How do mutant clones expand in the normal endometrium?

We demonstrate that rhizome structures, which are network-like glandular structures that run horizontally along the muscle layer at the bottom of the endometrium, are involved in the mechanism by which mutant clones expand their territories in the human endometrium.

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The human uterine endometrium is a unique tissue that undergoes cyclic shedding and regeneration every month by menstruation. Menstruation, which is familiar to women, is an extremely rare physiological phenomenon among mammals. Although the uterine endometrium is essential for human reproduction, it is also associated with ‘endometrium-related diseases’, such as endometriosis, adenomyosis, endometrial hyperplasia, and endometrial cancer. These diseases significantly impact the physical, mental, and social health of women throughout their lives. In particular, due to the social advancement of women and lifestyle changes, it is estimated that the number of menstrual cycles in the lifetimes of women in developed countries has increased tenfold compared to 100 years ago1. The increase in the incidence of endometrial-related diseases with increasing menstrual frequency is a serious problem in women's health.

 We previously discovered that several cancer-associated genes, such as KRAS and PIK3CA, were frequently mutated in both endometriotic epithelium and normal uterine endometrial glands2. Remarkably, the high mutant allele frequencies of somatic mutations in each endometrial gland demonstrated the monoclonal composition of each gland. The presence of cancer-associated gene mutations in histologically normal endometrial glands provides important clues regarding the pathogenesis of endometrium-related diseases. Moreover, several studies have recently reported that the burden of cancer-associated gene mutations in normal endometrium accumulates with age3,4. However, the etiology of cancer-associated gene mutation in the uterine endometrium, which is repeatedly shed with menstruation, is not clearly understood. In this study, we investigated the distribution of cancer-associated gene mutations across time and space in normal endometrium using large-scale genome analysis and 3D mapping of mutant clones in the human endometrium.

 First, we performed genomic analysis of 891 normal single endometrial glands that were randomly collected from 32 women aged 20-50 years. More than half of the normal endometrial glands acquired numerous somatic mutations in genes that are frequently mutated in endometrial cancer and endometriosis-associated ovarian cancer. The burden of somatic mutations for each subject over glands not only correlated with age but also correlated more strongly with the cumulative number of menstrual cycles. We also revealed that pan-gynecologic cancer-associated genes5, such as KRAS, PIK3CA, and PTEN and so on, underwent strong positive selection in the normal endometrium. Next, to investigate how endometrial glands with somatic mutations are distributed in the endometrium, we conducted genomic analysis for spatially resolved single endometrial glands, in which surgically resected specimens of the normal endometrium were subdivided into 6.25-25mm2 square grids, and the endometrial glands were collected from the grids. We demonstrated that multiple glands originating from the same ancestral clone formed clusters across adjacent grids and underwent clonal expansion in the endometrium. Furthermore, we estimated the chronological ages at which genomic events occurred in the endometrium and found that clonal expansions of cancer-associated gene mutations and copy neutral loss-of-heterozygosity events occurred early in life. In particular, bi-allelic PTEN loss, which has been considered as the important step for endometrial carcinogenesis, was estimated to occur during teenage years in the endometrium of a 50-year-old woman. The results show that such genomic events were tolerated for decades in the normal endometrium. Our findings suggest that cancer-associated gene mutations are not sufficient to trigger tumorigenesis immediately but might enhance cellular proliferation and benefit endometrial regeneration after menstruation.

