Single-cell characterization of menstrual fluid at homeostasis and in endometriosis

Reviewer #1 (Public review):

Summary:

The characteristics of endometrium health are an increasing topic in women's health issues, especially in the context of endometriosis. In this respect, having access to information is hampered by the inaccessibility of the uterine tissue. The authors propose here using the menstrual fluid (easily accessible by non-invasive methods) as an access door towards getting relevant information.

Overall, the paper is divided into two parts:
(1) The comparison between menstrual fluid samples and biopsies of the endometrium.

1. As a proof of concept, the authors then compared 11 controls and 7 endometriosis cases in this way, from different severity stages.

Strengths:

In Figure 1, general features of the 15 samples are presented (volume/number of cells/hematopoietic cells - cd45 labeling). The authors then used single-cell RNA-seq to characterize the different samples. Through having access to endometrium biopsies, they were able to compare the profiles obtained.

In the MF samples from the second part of the paper - aiming at comparing endometriosis and controls - one question is raised about the effect of culture. The authors compared freshly isolated and cultured tissues (ex vivo vs in vitro) by bulk RNA seq. Biases induced by the culture procedure were identified. Deconvolution was applied to strengthen this observation, with an important increase of seemingly stromal and unknown cells, especially in the unsorted cells and the CD45+ cells.

Interestingly, since the authors got successive samples from the same donor, they could evaluate the consistency of the samples and reveal indeed an overall stability of the molecular profile of the samples in a given patient.

The authors then attempted - quite originally - to characterize biomarkers in two major cell compartments that they studied - CD45- (stromal-like) and CD45+ (immune cells).

Weaknesses:

A potential problem is the justification of the a priori mix of cell types of three different phenotypes (CD45+, CD45- EPCAM+, and CD45- EPCAM-) from each patient before moving to the scRNAseq. It is not clear to me why this has been done, I guess that using directly the samples would supposedly bias the result. But in this case, why is it supposed that three categories are enough (immune cells, epithelial cells, and stromal cells)? I suppose that other markers could characterize other subtypes of the cells, and take into account the possibility of other cell types, for instance, connected to pain sensitivity, such as neuron precursor. Hence, the justification of the organized mixes should be much more detailed in my opinion.

It is a bit unclear to me when the biopsies were collected in the cycle of the donor patients.

The description of these markers that are deregulated is presented as a list, and connected with existing publications, which could rather be presented in discussion than in the results. The authors do tend to demonstrate that the Menstrual Fluid is a good proxy to analyse the endometrium health status of the women affected with endometriosis.

The identification of MTRNR2L1 seems to be a major discovery of the paper, as well as in a lesser measure HBG2, and it is a bit strange why these putative markers were not emphasized in the abstract. HBG2 was certainly identified previously in endometriosis endothelial cells but seems extremely variable from one sample to another - Geo profile (GDS3060, GDS3060 / 213515_x_at (inist.fr)).

Overall, the transcriptome analysis is a bit shallow, with no effort made to try to find potential transcription factors or miRNA that could activate/inhibit a series of modified genes; it could be relevant to identify such master genes or master regulators through bioinformatics analyses and wet-lab validations, to understand better the cascade of events.

Another issue that was overlooked is the presence of 'stem-cells' in the MF obtained. Since endometriosis is supposed to occur from the implantation of uterine stem cells, this category could be a major topic of scrutiny, in terms of quantity in the MF, as well as in terms of their specific molecular properties.

Reviewer #2 (Public review):

Summary:

The authors provided further evidence that menstrual fluid (MF) can be used as a non-invasive source of endometrial tissue for studying its normal physiological state and when it is abnormal such as in endometriosis. Single-cell RNA sequencing confirmed the presence of the major cell types -blood and tissue immune cells and endometrial stromal, epithelial, and vascular cells. The major new finding was that interindividual variation for the blood immune cells was minimal between multiple MF samples from an individual. A comparison between the ex vivo MF gene profile and cultured MF showed the expected attachment and culture of stromal (and a small number of epithelial) cells, but the immune cells failed to attach. Several differentially expressed genes between controls and endometriosis were suggested as potential biomarkers of the disease, however, these were a mitochondrial pseudogene and a hemoglobin subunit, both very unlikely related to endometriosis pathogenesis.

Strengths:

The Spearman correlation analysis between the control MF gene profiles of multiple samples from the same individual and its graphic presentation provided strong evidence that there is little variation between MF samples. Together with another study which showed similar findings for endometrial stem cells and a number of proteins in MF supernatant, this important data shows MF as a promising biofluid for pathology testing.

The bioinformatic analyses conducted by bioinformatic and computational experts are a major strength of the manuscript and in particular the comparison between MF and endometrial biopsy data obtained from published scRNAseq studies. This is an important finding, particularly if comparisons included late secretory and early proliferative stage biopsy tissue which would be most similar to shedding menstrual endometrium.

The inclusion of workflows in the Figures for the various studies and the use of symbols in the various panels is very helpful for the reader.

MF cell suspensions were enriched for stromal and epithelial cells to enable a detailed bioinformatic analysis of their respective gene profiles

Weaknesses:

Two patient cohorts from different institutions were used in the study and somewhat different methods were used to extract the cellular fraction from these cohorts for further study: (1) sample dilution and differential filtration to separate blood-derived immune cells from endometrial tissue then dissociated into single cells and separated into CD45+, CD45-EpCAM+ and CD45-EpCAM- cells, and (2) gradient density separation to generate unsorted, CD45+, CD45- and putative mesenchymal stem cells (MSC) CD45-CD105+ which were also cultured. In addition, questions on pelvic pain and proven fertility would have addressed the 2 key symptoms of endometriosis.

The use of CD105 to purify MSC from MF rather than well-characterised markers of clonogenic, self-renewing, and mesodermal differentiating endometrial MSC such as CD146+PDGFRB+ or SUSD2 (both mentioned in references 22 and 23) is a weakness. The ISCT markers are not specific and are also found on stromal fibroblasts of many tissues (Phinney and Sensebe Cytotherapy 2013; Demu et al Acta Haematologica 2016).
The UMAPs generated from the scRNAseq were at low resolution and more individual immune and endometrial cell types have previously been identified and reported in MF. More comparisons with these studies would also have enhanced the Discussion.

It was not always possible to work out how the data was reported in the gene expression tables (Supplementary Tables 2, 4-10) as they were not in adjusted P value order and sometimes positive log2 fold change values appeared amongst the negative log2FC. In some comparisons described, the adj P values were not significant but were described as up or down-regulated in the text.

The 2 DEGs highlighted in the endometriosis and control arm of the study appear as poor choices from many others that could have been chosen as MTRNR2L1 is a mitochondrial pseudogene and HBG2 is a hemoglobin subunit. Neither are likely indicators of endometriosis pathogenesis.

The manuscript format and organisation could be improved by reducing the discussion in the Results section and providing a more in-depth Discussion. More references need to be included in the Discussion and other work in the MF analysis field that supports - or not - the authors' findings or at least puts them into context, and should be included and referenced.

The potential to use MF as a non-invasive source of endometrial tissue for potential diagnosis is a very important avenue of research that is currently in its infancy and could have a major impact in the endometriosis research arena.