexplained infertility. Registration number PACTR201604001405465


Abstract Background
Endometrial natural killer (NK) is thought to play a role in implantation and maintenance of a healthy pregnancy. However, their immunological role in unexplained infertility is yet unknown. The aim of the current study was to investigate the role of endometrial injury in treatment of unexplained infertility and to have more insight on the potential underlying mechanisms of its action methodology randomized controlled trial (RCT) done at Minia university hospital, diagnostic laparoscopy was done for both study and control group, study group had gentle scratch and specimens were sent for histopathological examination while control group had Laparoscopy only .Patients in both groups were followed up at the outpatient clinic for 3 months to assess if pregnancy achieved or not. Patients in the study group who had not achieved pregnancy had a repeated scratch using a pipelle sampler which was also sent for histopathology. Patients were counselled for intrauterine insemination (IUI) with ovulation induction while patient in control group had been counselled for IUI and ovulation induction only. Patients in both groups were followed up for further 3 months to assess patients who achieved pregnancy either naturally or after IUI .

Results
Baseline characteristics were comparable in both groups. Clinical pregnancy rate (CPR) was signi cantly higher in scratch group compared with the control group (22%; vs. 7.4%, P = 0.001) in the rst three months of follow up. After second scratch and IUI cycles cumulative pregnancy rate was signi cantly higher in the scratch group compared with the control group (36.7% , vs. 16.2%,, P = 0.001). The

Introduction
Endometrial curettage (injury) has been shown to improve the pregnancy and live birth rate following recurrent implantation failure with 3 systematic reviews showing consistently improved pregnancy and live birth rate when endometrial injury (EI)or scratch was done in the luteal phase preceding IVF treatment.(Ref) The exact mechanism of action of EI is however not clearly understood. Proposed mechanism of action includes promoting recruitment of a more favourable repertoire of immunological cells in the endometrium with a pro le of growth factors and in ammatory cytokines conducive of implantation. Although the role of endometrial injury has been investigated in ART population its role as a primary tool for infertility treatment outside ARTtreatment is not clearly established. In patients with unexplained infertility it is possible that one of the underlying mechanisms is a defective implantation process which could be corrected by endometrial injury Endometrial scratching was favorable practice has been widely implemented in daily procedure to diagnose or treat infertility (1)(2)(3)(4).It is de ned as an intentional damage caused to the endometrium (5). The connection between local injury to endometrium and increased pregnancy rates in IVF cycles was rst described in 2003 (6).
However it is still unclear if endometrial scratching improves the chance of a live birth and, if so, which population bene ts most (7)(8)(9) (10) (11) Natural killer (NK) cells could be considered the single most important determinants of the success of human reproduction. While measuring them and attempting to manipulate their numbers and function are causes of much controversy, (12) A recent systematic review of available literature suggests that the prognostic value of pNK or uNK cell testing is uncertain when predicting IVF treatment outcome. the lack of appropriately powered studies is due to varying inclusion criteria, different NK cell testing methodology and varying outcome measures (13).
In this study we aim to investigate the role of endometrial injury in treatment of unexplained infertility and to have more insight on the potential underlying mechanisms of its action. Patients who are undergoing infertility investigation in Minia university hospital were recruited for the study if they ful l the following eligibility criteria -Female age is < 35 years -Duration of subfertility <3 years -Unexplained infertility (con rmed with semen analysis, basic hormonal pro le, pelvic ultrasound and diagnostic laparoscopy and dye test) -No history of previous ART Patients who ful l 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 con rming unexplained infertility, patients were randomised to two groups Group A 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.

Group B
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 rst 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.
Histopathological examination: 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 xed for 24h in 10% formaldehyde, processed and para n 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 e ciency 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).

