Effect of aging on the human myometrium at single-cell resolution

Age-associated myometrial dysfunction can prompt complications during pregnancy and labor, which is one of the factors contributing to the 7.8-fold increase in maternal mortality in women over 40. Using single-cell/single-nucleus RNA sequencing and spatial transcriptomics, we have constructed a cellular atlas of the aging myometrium from 186,120 cells across twenty perimenopausal and postmenopausal women. We identify 23 myometrial cell subpopulations, including contractile and venous capillary cells as well as immune-modulated fibroblasts. Myometrial aging leads to fewer contractile capillary cells, a reduced level of ion channel expression in smooth muscle cells, and impaired gene expression in endothelial, smooth muscle, fibroblast, perivascular, and immune cells. We observe altered myometrial cell-to-cell communication as an aging hallmark, which associated with the loss of 25 signaling pathways, including those related to angiogenesis, tissue repair, contractility, immunity, and nervous system regulation. These insights may contribute to a better understanding of the complications faced by older individuals during pregnancy and labor.


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Replication Randomization Blinding Our research employs the term "women" to refer to individuals with a uterus that undergo menopause while acknowledging that not all individuals identifying as women have a uterus and/or experience menopause, and not all individuals undergoing menopause identify as women.

Not applicable
This prospective, multicenter, descriptive case series included twenty females, both living and brain death donors , with ages ranging from 46 to 79 years old.
There was no self-selection bias in this study.Samples are equal or above 46 years old due to the occurence of menopause itself, as mentioned in the introduction ("Menopause, which typically occurs between 45 and 55").
The list of samples recruited per hospital is also detailed in the methods section, as well as the clinical variables used to exclude patients from the study: -The Hospital La Fe provided fifteen uteri from women undergoing hysterectomy for pelvic prolapse, while a total of five uteri (two from Hospital Clinico Universitario and three from Hospital General) were obtained from patients with brain death under the Organ Donor Program with non-cancer-related causes or traumatic injury.These five uteri were acquired through the surgical extraction of the entire uterus after meeting the criteria for brain death, following the protocol approved by the ethics committee.
-Patients were not considered for inclusion in the study if they had previously been diagnosed with malignancies related or unrelated to the uterus, and if such malignancies were also identified during the surgical procedure.
All patients and donor families provided written informed consent, and those with gynecological disorders such as endometriosis, uterine malformations, uterine leiomyoma, endometrial polyp, hyperplasia, uterine septum, Asherman's syndrome or hydrosalpinx, were excluded from the study.Patients were not considered for inclusion in the study if they had previously been diagnosed with malignancies related or unrelated to the uterus, and if such malignancies were also identified during the surgical procedure.To mitigate the potential effects of previous infections, inclusion in the study also necessitated negative serological tests for HIV, HBV, HCV, and RPR.
We have biological replicates for all the procedures shown through the manuscript.
All samples within the same menopausal group (peri or postmenopause) were considered biological replicates.Thus, for the single-nuclei/ single-cell experiments we had 6 biological replicates of perimenopause and 14 biological replicates of postmenopause.For spatial transcriptomics analysis we had 3 biological replicates of perimenopause and 5 biological replicates of postmenopause.
All experiments included in the study were succesful and no technical replicates were performed in this study.
Patients were divided into two groups: the perimenopausal group (n=6; age 46-54) and the postmenopausal (n= 15; age >54).The main clinical variables (site of collection, vital status, parity or live births and c-section) that might affect the expression patterns in any cell population were also considered for further analysis.
Blinding was not relevant in our case, since this is a descriptive study.

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