Immunoprofiling of human uterine mast cells identifies three phenotypes and expression of ERβ and glucocorticoid receptor

Background: Human mast cells (MCs) are long-lived tissue-resident immune cells characterised by granules containing the proteases chymase and/or tryptase. Their phenotype is modulated by their tissue microenvironment. The human uterus has an outer muscular layer (the myometrium) surrounding the endometrium, both of which play an important role in supporting a pregnancy. The endometrium is a sex steroid target tissue consisting of epithelial cells (luminal, glandular) surrounded by a multicellular stroma, with the latter containing an extensive vascular compartment as well as fluctuating populations of immune cells that play an important role in regulating tissue function. The role of MCs in the human uterus is poorly understood with little known about their regulation or the impact of steroids on their differentiation status. The current study had two aims: 1) To investigate the spatial and temporal location of uterine MCs and determine their phenotype; 2) To determine whether MCs express receptors for steroids implicated in uterine function, including oestrogen (ERα, ERβ), progesterone (PR) and glucocorticoids (GR). Methods: Tissue samples from women (n=46) were used for RNA extraction (n=26) or fixed (n=20) for immunohistochemistry. Results: Messenger RNAs encoded by TPSAB1 (tryptase) and CMA1 (chymase) were detected in endometrial tissue homogenates. Immunohistochemistry revealed the relative abundance of tryptase MCs was myometrium>basal endometrium>functional endometrium. We show for the first time that uterine MCs are predominantly of the classical MC subtypes: (positive, +; negative, -) tryptase+/chymase- and tryptase+/chymase+, but a third subtype was also identified (tryptase-/chymase+). Tryptase+ MCs were of an ERβ+/ERα-/PR-/GR+ phenotype mirroring other uterine immune cell populations, including natural killer cells. Conclusions: Endometrial tissue resident immune MCs have three protease-specific phenotypes. Expression of both ERβ and GR in MCs mirrors that of other immune cells in the endometrium and suggests that MC function may be altered by the local steroid microenvironment.


Abstract
Human mast cells (MCs) are long-lived tissue-resident immune Background: cells characterised by granules containing the proteases chymase and/or tryptase. Their phenotype is modulated by their tissue microenvironment. The human uterus has an outer muscular layer (the myometrium) surrounding the endometrium, both of which play an important role in supporting a pregnancy. The endometrium is a sex steroid target tissue consisting of epithelial cells (luminal, glandular) surrounded by a multicellular stroma, with the latter containing an extensive vascular compartment as well as fluctuating populations of immune cells that play an important role in regulating tissue function. The role of MCs in the human uterus is poorly understood with little known about their regulation or the impact of steroids on their differentiation status. The current study had two aims: 1) To investigate the spatial and temporal location of uterine MCs and determine their phenotype; 2) To determine whether MCs express receptors for steroids implicated in uterine function, including oestrogen (ERα, ERβ), progesterone (PR) and glucocorticoids (GR).
Tissue samples from women (n=46) were used for RNA extraction Methods: (n=26) or fixed (n=20) for immunohistochemistry.
Endometrial tissue resident immune MCs have three Conclusions: protease-specific phenotypes. Expression of both ERβ and GR in MCs mirrors that of other immune cells in the endometrium and suggests that MC function may be altered by the local steroid microenvironment.

