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In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell differentiation in restoration of an intact epithelial cell layer. These insights may inform development of new therapies to induce rapid healing in the endometrium and other tissues and offer hope to women who suffer from heavy menstrual bleeding.

The human endometrium undergoes approximately 450 cycles of proliferation, differentiation, shedding and regeneration over a woman’s reproductive lifetime. The regenerative capacity of the endometrium is attributed to stem/progenitor cells residing in the basalis layer of the tissue. Mesenchymal stem cells have been extensively studied in the endometrium, whereas endometrial epithelial stem/progenitor cells have remained more elusive. This review details the discovery of human and mouse endometrial epithelial stem/progenitor cells. It highlights recent significant developments identifying putative markers of these epithelial stem/progenitor cells that reveal their in vivo identity, location in both human and mouse endometrium, raising common but also different viewpoints. The review also outlines the techniques used to identify epithelial stem/progenitor cells, specifically in vitro functional assays and in vivo lineage tracing. We will also discuss their known interactions and hierarchy and known roles in endometrial dynamics across the menstrual or estrous cycle including re-epithelialization at menses and regeneration of the tissue during the proliferative phase. We also detail their potential role in endometrial proliferative disorders such as endometriosis.

Transvaginal meshes for the treatment of Pelvic Organ Prolapse (POP) have been associated with severe adverse events and have been banned for clinical use in many countries. We recently reported the design of degradable poly L-lactic acid-co-poly ε-caprolactone nanofibrous mesh (P nanomesh) bioengineered with endometrial mesenchymal stem/stromal cells (eMSC) for POP repair. We showed that such bioengineered meshes had high tissue integration as well as immunomodulatory effects . This study aimed to determine the key molecular players enabling eMSC-based foreign body response modulation. SUSD2 eMSC were purified from single cell suspensions obtained from endometrial biopsies from cycling women by magnetic bead sorting. Electrospun P nanomeshes with and without eMSC were implanted in a NSG mouse skin wound repair model for 1 and 6 weeks. Quantitative PCR was used to assess the expression of extracellular matrix (ECM), cell adhesion, angiogenesis and inflammation genes as log fold changes compared to sham controls. Histology and immunostaining were used to visualize the ECM, blood vessels, and multinucleated foreign body giant cells around implants. Bioengineered P nanomesh/eMSC constructs explanted after 6 weeks showed significant increase in 35 genes associated with ECM, ECM regulation, cell adhesion angiogenesis, and immune response in comparison to P nanomesh alone. In the absence of eMSC, acute inflammatory genes were significantly elevated at 1 week. However, in the presence of eMSC, there was an increased expression of anti-inflammatory genes including and by 6 weeks. There was formation of multinucleated foreign body giant cells around both implants at 6 weeks that expressed CD206, a M2 macrophage marker. This study reveals that eMSC modulate the foreign body response to degradable P nanomeshes by altering the expression profile of mouse genes. eMSC reduce acute inflammatory and increase ECM synthesis, angiogenesis and anti-inflammatory gene expression at 6 weeks while forming newly synthesized collagen within the nanomeshes and neo-vasculature in close proximity. From a tissue engineering perspective, this is a hallmark of a highly successful implant, suggesting significant potential as alternative surgical constructs for the treatment of POP.

The endometrium is a complex, steroid-dependent tissue that undergoes dynamic cyclical remodelling. Transformation of stromal fibroblasts (ESC) into specialised secretory cells (decidualization) is fundamental to the establishment of a receptive endometrial microenvironment which can support and maintain pregnancy. Androgen receptors (AR) are present in ESC; in other tissues local metabolism of ovarian and adrenal-derived androgens regulate AR-dependent gene expression. We hypothesised that altered expression/activity of androgen biosynthetic enzymes would regulate tissue availability of bioactive androgens and the process of decidualization. Primary human ESC were treated in vitro for 1–8 days with progesterone and cAMP (decidualized) in the presence or absence of the AR antagonist flutamide. Time and treatment-dependent changes in genes essential for a) intra-tissue biosynthesis of androgens (5α-reductase/SRD5A1, aldo-keto reductase family 1 member C3/AKR1C3), b) establishment of endometrial decidualization (IGFBP1, prolactin) and c) endometrial receptivity (SPP1, MAOA, EDNRB) were measured. Decidualization of ESC resulted in significant time-dependent changes in expression of AKR1C3 and SRD5A1 and secretion of T/DHT. Addition of flutamide significantly reduced secretion of IGFBP1 and prolactin and altered the expression of endometrial receptivity markers. Intracrine biosynthesis of endometrial androgens during decidualization may play a key role in endometrial receptivity and offer a novel target for fertility treatment.

