Abstract

Study question
Does the administration of DPPIV inhibiting medication during the window of implantation adversely affect trophoblast attachment to endometrial cells?

Summary answer
In this study, the in vitro spheroid attachment onto endometrial cells was significantly reduced using non-prescribed Diprotin-A compared to prescribed gliptins at serum pharmacological level.

What is known already
Type-2 diabetes incidence is rising globally in women of reproductive age and infertility affects one in six couples, with around 10% experiencing recurrent implantation failure. Clinical pregnancy success has remained stable for decades, partly due to limited understanding of the molecular events governing embryo implantation.

Therefore, the receptor dipeptidyl peptidase-IV (DPPIV) was selected for its endometrial upregulation at the window of implantation, downregulation in recurrent implantation failure patients compared to fertile patients and role in cell–cell adhesion during implantation via fibronectin- and collagen-binding. However, neither the role of DPPIV enzymatic activity nor its inhibition using prescribed inhibitors have been investigated.

Study design, size, duration
The experimental in vitro study was carried out by cultures of human endometrial epithelial Ishikawa cell line and human trophoblastic choriocarcinoma BeWo cell line. Non-prescribed DPPIV inhibitor, diprotin A, and commonly prescribed DPPIV inhibitors in the treatment of type-2 diabetes mellitus [sitagliptin, linagliptin, saxagliptin and vildagliptin] were tested. An in vitro spheroid attachment assay was used to investigate the role of DPPIV inhibitors on attachment at implantation. Experiments were replicated at least 3 times.

Participants/materials, setting, methods
Endometrial cells were treated with DPPIV inhibitors (1nM to 1000µM), vehicle or [oestradiol (10mM) and progesterone (1µM)] for 96 hours. DPPIV detection and activity were carried out by immunofluorescence, ELISA and enzymatic fluorometric assays. Cell count and PrestoBlue cell viability were carried out.

Fluorescent single cell attachment was performed in fibronectin- and collagen-coated wells. Ishikawa monolayers were pre-treated 1 hour before BeWo spheroid attachment assay. After 1h of co-culture, attachment percentages were determined using microscopy.

Main results and the role of chance
Using immunofluorescence, DPPIV was heterogeneous localised at the membrane of endometrial Ishikawa monolayer - predominantly at the apical side. Moreover, at BeWo spheroid attachment sites, cluster of DPPIV-positive cells were observed suggesting a potential upregulation of DPPIV expression during early trophoblast–endometrial interactions.

Endometrial DPPIV was detected in both cell lysates and spent media, and its abundance was significantly increased in both fractions following hormonal treatment. DPPIV was determined enzymatically active in endometrial cell lysates using enzymatic fluorometric assays. Moreover, its activity was significantly reduced by all DPPIV inhibitors at serum-relevant Cmax concentrations, except for linagliptin.

Compared to all DPPIV inhibitors, only linagliptin impaired endometrial cell viability at supra-pharmacological concentrations (>25 µM, p < 0.05). However, none of the inhibitors altered Ishikawa total cell number after 96-hour cultures with drug spiking every 24 hours.

Using single cell attachment assays, the non-prescribed inhibitors Diprotin A and prescribed sitagliptin significantly reduced Ishikawa cell adhesion to fibronectin and collagen I at 10 µM (p < 0.05). Furthermore, only diprotin A induced a significant 36% reduction in BeWo spheroid attachment onto Ishikawa monolayer, although no prescribed DPPIV inhibitor produced a significant impact on spheroid attachment in vitro.

Limitations, reasons for caution
The use of cell lines in a simplified model is a limitation of the study, compared to the more complex in vivo environment. Use of trophoblast spheroids may not fully represent the response of human embryos. However, this model is widely used in implantation model studies.

Wider implications of the findings
Although the upregulation of enzymatic-active endometrial DPPIV arises at implantation window, a prescribed DPPIV-targeted inhibition of enzymatic activity did not impair spheroid attachment in vitro suggesting that DPPIV enzymatic activity is not crucial for embryo attachment and setting the scene for further investigations on the gliptin safety in early pregnancy.

Trial registration number
No