Introduction
Mast cells (MCs) are long-lived tissue resident immune cells, derived from CD34 + /c-kit + pluripotent progenitors, that reside in the bone marrow (Kirshenbaum et al., 1999). MC progenitors are recruited into peripheral tissues by chemokines secreted by stromal cells, which together with stem cell factor, a complex array of cytokines, and a range of micro-environmental factors, are reported to stimulate the development of tissue resident mature MCs (Valent et al., 1992). Mature MCs are usually classified according to the presence of one or more serine proteases (tryptase and/or chymase) in prominent cytoplasmic granules.
MCs are typically phenotyped as MC TC , with granules containing both tryptase (TPSAB1) and chymase (CMA1), or MC T , with granules only containing tryptase alone (Collington et al., 2011;Wernersson & Pejler, 2014). It has been reported that MCs maturing in different tissue microenvironments can vary widely in the amount of tryptase and chymase they contain (Caughey, 2007). When MCs are activated they de-granulate by exocytosis, releasing these serine proteases together with other inflammatory meditators (Lorentz et al., 2012;Tiwari et al., 2008). The female sex hormones, oestradiol and progesterone, are thought to have an impact on MCs because many pathophysiological conditions attributed to MC activity have a higher prevalence in females than males (Narita et al., 2007). Studies in non-reproductive tissue systems and those using the HMC-1 cell line (human MC line, (Butterfield et al., 1988)) have reported that MCs express the oestrogen receptor α isoform (ERα) and progesterone receptor (PR) (Jensen et al., 2010;Nicovani & Rudolph, 2002;Zaitsu et al., 2007). Some authors found evidence that MCs can be rapidly stimulated to degranulate by oestradiol via ERα (Zaitsu et al., 2007). Glucocorticoid treatments are reported to reduce the number of tissue resident MCs by reducing concentrations of stem cell factor that are required for MC survival (Finotto et al., 1997). Glucocorticoids are also reported to prevent MC activation via their high-affinity IgE receptor (Smith et al., 2002).
The human endometrium undergoes physiological cycles of cellular proliferation, differentiation and secretory activity during each menstrual cycle (Johannisson et al., 1987). In the absence of embryo implantation, the upper functional layer of the endometrium breaks down and is shed at menstruation, which is considered to bear the hallmarks of an inflammatory process (Jabbour et al., 2006). Endometrial tissue adjacent to the myometrium (basal compartment) is not shed during menstruation and is implicated in the rapid re-epithelialisation, cessation of bleeding and restoration of tissue homeostasis facilitating regeneration of the functional layer. This monthly tissue remodelling is regulated by changes in cyclical ovarian steroid hormones with oestrogen increasing cell proliferation, progesterone inducing functional maturation of stromal cells in preparation for implantation, and progesterone withdrawal precipitating a cascade of changes culminating in tissue breakdown and menstruation (Kelly et al., 2001;Maybin & Critchley, 2015;Salamonsen & Lathbury, 2000). Endometrial tissue contains stromal, epithelial, and endothelial cells, as well as a diverse population of immune cells, the most abundant of which are uterine natural killer cells (uNK) and macrophages (Evans & Salamonsen, 2012;Thiruchelvam et al., 2013). We, and others, have investigated the impact of steroids on uNK cells and macrophages and shown that they contain both receptors that can bind oestrogens (ERβ isoform) and glucocorticoids (Bombail et al., 2008;Henderson et al., 2003), but are immmuno-negative for ERα and PR. The concentrations of steroids in endometrial tissue are subject to local modulation by enzymes that metabolise sex steroids (androgens, oestrogens), as well as glucocorticoids (Bamberger et al., 2001;Gibson et al., 2013;Gibson et al., 2016;McDonald et al., 2006). The creation of a steroid rich microenvironment has an impact on the function of immune cells and the vasculature. For example, exposure of uNK to oestrogens increases their secretion of CCL2, which has an impact on vascular endothelial cells (Gibson et al., 2015), and likewise exposure of macrophages to cortisol results in the release of factors that induce altered endometrial endothelial cell expression of angiogenic genes (Thiruchelvam et al., 2016).
The basal portion of the endometrium sits directly on the myometrium, which is made up of three layers consisting of smooth muscle fibres and associated vasculature and stroma. The inner layer, adjacent to the endometrium, is also known as the junctional

Amendments from Version 1
• We have referenced the study by Mori et al. (1997) in the Introduction and the study by Engemise et al. (2011) in the Discussion.
• We have changed the text of the Introduction so that the abbreviations for TPSAB1 and CMA1 are introduced during the first mention of their importance in defining the mast cell phenotype.
• The text describing the samples has been changed to clarify that although 46 patients were recruited during this study their samples were split between RNA (n=20) and fixation for immunostaining (n=26).
• Although the delta delta CT method is one of the most popular it is based on comparable amplification efficiencies of the endogenous control and gene of interest. As determined by the standard curves the efficiencies of the TPSAB1 and CMA1 were not suitable for analysis by this method and therefore a more appropriate method was the relative standard curve method.
• The tonsil sample was chosen as a positive control as this tissue has mast cells which contain tryptase and chymase: we chose this tissue because we were able to access fixed material from our hospital to use for control experiments to validate the antibodies.
• For dual immunostaining primary antibodies were applied to sections overnight (16h) and incubated in a fridge -secondary antibodies were applied for 1h at room temperature. This is a standard method used in our laboratory and in our hands it leads to lower rates of nonspecific background staining than application of primary antibody at room temperature.
• The limitations on the amount of individual fixed samples meant that not all of the fixed samples (n=20) were stained with all antibodies and this is reflected in the numbers stated in the figure legends.

