Human receptive endometrial organoid for deciphering the implantation window

Reviewer #1 (Public Review):

Summary:

This study generated 3D cell constructs from endometrial cell mixtures that were seeded in the Matrigel scaffold. The cell assemblies were treated with hormones to induce a "window of implantation" (WOI) state. Although many bioinformatic analyses point in this direction, there are major concerns that must be addressed.

Strengths:

The addition of 3 hormones to enhance the WOI state (although not clearly supported in comparison to the secretory state).

Weaknesses:

First of all, the term organoid must be discarded. The authors just seed the endometrial cell mixture which assembles and aggregates into a 3D structure which is then immediately used for analysis. Organoids grow from tissue stem cells and must be passage-able (see their own description in lines 69-71). So, the term organoid must be removed everywhere, to not confuse the organoid field. It is not shown that the whole 3D assembly is passageable, which would be very surprising given the fact that immune and stromal cells do not grow in Matrigel because of the unfavorable growing conditions (which are targeted to epithelial cell growth).

Second, the study remains fully descriptive, bombing the reader with a mass of bioinformatic analyses without clear descriptions and take-home messages. The paper is very dense, meaning readers may give up. Moreover, functional validation, except for morphological and immunostaining analyses (which are posed as "functional" but actually are only again expression) is missing, such as in vivo functionality (after transplantation e.g.) and embryo interaction. Importantly, the 3D structure misses the right architecture with a lining luminal epithelium which is present in the receptive endometrium in vivo and needed as the first contact site with the embryo. So, in contrast to what the authors claim, this is not the best model to study embryo interaction, or the closest model to the in vivo state (line 318, line 326).

Third, receptive endometrial organoids (assembloids; Rawlings et al., eLife 2021) and receptive organoid-derived "open-faced endometrial layer" (Kagawa et al., Nature 2022) have already been described, which is in contrast to what the authors claim in several places that "they are the first" (e.g. lines 87-88, 316-319, etc). These studies used real organoids to achieve their model (and even showed embryo interaction), while in the present study, different cell types are just seeded and assembled. Hence, logically, immune cells are present which are never found in real organoid models. The only original aspect in the present study is the use of hormones to enhance the WOI phenotype. However, crucial information on this original aspect is missing such as concentration of the hormones, refreshment schedule, all 3 hormones added together or separately, and all 3 required?

Moreover, it is not a "robust" model at all as the authors claim, given the variability of the initial cell mixture (varying from patient to patient). Actually, the reproducibility is not shown. The proportions of the different cell types seeded in the Matrigel droplet will be different with every endometrial biopsy. It would be much better to recombine epithelial (passageable) organoids with stromal and immune cells in a quantified, standardized manner to establish a "robust" model.

Reviewer #2 (Public Review):

A wide variety of assays are used to describe the new culture system and compare it both with those previously described and with the endometrial tissue itself. The three different cultures they used are control organoids (CTRL) cultured with described expansion media, secretory organoids (SEC, cultured with E2, MPA and cAMP inducing secretory phase as previously reported) and WOI organoids (cultured with E2, MPA, cAMP, prolactin (PRL), human chorionic gonadotropin (hCG) and human placental lactogen (hPL)). First, they performed morphological characterization of cultures using different antibodies, showing the presence of epithelial glandular cells and stromal cells, as well as their proliferation and absence of apoptosis. Glycogen secretion and progesterone receptor expression complete organoid characterization at the functional and hormone response levels respectively.

Then, they performed single-cell transcriptomics to analyse its composition in terms of cell type, comparing with different databases, but with an unknown "n". They detect stromal, epithelial, and immune cells (also by microscopy), and analyse gene expression and transcription regulation, showing similarities between WOI organoids and mid-secretory endometrium. With endometrial receptivity analysis, they suggest a successful formation of the implantation window in vitro, but this result is difficult to interpret.

Analyzing transcriptome and proteome information of WOI organoids, authors demonstrate a strong response to estrogen and progesterone, but some comparisons are made with CTRL and SEC, and others only with CTRL, which limits the power of some results. In the same way, some genes related to Cilia and pinopodes appear dominant in WOI organoids, but the comparison by electron microscopy is made only against CTRL organoids.

In subsequent analysis, WOI organoids showed a marked differentiation from proliferative to secretory epithelium, and from proliferative epithelium to EMT-derived stromal cells than SEC organoids. These statements are based on their upregulation of monocarboxylic acid and lipid metabolism, their enhanced peptide metabolism and mitochondrial energy metabolism, or their pseudotime trajectories. However, other analyses (such as the accumulation of secretory epithelium or decreased proliferative epithelium, the increased ciliated epithelium after hormonal treatment, or the presence of EMT-derived stromal cells) show only small differences between SEC and WOI organoids.

In summary, the development of an endometrial organoid culture methodology that allows targeting the endometrial situation in the window of implantation could change the experimental approaches of many studies, but more evidence is needed, and above all, more approaches on how different WOI organoids are from SEC organoids, to be sure if it is worth using them in implantation.