O-097 Implantation-on-chip: precise quantification for functional implantation failure studies
Authors: Ak A , Luijkx D , Carvalho D , Romano A , Stevens-Brentjens L , Voncken W , Giselbrecht S , Van Golde R , Vrij E 
Published: July 2024

Abstract

Study question
How can we obtain precise quantitative information about what factors causally affect the first steps of embryo implantation and Repeated Implantation Failure (RIF)?

Summary answer
We have developed a personalized implantation-on-chip model, based on novel in vitro models of the endometrium and the embryo (i.e., blastoids), to quantify functional attachment.

What is known already
RIF is a multifactorial condition characterized by the persistent inability to achieve clinical pregnancy, despite the transfer of high-quality embryos. Typically diagnosed within the context of assisted reproductive technology (ART) treatment, RIF arises due to disruptions in the endometrium receptivity, the interaction between the endometrium and early embryo, or both. The endometrial factors of RIF can be linked to disrupted hormone signaling and timing, impacting endometrial receptivity and therefore the chance of conceiving. Synchronization between the blastocyst and receptive endometrium is critical for successful implantation. Ethical and technical limitations of in vivo patient research restricts our understanding of implantation failure.

Study design, size, duration
Organoids were transformed into endometrial monolayers within custom-made microfluidic chips. These monolayers on chip were either treated with EPCX; β-estradiol (E2), progesterone (P4), 8-Br-cAMP (C) and XAV939 (X) (referred as induced), or not treated (referred as control) for 6 days. Large number of blastoids (&gt;100) were then infused per chip followed by 48 hours of culture. Then, the rate of adhered blastoids was measured under exposure to controlled flow rates (50, 100 and 400 µl/min).

Participants/materials, setting, methods
Human naïve induced pluripotent stem cells were used to form blastoids, stem cell-based models of the blastocyst, to alleviate the ethical concerns and limited access of blastocysts. Blastoids present the three blastocyst lineages required for development and offer limitless production in number. Endometrium organoids, derived from parous fertile patients (N = 3), was used to recapitulate molecular and functional receptivity by RT-qPCR-based gene expression measurements and microfluidic flow-dependent adhesion rates of blastoids, respectively.

Main results and the role of chance
2-D monolayers that maintained their epithelial identity and responsiveness to hormones as measured by expression of receptivity-related genes (PAEP, SPP1 and GPX3), which were upregulated in EPCX condition. Further characterization of the epithelial monolayers showed reduced MUC1 expression in the EPCX condition, suggesting increased receptivity. Additionally, reduced acetylated α-tubulin, indicating a reduction in the number of ciliated cells, aligning with our observations. Through controlled exposure to increasing flow, functional receptivity through blastoid adhesion was precisely quantified, which showed 35% of the blastoid remained adhered in the EPCX condition, whereas only 15% remained adhered in the control. A 48 hours culture period presented a blastoid-induced endometrial gap in 45.54% of the remaining blastoids, with an average of 110.287,3 µm2 in surface area, which was associated with trophoblast cells infiltration, measuring an average migration distance of 174.8 µm from the pluripotent cell core. In addition, the substantial difference in receptivity profiles established a baseline for subsequent experiments, including the study of temporal alterations in hormone signaling, a potential problem associated with RIF. Hormone administration variations, timing and a progesterone-deficient condition are currently being tested. These results will underscore the sensitivity of our system and demonstrate its potential for applications in drug screening.

Limitations, reasons for caution
Currently our endometrium model consists of epithelial cells, which are only representative for implantation to a limited extent. A more complex endometrium including stromal, endothelial and immune cells could highly increase the physiological relevance of the model.

Wider implications of the findings
This platform has the potential to provide a diagnostic platform to identify patients with endometrium epithelium-associated RIF and factors that may improve embryo receptivity and implantation. Moreover, this approach has the potential to be pivotal for toxicological studies, and furthering our understanding of human reproductive biology.

Trial registration number
not applicable