 However, how do the mutant endometrial glands expand their clusters within the endometrium during the menstrual cycle? The key to solving this mystery is the 3D structure of the endometrial glands. It has been widely assumed that the bottom of human endometrial glands forms a crypt based on previous 2D histological studies6,7. However, our previous 3D imaging study revealed that the “rhizome structure”, which is a network-like glandular structure that runs horizontally along the muscle layer at the bottom of the endometrium, is generated in the human endometrium8., Although the vertical glands which grow from the rhizome are shed during menstruation, the rhizome structure remains at the stratum basalis. We hypothesized that the rhizome structure might be monoclonal in origin. To demonstrate this hypothesis, we integrated genomic sequencing and 3D imaging techniques. To analyze the genomes of rhizome structures and the vertical glands derived from them, we examined 70 serial cryosections to identify 3D structures of the endometrial glands. We also collected three sets of rhizome with vertical glands growing from the rhizome separately by laser-capture microdissection. The results of whole-genome sequencing showed that all endometrial glands sharing the same rhizome originated from the same ancestral clone. Moreover, mutant clones detected in the vertical glands diversified by acquiring additional mutations. These results suggest that clonal expansions through the rhizome structures are involved in the mechanism by which mutant clones extend their territories.

 Here, we propose a new model for clonal expansion in the normal endometrium. The presence of rhizome structures might be beneficial for postmenstrual endometrial repair by protecting endometrial stem/progenitor cells from shedding at the menstrual phase8,9. During menstruation, residual basal glands extend horizontally along the muscular layer to shape monoclonal rhizomes. Then, the rhizome gives rise to vertical glands with the same clonal origin. Some rhizome structures persist for many cycles of repair and regeneration during menstrual cycles and further expand their territories. We presume that clones with cancer-associated gene mutations may confer a proliferative advantage and contribute to stable tissue regeneration by expanding the area of rhizome structure. However, rhizome structures might confer deleterious effects that predispose women to endometrium-related diseases by accumulating cancer-associated gene mutations. The rhizome structures might act as a double-edged sword for women’s health.

 Our results of the evolutionary dynamics of mutant clones in the human endometrium will provide a molecular basis for better understanding the mechanisms of endometrial regeneration during the menstrual cycle and the pathogenesis of all diseases involving the endometrium. This ‘new normal’ knowledge will lead to the development of therapies to prevent and treat endometrium-related diseases. 

Left panel: Schematic diagram of clusters of endometrial glands in the normal uterine endometrium. The colors of the clusters indicate clones derived from different ancestral origins.

Right panel: Schematic diagram of histological morphology of endometrium. The horizontally expanding plexus morphology of the basal glands in the stratum basalis is referred to as the rhizome structure. All endometrial glands sharing the same rhizome are originated from the same ancestral clone. Thus, the rhizome structure is involved in clonal expansions of cancer-associated mutations in the human endometrium.

  1. Critchley, H.O.D. et al. Menstruation: science and society. Am J Obstet Gynecol 223, 624-664 (2020).
  2. Suda, K. et al. Clonal Expansion and Diversification of Cancer-Associated Mutations in Endometriosis and Normal Endometrium. Cell Rep 24, 1777-1789 (2018).
  3. Moore, L. et al. The mutational landscape of normal human endometrial epithelium. Nature 580, 640-646 (2020).
  4. Lac, V. et al. Oncogenic mutations in histologically normal endometrium: the new normal? J Pathol 249, 173-181 (2019).
  5. Berger, A.C. et al. A Comprehensive Pan-Cancer Molecular Study of Gynecologic and Breast Cancers. Cancer Cell 33, 690-705.e9 (2018).
  6. Cooke, P.S., Spencer, T.E., Bartol, F.F. & Hayashi, K. Uterine glands: development, function and experimental model systems. Mol Hum Reprod 19, 547-58 (2013).
  7. Gray, C.A. et al. Developmental biology of uterine glands. Biol Reprod 65, 1311-23 (2001).
  8. Yamaguchi, M. et al. Three-dimensional understanding of the morphological complexity of the human uterine endometrium. iScience 24, 102258 (2021).
  9. Tempest, N. et al. Histological 3D reconstruction and in vivo lineage tracing of the human endometrium. J Pathol 251, 440-451 (2020).

MANAKO YAMAGUCHI

Obstetrics and Gynecology, Niigata University Graduate School of Medical and Dental Sciences