In women, endometrial breakdown, which is experienced as menstruation, is characterised by high concentrations of inflammatory mediators and immune cells which account for ~40% of the stromal compartment during tissue shedding. These inflammatory cells are known to play a pivotal role in tissue breakdown but their contribution to the rapid scarless repair of endometrium remains poorly understood. In the current study we used a mouse model of menstruation to investigate dynamic changes in mononuclear phagocytes during endometrial repair and remodelling. Menstruation was simulated in MacGreen mice to allow visualisation of CSF1R + mononuclear phagocytes. Immunohistochemistry revealed dynamic spatio-temporal changes in numbers and location of CSF1R-EGFP + cells and Ly6G + neutrophils. Flow cytometry confirmed a striking increase in numbers of GFP + cells during repair (24 h): influxed cells were 66% F4/80 + Gr-1 + and 30% F4/80 − Gr-1 + . Immunostaining identified distinct populations of putative ‘classical’ monocytes (GFP + F4/80 − ), monocyte-derived macrophages (GFP + F4/80 + ) and a stable population of putative tissue-resident macrophages (GFP - F4/80 + ) localised to areas of breakdown, repair and remodelling respectively. Collectively, these data provide the first compelling evidence to support a role for different populations of monocytes/macrophages in endometrial repair and provide the platform for future studies on the role of these cells in scarless healing.

To develop a patient derived xenograft (PDX) model of cervical cancer and cervical dysplasia using the subrenal capsule. Cervical cancer (12 Squamous Cell Carcinoma, 1 Adenocarcinoma, 1 Adenosquamous Carcinoma), 7 cervical dysplasia biopsy and normal cervical tissues were transplanted beneath the renal capsule of immunocompromised NOD/SCID/gamma mice. Resulting tumours were harvested and portions serially transplanted into new recipient mice for up to three in vivo passages. Parent and xenograft tumours were examined by immunohistochemistry for p16INK41, HPV, and CD-45. Single cell suspensions of mixed mouse and human, or human only cell populations were also transplanted. The overall engraftment rate for the primary cervical cancer PDX model was 71.4 ±12.5% (n = 14). Tumours maintained morphological, histoarchitecture and immunohistochemical features of the parent tumour, and demonstrated invasiveness into local tissues. Single cell suspensions did not produce tumour growth in this model. Mean length of time (32.4 +/- 3.5 weeks) for the transplanted tissue to generate a tumour in the animal was similar between successive transplantations. Three of four xenografted cervical dysplasia tissues generated microscopic cystic structures resembling dysplastic cervical tissue. Normal cervical tissue (4 of 5 xenografted) also developed microscopic cervical tissue grafts. The subrenal capsule can be used for a PDX model of human cervical cancer with a good engraftment rate and the ability to model in vivo characteristics of cervical cancer. For the first time we have demonstrated that cervical dysplasia and normal cervical tissue generated microscopic tissues in a PDX model.

Mesenchymal stem cells (MSCs) that meet the International Society for Cellular Therapy (ISCT) criteria are obtained from placental tissue by plastic adherence. Historically, no known single marker was available for isolating placental MSCs (pMSCs) from the decidua basalis. As the decidua basalis is derived from the regenerative endometrium, we hypothesised that SUSD2, an endometrial perivascular MSC marker, would purify maternal perivascular pMSC. Perivascular pMSCs were isolated from the maternal placenta using SUSD2 magnetic bead sorting and assessed for the colony-forming unit-fibroblasts (CFU-F), surface markers, and in vitro differentiation into mesodermal lineages. Multi-colour immunofluorescence was used to colocalise SUSD2 and α-SMA, a perivascular marker in the decidua basalis. Placental stromal cell suspensions comprised 5.1%SUSD2+ cells. SUSD2 magnetic bead sorting of the placental stromal cells increased their purity approximately two-fold. SUSD2+ pMSCs displayed greater CFU-F activity than SUSD2− stromal fibroblasts (pSFs). However, both SUSD2+ pMSC and SUSD2− pSF underwent mesodermal differentiation in vitro, and both expressed the ISCT surface markers. Higher percentages of cultured SUSD2+ pMSCs expressed the perivascular markers CD146, CD140b, and SUSD2 than SUSD2− pSFs. These findings suggest that SUSD2 is a single marker that enriches maternal pMSCs, suggesting they may originate from eMSC. Placental decidua basalis can be used as an alternative source of MSC for clinical translation in situations where there is no access to endometrial tissue.

Female cancer survivors are significantly more likely to experience infertility than the general population. It is well established that chemotherapy and radiotherapy can damage the ovary and compromise fertility, yet the ability of cancer treatments to induce uterine damage, and the underlying mechanisms, have been understudied. Here, we show that in mice total-body γ-irradiation (TBI) induced extensive DNA damage and apoptosis in uterine cells. We then transferred healthy donor embryos into ovariectomized adolescent female mice that were previously exposed to TBI to study the impacts of radiotherapy on the uterus independent from effects to ovarian endocrine function. Following TBI, embryo attachment and implantation were unaffected, but fetal resorption was evident at midgestation in 100% of dams, suggesting failed placental development. Consistent with this hypothesis, TBI impaired the decidual response in mice and primary human endometrial stromal cells. TBI also caused uterine artery endothelial dysfunction, likely preventing adequate blood vessel remodeling in early pregnancy. Notably, when pro-apoptotic protein Puma-deficient ( Puma –/– ) mice were exposed to TBI, apoptosis within the uterus was prevented, and decidualization, vascular function, and pregnancy were restored, identifying PUMA-mediated apoptosis as a key mechanism. Collectively, these data show that TBI damages the uterus and compromises pregnancy success, suggesting that optimal fertility preservation during radiotherapy may require protection of both the ovaries and uterus. In this regard, inhibition of PUMA may represent a potential fertility preservation strategy.