P-539 Modelling embryo implantation in vitro using 3D ‘instant’ assembloids and human blastocysts
Authors: Van de Velde H , Tryfonos M , Rawlings T , Wafaa E , De Munck N , De Brucker M , Tournaye H , Brosens J 
Published: June 2023

Abstract

Study question
Can embryo implantation be recapitulated in vitro using human blastocysts and a new 3D model of the human endometrium representing the architecture of the tissue?

Summary answer
We present an innovative implantation model consisting of blastocysts attaching on endometrial assembloids containing stromal cells, gland-like epithelial organoids, and overlaid with an epithelial monolayer.

What is known already
A successful pregnancy denotes the implantation of an embryo to the receptive, luminal lining of the uterus, the endometrium. The tissue transforms into the semi-permanent decidua which provides an optimal microenvironment and nourishes a healthy embryo. The transformation of the tissue is driven by a differentiation process, known as decidualisation, which accounts for the change of tissue-resident stromal cells to specialised decidual cells or senescent decidual cells. Simultaneously, endometrial glands of epithelial origin become secretory and provide histotrophic nutrition to the implanting embryo. Perturbations in this process of decidualisation may also result in an implantation failure or a pregnancy loss.

Study design, size, duration
Endometrial 'instant' assembloids, consisting of gland organoids and primary stromal cells in collagen hydrogels, were established from freshly isolated cells from endometrial biopsies. Additional cell types were incorporated including immune and endothelial cells creating a layer of epithelial cells recapitulating the luminal epithelium. Hormonal stimulated assembloids were further co-cultured with human blastocysts at 37 °C, 5% O2 and 6% CO2. Attachment of the blastocysts to the luminal epithelium was assessed upon overnight co-culture.

Participants/materials, setting, methods
Endometrial biopsies (n = 3) were obtained from consenting hormonally stimulated, healthy oocyte donors. After informed consent, human embryos (n = 14) were donated to research after 5 years of cryopreservation. Decidualisation was monitored by RT-qPCR analysis using epithelial and stromal cells from undifferentiated or decidualised assembloids. Attachment of the embryos (n = 11) to the model was assessed by disturbance of the liquid and this was further confirmed by immunofluorescence antibody labelling.

Main results and the role of chance
'Instant' assembloids mimic the endometrium morphologically and functionally since hormonal stimulation results in the induction of decidual stromal (PRL, SCARA5 and DIO2) and epithelial (PAEP and SPP1) marker genes. The presence of immune and endothelial cells was confirmed with the induction of IL2RB and VWF respectively. Immunofluorescent antibody labelling confirmed the similarity in morphology of the model to the human endometrium. Aiming to mimic implantation, assembloids underwent a 3-day long differentiation in a chemically defined medium, 8-bromo-cAMP, estradiol, and a progestin, that induced in vitro decidualisation. Following the 3-day treatment, instant assembloids were overlaid with single epithelial cells, isolated from instant assembloids which had undergone prolonged decidualisation treatment to imitate the physiology of the endometrium. Following a 24-hour long incubation period, human hatched day 6 blastocysts were positioned on the top epithelial layer of the instant assembloid. Immunofluorescent antibody labelling using makers for the epiblast (NANOG), primitive endoderm (GATA4) and epithelial cells (E-cadherin) were used to visualise the co-culture. Our findings signify the newly established model as the most advanced method to study embryo implantation in vitro. The uniqueness of the model is owned by the preservation of endometrial cell types and the potential to model disease through patient specificity.

Limitations, reasons for caution
The proposed system is an innovative method to recapitulate apposition and adhesion during implantation. Our results are preliminary and further experiments will be performed to increase our repeats and therefore reliability. Moreover, due to the complex nature of the co-culture system, imaging remains a challenge that requires further optimisation.

Wider implications of the findings
The embryo-assembloid co-culture system may provide a useful tool to aid our understanding of the mechanisms of implantation. Future work will include the labelling of the embryos with a nuclear dye to enable tracking using time-lapse microscopy. We further aim to expand our protocol to study the process of invasion.

Trial registration number
N/A