REVISED
zone. This zone has circular muscle fibres and like the endometrium it is derived from the Mullerian duct (Uduwela et al., 2000), whilst the other layers develop from non-Mullerian tissue. The smooth muscle cells of the myometrium (myocytes) are active during the non-pregnant menstrual cycle, with uterine peristalsis constituting one of their fundamental functions (Kunz & Leyendecker, 2002). Like the endometrium, the myometrium is a steroid target organ with myocytes expressing receptors for oestrogens, progestagens and androgens, all of which can induce changes in gene expression (Chandran et al., 2016;Makieva et al., 2016).
MCs have been identified in the human uterus, with reports that their phenotype is similar to lung MCs in terms of a response to secretagogues and release of prostaglandins, but a granule phenotype distinct to that of gut MCs (Massey et al., 1991). There has also been interest in the role played by MCs in myometrial contractions and in fibroids, although whether they play an important role in either has been questioned (Garfield et al., 2006;Menzies et al., 2011;Protic et al., 2016). A detailed study on uterine MCs was published by Jeziorska et al. (1995), who used immunohistochemistry to identify tryptase and chymase positive cells in 107 uterine samples taken across the menstrual cycle. They reported that there were similar MC numbers throughout the functional, basal layers of the endometrium, and myometrium during the menstrual cycle. In a smaller study Mori et al. (1997) immunostained uterine samples from 24 women with a variety of gynaecological disorders using antibodies against tryptase and chymase. They reported that the MCs were most abundant in the inner half of the myometrium and most endometrial MCs were of the MC T phenotype.
In summary, the role of MCs in the human uterus is poorly understood and little is known about their regulation, or the impact of steroids within the uterine microenvironment on their differentiation status. The current study used tissue sections of human uterus to define the spatial and temporal location of MCs in the myometrium and endometrium, and explored their phenotype by examining the pattern of expression of the proteases tryptase and chymase using fluorescent co-staining. We also examined expression receptors for oestrogen (ERα, ERβ), progesterone (PR) or glucocorticoids (GR) to determine their ability to respond directly to steroids.

Patients and tissue recovery
Ethical committee approval was obtained from the Lothian Research Ethics Committee (LREC; approval numbers, 10/ S1402/59 and 16/ES/0007). Patients were recruited by dedicated research nurses from clinics treating women for benign gynaecological conditions, including heavy menstrual bleeding and fibroids. In all cases written patient consent was obtained prior to tissue collection. Full details of patients are provided in Supplementary Table 1. The total number of women from which samples were obtained was 46. However, due to limited amount of tissue available, some samples were either fixed (n=20) or used for RNA extraction (n=26), but not for both. Patients were aged between 25-50 years (average of 39.8 years), reported regular menstrual cycles and had not taken any exogenous hormones in the three months prior to surgery. Stage of the menstrual cycle was evaluated by analysis of circulating steroid concentrations (P 4 , E 2 ) using blood samples obtained at the time of surgery. Assays were performed by the Specialist Assay Service (Surf Facility, University of Edinburgh) and cycle stage was further confirmed by examination of tissue sections by an expert pathologist, Professor A.R.W. Williams (NHS, Royal Infirmary, Edinburgh). Critical inclusion criteria were the absence of pelvic pain, such as dysmenorrhea, absence of fibroids or presence of small fibroids only (<3 cm): none of these women had a diagnosis of endometriosis.
RNA extraction, cDNA synthesis and qRT-PCR Total RNA was extracted using the RNeasy Mini Kit (Qiagen, UK), according to manufacturer's instructions. RNA concentration and purity was measured using the Nanodrop (LabTech International, UK) and standardised to 100ng/µl for all samples. Reverse transcription was performed using 100ng of RNA with 0.125× Superscript Enzyme in 1× VILO reaction mix (Thermo Fisher Scientific, UK) at 25°C for 10 minutes, followed by 42°C for 60 minutes and finally 85°C for 5 minutes. Quantitative PCR was performed using FAM labelled probes for TPSAB1 (number 20) and CMA1 (number 81) from the Universal Probe Library (Roche Diagnostics, UK) and VIC labelled human PPIA (Cyclophilin A) endogenous control (Thermo Fisher Scientific), with specific primers for TPSAB1 (forward 5'-cctgcctcagagaccttcc-3'; reverse 5'-acctgcttcagaggaaatgg-3') and CMA1 (forward 5'-ttcacccgaatctcccatta-3'; reverse 5'-tcaggatccaggattaatttgc-3') (Eurofins Genomics, UK Statistical analysis was carried out using GraphPad Prism 6.0 (GraphPad Software, USA). Data are presented as the median. One-way ANOVA was used, and Kruskal-Wallis was performed as a secondary test with Dunn's multiple comparisons test. Criterion for significance was p<0.05.