Immunohistochemistry:
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 depara nization followed by descending grades of alcohol for rehydration. Blocking of endogenous peroxidase activity was done using 3% H 2 O 2 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.
Immunohistochemical scoring: Sections were examined under Olympus light microscope to count the number of positive cells in at least nine 400x high power elds and at three different sites per slide if su cient area was available (14) Sample calculation: 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 Results A total of 136 patients presenting with primary or secondary infertility (unexplained infertility ) were screened and assessed for eligibility criteria. Of these, patients were randomized into intervention group (n = 68) and control group (n = 68)   Discussion uNK cells are the numerous types of leukocytes in the human endometrium during the window of implantation and considered to play a fundamental role in implantation and maintenance of pregnancy. It supposed to release a unique pattern of immune and angiogenic factors during their interactions with the trophoblast during the early gestation. natural killer (NK) cells in the uterus promote successful pregnancies by regulating the depth of placental trophoblast invasion. (15) The present study was conducted to evaluate the role of endometrial scratching and uterine NK cells in cases with unexplained infertility. Our results showed signi cantly higher clinical pregnancy rate in patients undergoing endometrial scratching than control cases that undergo same management without scratch 43.1% versus 17.7% respectively with signi cant (P value 0.001).
Endometrial NK cells have little cytotoxic activity, but are a rich source of cytokines, particularly angiogenic ones, which are possibly involved in tissue remodelling and angiogenesis.a reliable method to determine which women might bene t from manipulation of the maternal immune system are not yet done and is urgently needed (16) In cases had endometrial scratch the endometrial CD56+ NK cells are stimulated for differentiation in the endometrium. and involved in the regulation of trophoblasticinvasion that explain the high pregnancy rate in the study group .The role of endometrial CD56+ NK cells in the immunological mechanism of unexplained infertility is still under research. Quanti cation of endometrial CD56+ NK cells, in the proliferative phase and its correlation to occurrence of pregnancy in cases of unexplained infertility was rst done in our research. We noticed that the median scoring of CD56 NK cells increased in study group who had pregnancy( median CD 56 scoring 2) than study group hadn't (median CD 56 scoring zero) with signi cant P value(<0.001) Our study shows strong positive association between CD56 scoring and occurrence of clinical pregnancy this supported by Yang et al who reported successful implantation requires a ne-tuned and highly regulated balance between the maternal immune system and the fetus .The maternal-fetal interface consists of different immune cells, such as (NK) cells, macrophages, T cells, dendritic cells, B cells.. Dynamic changes in immune cells at the maternal-fetal interface have not been clearly stated. (17) atching was suggested by Gnainsky et al. [18]. The authors observed that endometrial biopsy induced an in ammatory response that perhaps facilitates the preparation of the endometrium for implantation.
The pregnancy rate did not improved signi cantly with repeated scratch this was in consistence with ndings reported by Gnainsky et al. [18]who suggested that the effect of endometrial scratching is long lived as monocytes recruited to the injured sites are long lived and reside in tissues for a long time. Such bene cial effect even up to 6 months has been reported by Gibreel et al. [19] who reported increased cumulative pregnancy rates for 6 months.
One RCT has shown an impressive improvement in cumulative pregnancy rate of 25.9% within 6 months of endometrial injury as compared with 9.8% within 6 months without endometrial injury in cases of unexplained infertility this is consistent with results of our study as cumulative pregnancy rate in study group was ..versus ..in the control group. the total number of uterine CD56 + cells seems to play a relevant role in increase rate of pregnancy. the data reported above suggests CD56 + uNK cells, may be more important for maintaining a supportive environment for the establishment of pregnancy. However, further investigation on larger populations of women is needed to con rm this result. ( Giuliani et al 2014). This nding has not been corroborated by other RCTS. Contrary to previous data, did not nd any bene t from local injury to the endometrium this may attributed to difrrent inclusion citreia and different study procedure they concluded that Further studies are warranted to better de ne the target population of patients who may bene t from this procedure. (20) (21) Abbreviations NK natural killer randomized controlled trial (RCT) IUI intrauterine insemination uNK uterine natural killer Declarations Ethics approval and consent to participate the study was approved by the institutional ethical committee of Faculty of medicine, Minia University (Registration number: MUH16730). All patients were invited for involvement in this research and they gave an informed consent for their approval.

Consent for publication: not applicable
Availability of data and materials The datasets analyzed during the current study are available from the corresponding author on reasonable request Competing interests, the authors declare that they have no competing interest.

Funding none
Authors' contributions All the authors have made signi cant contribution to the manuscript. RE, AM, HB, and MM were involved in the conception and design of the study. All authors participated in the initial data collection, planning of the analysis and interpretation of results. AG, AE, and MI performed the literature review and drafted the manuscript. RE,AM,HB,MI prepared the nal version of the manuscript. All authors have read and approved the nal version of the manuscript.Nisreen Toni was responsible for the practical part of pathology and immunohistochemistry.