P-442 Genome edition in endometrial stroma cells leading to decreased expression of UDP-glucuronic acid decarboxylase I (UXS1) alters the molecular interactions of the implantation area
Authors: Söyler G , Yilmaz Duzgun İ , Albayrak O , Sahin G  N , Keskin E , Taskin A  C , Acilan Ayhan C , Oktem O , Gonen M , Ayla S , Karahuseyinoglu S 
Published: July 2024

Abstract

Study question
Does UDP-glucuronic acid decarboxylase 1 (UXS1) expression in endometrial stroma cells have a direct effect on the early stages of implantation?

Summary answer
Decreased expression of UXS1 was found to alter adhesion, invasion, development and function related genes of implantation in 3D cultures in a time dependent manner.

What is known already
The implantation process is governed by a complex interaction of genes and molecular pathways. Some of those genes are well-defined, however, as fertility problems increase with escalating rates of recurrent implantation failure or abortions, with an effort to reveal those genes that may be more significant than known for implantation RNA-seq analyses are used. UDP-glucuronic acid decarboxylase 1 (UXS1) is an enzyme involved in the detoxification and conversion of UDP-glucuronic acid to UDP-xylose. Loss of UXS1 led to defective glycosylation, increased protein degradation, and decreased overall protein levels, but no direct relation to has been stated before.

Study design, size, duration
Two different Matrigel based 3D culture systems were constructed with labeled mouse endometrium epithelial cells, stroma cells and blastocysts for 24-72 hours (n = 24). Sorted cells were sequenced for RNA for determination of differentially expressed genes. Of over 4000 genes, a highly expressed one was UXS1 in the endometrial stroma cells, The gene was knocked out via CRISPR/Cas9. The impact of decline of UXS1 in stroma cells on implantation parameters has been evaluated.

Participants/materials, setting, methods
CB6F1 female mice were superovulated. Blastocysts were obtained, endometrium was used for isolation of epithelial and stroma cells. 3D cultures were constructed for RNA-seq analysis. Three gRNAs were designed for CRISPR/Cas9 experiments. Cultures were constructed with knock out stroma and wild epithelial cells, wild blastocysts. On hours 24 and 72 of culture, gene and protein expression analyses for e-cadherin, MMP9, entactin, LIF; Hoxa10, Wnt4, ESR1, PR, fibronectin have been accomplished. Supported by TUBITAK 118S676.

Main results and the role of chance
All adherence molecules tested (e-cadherin, entactin, fibronectin) have shown significant increase compared to non targeted (the cell has Cas9, but no gRNA) groups (p &lt; 0.00001). As e-cadherin has decreased detrimentally from 24 to 72 hours, fibronectin has continued to increase. LIF, an important gene for embryo-blastocyst interphase, has increased steeply and then decreased in the long term culture (p &lt; 0.00001). ESR1 and PR (estrogen and progesterone receptors) increased compared to non-targeted groups but they were lower than the control groups with no genome edition (p &lt; 0.01). The invasion gene MMP9 has increased steeply in the first 24 hours, then declined suddenly at hour 72 (p &lt; 0.0001). The developmental genes Wnt4 and Hoxa10 were also affected significantly, as Wnt4 has decreased and Hoxa10 has increased compared to non-targeted groups (p &lt; 0.001, p &lt; 0.0001 respectively), however the expressions did not change from 24 to 72 hours. These findings were mostly supported by the protein expressions depicted by IF staining. The data can be interpreted as UXS1 has some fundamental effects during the implantation process as its decreased expression changes the adhesion and invasion patterns with unexpectedly high and low pattern, and it deteriorates differential expression of LIF during the critical interaction of endometrial cells and the blastocyst.

Limitations, reasons for caution
- This is a 3D in vitro implantation model constructed with mouse cells, so it cannot reflect in vivo process perfectly.
- The genome edition of primary cells can show lower yield than cell lines.
- There are numerous parameters of implantation, more tests can always be added.

Wider implications of the findings
- Data arising from various RNAseq analyses for implantation is accumulating and it is possible to find new genes and their connections and contribution can be revealed.
- The new genes tested in vitro can further be re-examined in knock out mouse models or can be scanned in the abortus materials.

Trial registration number
not applicable