DOI: 10.6084/m9.figshare.29083946.v1
title: Supplementary Material for: Endometrial metabolism of 17β-estradiol during the window of implantation in women with recurrent implantation failure

description: Objectives
Alterations in 17β-estradiol metabolism are known to potentially impair endometrial receptivity. Previous pioneering studies have investigated the role of endometrial steroid metabolism by determining steroid hormone levels and steroid-metabolizing enzyme activity in endometrial biopsies of patients undergoing IVF. The activity of oxidative and reductive 17β-hydroxysteroid dehydrogenases (17β-HSDs), which catalyse the interconversion between estrone and 17β-estradiol, was found to be similar between IVF patients who - after fresh embryo transfer in the cycle following endometrial biopsy - did and did not become pregnant. However, inhibition of the reductive enzyme 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), the most prominent 17β-HSD type in 17β-estradiol formation, was found to differ between groups. The primary objective of this study is to determine oxidative and reductive 17β-HSD enzyme activity in the endometrium of two well-defined groups: IVF patients with Recurrent Implantation Failure (RIF) and control patients.

Design
Prospective observational study of IVF patients with RIF (n=52) and controls (n=25). Patients undergoing treatment because of pre-implantation genetic testing, a severe male factor or bilateral tubal pathology were recruited as controls, since these conditions did not suggest an endometrial contribution to infertility.

Participants/Materials, Setting, Methods
Endometrial biopsies were obtained 5 to 8 days after a positive urine ovulation test in a natural cycle using a Pipelle catheter. Activity of oxidative and reductive enzymes, inhibition of 17β-HSD1, 5, 7 and 12 and immunostaining of 17β-HSD7 were performed. The formation of 17β-estradiol by reduction of estrone (reductive enzymes), formation of estrone by oxidation of 17β-estradiol (oxidative enzymes) and inhibition of specific 17β-HSD enzymes was determined using high performance liquid chromatography. Formalin fixed paraffin embedded tissue was used for immunostaining. The Student’s t-test and Mann-Whitney U test were used for statistical analysis. Multivariate analysis was used to determine the influence of confounders. 

Results
No differences were found in activity of oxidative and reductive 17β-HSD enzymes in RIF patients and controls. Combined inhibition of 17β-HSD5, 7 and 12 was significantly lower in the RIF group compared to controls (p=0.04). Inhibition of 17β-HSD1 and 17β-HSD7 combined was also significantly lower (more production of 17β-estradiol remained) in the RIF group compared to controls (p&lt;0.01). However, solely inhibiting 17β-HSD1 or 17β-HSD7 showed no significant difference between groups. Immunostaining revealed the expression of 17β-HSD7 in all endometrial samples.

Limitations
Results should be interpreted carefully due to possible cycle-to-cycle variation, challenges to translate in vitro findings biological conditions, and the heterogeneous aetiology of RIF.

Conclusions
Differences in formation of 17β-estradiol in the presence of two specific inhibitors of 17β-HSD1 and 7 between RIF patients and controls were found. Although 17β-HSD1, expressed at the fetal-maternal interface, has been associated with fertility, the potential role of 17β-HSD7 in human implantation has not been previously described. The observed differences between patients with RIF and controls warrant future research on the role of this main enzyme and its lesser-known 17β-HSD type in endometrial receptivity and implantation.