P-426 Comprehensive study of the endometrial early/mid secretory phase during natural cycles reveals conserved molecular dynamics involved in the window of implantation
Authors: Zambelli F , Pietroforte S , Gayete-Mor B , Guillén Quílez J  J , Trullenque M , López M  J , Corral Garrido M , Falco N , Miguel-Escalada I , Moustakas I , Chuva de Sousa Lopes S , Popovic M 
Published: July 2024

Abstract

Study question
Is there a common transcriptomic signature in the endometrial early/mid secretory phase of healthy women in natural cycles?

Summary answer
We observed a conserved transcriptomic regulation of the endometrium around the window of implantation (WOI), consistent across donors, and not influenced by the biopsy procedure.

What is known already
Extensive research has been conducted on the gene expression changes that characterize the WOI, aiming to identify signals that indicate its onset. However, much of this work has utilized whole-transcriptome techniques and has involved IVF patients, who require luteal phase support. The few studies performed in natural cycles often relied on restricted methods, such as qPCR that examines only a few targets simultaneously. As such, an extensive transcriptomics study in volunteer donors in true natural cycles is lacking. This may provide deeper insights into the physiological changes during the WOI in young women driven by the endogenous effects of progesterone.

Study design, size, duration
34 healthy donors were enrolled. Each donor provided one endometrial sample, on a day between LH + 2 and LH + 9 during their natural cycle. A minimum of two and a maximum of five donors were allocated to each day. Ovulation was detected via ecographic inspection and hormonal determination of LH/E2/P4. LH + 0 was established when the LH peak was detected (&gt;2.8 times basal levels), together with a subsequenc decrease in E2 (-30%) and increase in P4 (&gt;1.6ng/ml)

Participants/materials, setting, methods
When the biopsy was of sufficient size, we separated it in two parts and extracted RNA separately to assess concordance between different areas of the same biopsy. RNAseq libraries were prepared with NEBNextUltraII Directional RNA LibraryPrep and sequenced on a NovaSeq6000. Fastq files were aligned with STAR to GRCh38. Principal component analyses (PCA) were created with plotPCA, gene expression time course analysis was performed with DEseq2, and pseudotime analysis was performed using the slingshot package.

Main results and the role of chance
Of the 34 donors, we obtained good quality RNAseq data (minimum 20 million reads) from 41 samples, with 27 donors having one sample sequenced, while 2 independent samples from the same biopsy were obtained from 7 volunteers. Analysing same-biopsy concordance via PCA revealed a great degree of similarity between all the pairs, which clustered close to each other. Samples from different donors collected on the same day also showed a good clustering, apart from a single day LH + 4 sample. Pseudotime analysis revealed a distinct trajectory in transcriptomic profiles changing progressively from day LH + 2 to LH + 9. This analysis clearly delineated three phases: LH + 2 to LH + 5, LH + 6 to LH + 7, and LH + 8 to LH + 9. Accordingly, we categorized these into pre-receptive, receptive and post receptive groups, and conducted a time course experiment. We identified a total of 3,311 genes with varying expression across these three timepoints, primarily associated with progesterone signalling, metabolism and cell-to-cell communication. Based on their expression patterns, we isolated a subset of 538 genes which increased their expression during the receptive phase only. These genes could represent novel markers for a more in-depth characterization of the WOI.

Limitations, reasons for caution
The samples analysed are in bulk, and the genes identified could be expressed either in the stroma or in the epithelial compartment. These findings are not applicable to artificial cycles, as the absence of the corpus luteum and varying levels of exogenous progesterone administered may change the transcriptomic signatures.

Wider implications of the findings
Our study revealed good concordance of whole transcriptome profiles of donor’s endometria according to cycle progression. This may lead to the identification of novel markers involved in the regulation of the WOI that may not have been found in previous studies performed on artificial cycles.

Trial registration number
not applicable