Phenotyping mast cells by immunohistochemistry and dual colour immunofluorescence
Immunohistochemistry was carried out on "full thickness" (uterine lumen to endometrial-myometrial junction) human uterine sections to localize MCs to the different tissue layers: myometrium, basal endometrium and functional (luminal) endometrium. Uterine biopsies were fixed in 4% neutral buffered formalin, embedded in paraffin wax and cut to 5µm sections. Following dewaxing and rehydration, sections were blocked in methanol peroxide for 30 minutes on a rocker at room temperature (RT), followed by 30 minutes blocking in normal goat blocking serum (Sigma Aldrich, Dorset, UK), before primary antibody incubation at 4°C overnight (16h): Tryptase, rabbit monoclonal, Abcam, UK; Chymase, mouse monoclonal, AbSerotec, UK; ERα, mouse monoclonal, Vector Laboratories, UK; ERβ mouse monoclonal, AbSerotec, UK. After washing in 1× Tris Buffered Saline + 0.05% Tween, slides were incubated with secondary antibody for 1 hour at RT, followed by 1:50 tyramide signal amplification (TSA Fluorescein Tyramide Reagent Pack, PerkinElmer, USA) for 10 minutes. Antigen retrieval was performed at pH6 in citrate buffer, and then further blocked with serum to avoid cross-reactivity. The second primary antibody was then added and incubated overnight at 4°C. Incubation with an appropriate secondary antibody at 4°C for 16 hours and a further TSA amplification step were carried out before counterstaining the sections with DAPI (1:500 dilution in TBS) and mounting with permafluor (ThermoFisher Scientific). Fluorescent images were acquired with a Zeiss Axioscan Z1 or a Zeiss 710 confocal microscope, and analysed with Zen Blue or Black software (version 2; Zeiss, Jena, Germany). Full antibody details can be found in Supplementary Table 2.

Results
Messenger RNAs encoding mast cell proteases were detected in human endometrium In tissue homogenates of endometrium, total concentrations of messenger RNAs encoded by TPSAB1 (gene for tryptase α and βisoforms; Figure 1A) and CMA1 (chymase; Figure 1B Figure 4) phases, also identified a population of MCs that were chymase positive, but without co-incident expression of tryptase (arrows). These cells appeared less abundant than those that were immunopositive for both tryptase and chymase (arrowheads), and were confined to the basal compartment of the endometrium and the myometrium.
The data obtained from immunohistochemical analysis of the 20 patients are summarized in Table 1.
Tryptase-positive uterine mast cells were immunopositive for oestrogen receptor beta but did not express either oestrogen receptor alpha or progesterone receptor The uterus is an oestrogen target organ and detailed immunohistochemical studies conducted on menstrual cycle staged sections of endometrial tissue by ourselves (Bombail et al., 2008;Critchley et al., 2001) and others (Mylonas et al., 2004;Snijders et al., 1992) have documented cell and phase-dependent expression of both isoforms of the oestrogen receptor (ERα, ERβ). In the current study, in line with expectation, we identified ERα  Figure 4); however, although tryptase-positive cells were readily detected in the basal endometrium and myometrium, none of these had detectable ERα protein in their nuclei (Supplementary Figure 4). In contrast, immunopositive staining for ERβ protein was present in multiple cell types, including stromal fibroblasts and endometrial epithelial cells, as well as tryptase-positive (green cytoplasm) MCs in both the functional and basal regions of the endometrium and throughout the myometrium of the uterus (Figure 5, arrows). The results obtained with antibody directed against the progesterone receptor mirrored those of ERα, with no evidence of PR-positive MCs (Supplementary Figure 5).

