P–369 Three-dimensional co-culture of human endometrial cells with aneuploid and euploid embryos
Authors: Amiri S , Amjadi F , Ashrafi M , Aflatoonian R , Akbar. Sene A , Mehdizadeh M , Zandieh Z 
Published: August 2021

Abstract

Study question
Is implantation different in euploid and aneuploid embryos?

Summary answer
By simulating the human endometrium using a three-dimensional scaffold, aneuploid embryos were unable to attach to the endometrial cells, while euploid embryos attached.

What is known already
Although embryo selection for transfer is usually based on morphology, 70% of embryos with high morphological quality have chromosomal abnormalities. The results of implantation and pregnancy rate assessments following Preimplantation Genetic Screening (PGS) are controversial. There is still no in vitro study to compare the implantation of human euploid and aneuploid embryos.

Study design, size, duration
After informed consent, 10 endometrial biopsies were taken from fertile women. For scaffolding, the stromal cells were resided within the matrix, after 24 hours, the epithelial cells were seeded on the scaffold. Cell culture continued for 5 days to reach the appropriate confluence. The embryos were also examined by performing PGS following CGH Array. 10 euploid and 10 aneuploid blastocysts were selected and co-cultured for 72 hours with the 3D structure of human endometrial cells.

Participants/materials, setting, methods
Endometrial cells were isolated and expanded in 2D cultures to achieve enough cells. The fibrin-agarose scaffold was made and stromal and epithelial cells were cultured into and on the scaffold, respectively. Then, cell proliferation was assessed by MTT assay. The simulated endometrial construct was confirmed by H&E and IHC. Partial hatching of blastocysts was performed using a laser system. The blastocyst’s attachment to the endometrial-like structure was examined under a phase-contrast microscope and SEM.

Main results and the role of chance
The MTT OD of scaffolds increased during 5 days of cell culture (P &lt; 0.05). The histological evaluation of the co-culture systems was done under light microscopy by H&E staining. On the top of the 3D culture system, epithelial cells shaped a constricted cell monolayer. Stromal cells combined with the fibrin-agarose scaffold got lengthened and expanded, displaying that the 3D culture systems supplied a suitable environment for the growth of endometrial cells.
In the 3D culture, the origins and locations of epithelial and stromal cells were defined by cytokeratin and vimentin immunostaining, respectively. IHC for cytokeratin was only positive for epithelial cells in the surface epithelium. IHC for the vimentin was positive for the stromal cells located in the 3D matrix. These results showed that fibrin-agarose scaffold could simulate the human endometrial structure.
Using SEM and phase-contrast microscopy, it was found that only euploid embryos were able to attach to the endometrial construct while aneuploid embryos weren’t.

Limitations, reasons for caution
Since the co-culture does not contain a unique cell type, and the MTT OD standard curve against cell number is specified for cell type, the number of growing cells in the co-culture cannot be calculated; therefore it is reported as OD.
Wider implications of the findings: Our findings determined that PGS allows us to transfer top-quality embryos with higher implantation potential. It improves implantation and pregnancy rate during ART cycles, especially in patients with recurrent implantation failure.

Trial registration number
Not applicable