Uterine mast cells are immunopositive for the glucocorticoid receptor
We have previously identified GR in multiple cells within the endometrium, including endothelial cells and immune cells (Rae et al., 2009;Thiruchelvam et al., 2016), complemented by evidence that enzymes capable of the biosynthesis of cortisol, the natural ligand for GR, are present in the tissue (Thiruchelvam et al., 2016). In the present study, immunostaining for GR showed it was expressed within the stromal fibroblasts and other cells (putative immune cells), as well as being present in the nucleus of tryptase-positive cells in both endometrium and myometrium (arrowheads, Figure 6).
In summary, we detected co-immunoexpression of both the beta isoform of ER and GR in the nuclei of uterine MCs (tryptase positive staining in their cytoplasm), but no evidence of immunoexpression of ERα or PR. A photomontage of representative sections stained for each of the receptors is provided in Figure 7.

Discussion
This study has shed new light both on the phenotype of endometrial and myometrial MCs, as well as revealing the potential that they might respond in situ to both oestrogens and glucocorticoids.
MCs are known to arise from progenitors in the bone marrow, but adapt to their mature phenotype depending upon the tissue microenvironment in which they mature. To date, uterine MCs have received little attention compared to other immune cell populations, such as uNK cells and macrophages (Gibson et al., 2015; Analysis of mRNAs encoding proteases expressed by MCs has not previously been reported in endometrial tissue homogenates. We found that the concentrations of tryptase and chymase mRNAs in our samples did not vary significantly between different phases of the menstrual cycle. These results would be consistent with previous reports that MC numbers vary little throughout the menstrual cycle (Salamonsen et al., 2002). We speculate that these results may reflect the long life span of tissue resident MCs, with some studies reporting that MCs have a lifespan of weeks to months (Kiernan, 1979;Padawer, 1974). As tryptase and chymase mRNAs are constitutively expressed by MCs (Pejler et al., 2007), it is unsurprising they may remain unchanged if cell numbers are fairly constant.
Traditionally, human MCs are classified according to different phenotypes depending on their expression of tryptase and/or chymase in cellular granules (Irani et al., 1986). In line with expectations based on MC phenotype in multiple tissues, we readily detected both tryptase positive and chymase positive cells within both the endometrium and myometrium. Using immunofluorescence, we were able to co-stain for tryptase and chymase in the same cells revealing populations of cells that were of MC T and MC TC phenotypes. Weidner & Austin (1993) were the first to   Results in this study showed the phenotype of the uterine MCs varied between the different tissue layers of the uterus. Confirming previous studies, MC TC were predominantly resident in the basal endometrium and in the myometrium and MC T were found in functional endometrium. The rare MC C type was detected in the basal endometrium and myometrium and completely absent in the functional layer. These findings reinforce the principle that tissue specific phenotypes of MCs can exist within different regions within the same organ. It is already well known that the functional and basal compartments of the endometrium and myometrium vary with regard to cellular composition and cytokine/ chemokine concentrations. Interestingly the region of the tissue  where MCs appeared most abundant were close to the endometrial-myometrial junction. Previous studies have demonstrated that a large number of CD34 + MC progenitor cells reside in this area of the tissue and that their numbers are independent of phase of the menstrual cycle (Cho et al., 2004;Mai et al., 2008). Finding MCs in close proximity to smooth muscle fibres would be consistent with this cell type being a key source of stem cell factor, and a vital mediator for MC maturation and survival (Zhang et al., 1996).
The activation state of uterine MCs was also explored during the present study. Previously, endometrial MCs were reported to degranulate during the secretory phase, at a time when the tissue is in an oedematous state. This observation was based on detection of extracellular tryptase during oedema and weak intracellular tryptase staining detectable during the proliferative phase (Jeziorska et al., 1995). In this study, endometrial activation and the degranulation of MCs was documented during the early and mid-secretory stages of normal uterine tissue with detection of tryptase and chymase in the extracellular matrix. A 'recovery' state, characterised by weak immunostaining, was observed in tissue collected from patients during the proliferative (Supplementary Figure 1), late secretory (Supplementary Figure 3) and menstrual (Figure 4) stages. Within the myometrial compartment, MCs appeared to be in a 'resting' state during the proliferative phase (Supplementary Figure 1). Interestingly, in the myometrium, MCs appeared to be 'activated' with detection of immunopositive staining for chymase diffuse and spread beyond the margins of the individual cells during both mid secretory and menstrual phases (Figure 3 and Figure 4). We speculate that this might suggest a potential role for MC derived proteases in regulation of arteriole sprouting at early/mid secretory phases. A previous study also suggested the release of granules may play a role in smooth muscle contraction during menses (Sivridis et al., 2001) and our findings would be consistent with this suggestion.
Although, other authors have previously demonstrated a direct effect of female sex hormones on MC behaviour, activation and migration, in those studies they cited activation of MCs via ERα and PR (Jensen et al., 2010;Zaitsu et al., 2007). The study by Engemise et al. (2011) examined the number of mast cells, identified by toluidine blue, in the endometrium of 28 women with endometriosis before and after insertion of a Mirena coil secreting the progestogen levonorgestrel. They reported a reduction in mast cell number after 6 months but found no evidence of staining for PR or ERα. In the current study, based on detailed immunohistochemical analysis, using previously validated antibodies directed against oestrogen receptor subtypes (Critchley et al., 2002;Henderson et al., 2003), we found novel evidence for immunoexpression of ERβ, but no evidence of expression of ERα. These results mirror the oestrogen receptor phenotype of both uNK (Gibson et al., 2015;Henderson et al., 2003)) and macrophages (Thiruchelvam et al., 2013). This observation is also supported by the activation of uterine MCs during the secretory phase at a time in the menstrual cycle when intracrine biosynthesis of oestradiol has been shown to activate ERβ positive uNKs (Gibson et al., 2015).
In other studies, we have shown that in endometrial tissue expression of ERβ often parallels that of GR with co-expression in both uNK (Henderson et al., 2003) and endothelial cells (Critchley et al., 2002). In the current study, nuclear GR was detected in MCs in the functional, basal endometrium and myometrium. In the same samples, GR protein was also detected in endometrial stroma and smooth muscle fibres during the proliferative phase, but its expression was reduced during the progesterone-dominant secretory phase, a result which was in agreement with previous reports (Bamberger et al., 2001;Henderson et al., 2003). Several studies have reported that glucocorticoids may have an indirect anti-inflammatory impact on MCs, with the postulated mechanism being a reduction of stem cell factor production by fibroblasts (Da Silva et al., 2002). Alternatively, they may also have direct impacts by reducing IgE binding to the FcεRI receptors, thereby down regulating the expression of these receptors on the cell membrane of the MCs and inhibiting MC degranulation in vitro (Finotto et al., 1997;Yamaguchi et al., 2001;Zhou et al., 2008). Prior to the current study, the only report of expression of GR in human MCs was from Oppong et al. (2014). In their study, they localized GR to the plasma membrane in the RBL-2H3 MC line. The current study is the first to demonstrate that uterine MCs are immunopositive for nuclear GR. A glucocorticoid rich environment would favour activation of GR, with shuttling of ligand activated receptor from the cytoplasm towards the nucleus (Phuc Le et al., 2005). This observation would be consistent with expression 11-β hydroxysteroid dehydrogenase enzymes within the uterus, resulting in a cortisol rich microenvironment (Gibson et al., 2013;McDonald et al., 2006).
In summary, our study confirms that MCs are members of the leukocyte population of the human uterus, and that they are most abundant in the myometrial and basal endometrial compartments. Whilst uterine MCs predominantly belong to the classic MC subtypes: tryptase positive/chymase negative (MC T ) and tryptase/chymase positive (MC TC ), a rare third subtype (MC C ) was also identified in the uterus for the first time. We demonstrated that endometrial and myometrial MCs are immunopositive for both ERβ and GR, demonstrating that, like other immune cells present in the endometrium (uNK, macrophages), they may be a target for the direct actions of oestrogens and glucocorticoids, which are both synthesised within the endometrial tissue microenvironment. This study provides a framework for furture studies on the role of MCs in endometrial and myometrial disorders, including conditions associated with increased pain, such as endometriosis.

Ethical approval
Lothian Research Ethics Committee (LREC) approval was granted and written patient consent was obtained prior to tissue collection by dedicated research nurses (approval numbers, 10/S1402/59 and 16/ES/0007).

Competing interests
The authors have no competing interests.

Grant information
PTKS, AZ-E and FC were funded by MRC Programme (grant G1100356) to PTKS, which also paid for the costs of consumables used in this study. BD was in receipt of an MRC PhD studentship funded by the Centre Grant for Reproductive Health (G1002033). Salaries of nurses who recruited patients to the study were paid by MRC grants G1100356 (PI. PTKS) and G0500047 (PI. HODC).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
1. This is an observational, descriptive study describing the enzymatic and hormonal phenotype of the mast cells in full thickness normal human endometrium and the adjacent myometrial layers. The data from the modest sample size presented in the manuscript confirms previous reports of spatio-temporal distribution of MC and their phenotype throughout the menstrual cycle; highlighting the novel finding, the glucocorticoid receptor expression in these cells. The authors have also examined the GR expression in the context of the expression of a selected panel of other steroid receptors. The manuscript is well written and presented; however, there are some points detailed below which warrants further consideration/ clarification.

General comments:
The conventional histopathological description of the endometrial layers as endometrial basalis layer and functionalis layer, or stratum basalis or stratum functionalis throughout the manuscript may provide more clarity than the inconsistent use of less conventional descriptive terms of basal compartment etc. in the present MS.
Referral to some pivotal previous references could make the manuscript more comprehensive, such as , 2011; Engemise SL et al, Eur J Obstet Gynecol Reprod Biol.
; Mori A , Hum Reprod., 1997et al. Drudy L et al., Eur J Obstet Gynecol Reprod Biol., 1991 The description of the genes and first appear in the results section but should be stated at TPSAB1 CMA1 the initial mention for clarity.

Methods:
Authors have analysed the and mRNA expression by relative standard curve using TPSAB1 CMA1 a tonsil mRNA as a reference. What is the reason for this and why was this method utilised in place of the usual comparative Ct (delta delta Ct) method? The reasons need to be explained with reference to the more widely known method.
In dual IHC, was the 2RT antibody incubation for 16h? This appears to be relatively longer than the We thank Dr Kamal for her comments on our paper which we have edited to address some of her concerns.

Specific responses:
We have now referenced the small study by Mori (1997) in the Introduction and the study by et al Engemise in the Discussion.

et al
We have changed the text of the Introduction so that the abbreviations for TPSAB1 and CMA1 are introduced during the first mention of their importance in defining the mast cell phenotype.

Methods.
The text describing the samples has been changed to highlight the patient information provided in Suppl Table 1 and clarify that although 46 patients were recruited during this study their samples were split between RNA (n=29) and fixation for immunostaining (n=26).
Analysis methods for RTPCR data: Although the delta delta CT method is one of the most popular it is based on comparable amplification efficiencies of the endogenous control and gene of interest. As determined by the standard curves the efficiencies of the TPSAB1 and CMA1 were not suitable for analysis by this method and therefore a more appropriate method was the relative standard curve method. The text in M&M has been revised to make this clearer. These methods are also described in detail in Applied Biosystems manual online http://www6.appliedbiosystems.com/support/tutorials/pdf/performing_rq_gene_exp_rtpcr.pdf The tonsil sample was chosen as a positive control as this tissue has mast cells which contain tryptase and chymase, the RNA sample was purchased from a commercial supplier and we chose this tissue because we were able to access fixed material from our hospital to use for control experiments to validate the antibodies.
For dual immunostaining primary antibodies were applied to sections overnight (16h) and incubated in a fridge -secondary antibodies were applied for 1h at room temperature. This is a standard method used in our laboratory and in our hands it leads to lower rates of non-specific background staining than application of primary antibody at room temperature. We did not quantify the fluorescent staining.

Results.
The limitations on the amount of individual fixed samples meant that not all of the fixed samples