human endometrial programming and lessons in health and disease

In this Review, we describe the remodelling of the endometrium before it becomes receptive for embryo implantation, the dynamic fetal–maternal communi-cation that contributes to successf

The transformative processes that prepare the endome-trium for embryo implantation are unique to menstruating species, and are thought to underlie the evolution of menstruation Although rodent species, which are easy

to manipulate, are common experimental models for studies of endometrial receptivity and embryo implanta-tion, findings obtained with these animals often cannot

be directly translated to humans

Biological processes that have developed in the human endometrium during the evolution of menstru-ation are specialized versions of processes that are found

in other tissues, altered to regulate endometrial biology

Understanding how the human endometrium under-goes controlled and spatially limited tissue destruction, resolution of inflammation, scar-free repair and re- epithelialization followed by regeneration and transfor-mation can inform our understanding of processes that occur in other tissues

In this Review, we describe the remodelling of the endometrium before it becomes receptive for embryo implantation, the dynamic fetal–maternal communi-cation that contributes to successful implantation, the endometrial defects that result in infertility and miscar-riage and the detection and treatment of these disorders

We also identify missing links, both experimental and clinical, which should be investigated to enable progress

in the field, and areas where understanding of endo-metrial biology might influence other fields and the develop ment of therapeutics

Evolution of human menstruation

Unlike other organs, the human endometrium does not have a single, constant function from birth to death The endometrium exists to provide a ‘fertile ground’ for implantation of an embryo and development of a highly invasive placenta, which is achieved by an orderly sequence of development and transformation within each menstrual cycle, under the influence of the ovarian steroid hormones1 The endometrial cells become terminally dif-ferentiated during each menstrual cycle; in the absence of conception, tissue shedding and regeneration for subse-quent fertile cycles occurs In menstruating species, decid-ualization is spontaneous, rather than embryo-mediated Decidualization is the process of the transformation or differentiation of human endometrial stromal fibroblasts

to secretory ‘epithelioid’ cells, which occurs under the influence of the hormones oestrogen and progesterone, along with cAMP and local paracrine factors

The evolution of spontaneous decidualization is thought to have occurred when genes that were ances-trally expressed in other organs and tissue systems were expressed in the endometrium Transposable elements,

1 Centre for Reproductive

Health, Hudson Institute of

Medical Research, Clayton,

3168, Australia.

2 Department of Molecular

and Translational Medicine,

Monash University, Clayton,

3800, Australia.

3 Department of Physiology,

Monash University, Clayton,

3800, Australia.

4 Department of Obstetrics

and Gynaecology, Monash

University, Clayton, 3800,

Australia.

5 Department of Biochemistry

and Molecular Biology,

Monash University, Clayton,

3800, Australia.

6 The Ritchie Centre, Hudson

Institute of Medical Research,

Clayton, 3168, Australia.

7 Department of Anatomy and

Developmental Biology,

Monash University, Clayton,

3800, Australia.

Correspondence to E.D

evdokia.dimitriadis@hudson.

org.au

doi: 10.1038/nrendo.2016.116

Published online 22 Jul 2016

Fertile ground: human endometrial programming and lessons in health and disease

Jemma Evans1–3, Lois A. Salamonsen1,2,4, Amy Winship1,2, Ellen Menkhorst1,2, Guiying Nie1,2,5, Caroline E. Gargett4,6 and Eva Dimitriadis1,2,7

Abstract | The human endometrium is a highly dynamic tissue that is cyclically shed, repaired, regenerated and remodelled, primarily under the orchestration of oestrogen and progesterone,

in preparation for embryo implantation Humans are among the very few species that menstruate and that, consequently, are equipped with unique cellular and molecular mechanisms controlling these cyclic processes Many reproductive pathologies are specific to menstruating species, and studies in animal models rarely translate to humans Abnormal remodelling and regeneration of the human endometrium leads to a range of reproductive complications Furthermore, the processes regulating endometrial remodelling and implantation, including those controlling hormonal impact, breakdown and repair, stem/progenitor cell activation, inflammation and cell invasion have broad applications to other fields This Review presents current knowledge regarding the normal and abnormal function of the human endometrium The development of biomarkers for prediction of uterine diseases and pregnancy disorders and future avenues

of investigation to improve fertility and enhance endometrial function are also discussed.

REVIEWS

for instance, contributed to the origin of decidualization

by conferring progesterone responsiveness to numerous genes across the genome2 The evolutionary transforma-tion of the endometrial regulatory landscape has been mapped and found to explain the developments within the human uterus that support its unique pregnancy phenotype, of which decidualization and menstruation are central2

Decidualization probably evolved because it pro-vided protection to uterine tissues from the hyper-inflammation and oxidative stress associated with deep haemochorial placentation3,4 However, menstruation as

a consequence of decidualization is equally important

in the human adaptation to haemochorial placentation

Repeated cycles of decidualization and shedding prepare human uterine tissues by physiological preconditioning for the stress of haemochorial placentation4 In an adolescent who is pregnant (and has experienced few menstrual cycles), extensive preconditioning has not occurred, which results in a higher risk of major obstetric complications associated with defective placentation than is seen in older pregnant women3 Menstrual cycles are hypothesized to undergo their own ‘evolu-tion’ throughout the reproductive lifespan, with the endometrium transitioning from a fairly progesterone- resistant, immature tissue at menarche to become more responsive because of the cumulative effects of cyclic menstruation and inflammatory signalling3,5 The lack

of preconditioning and, thus, the absence of these cycle- derived changes is proposed to contribute to the aetiol-ogy of pregnancy complications in adolescents who have not yet developed progesterone responsiveness

This evolution of spontaneous decidualization and menstruation, and the dysfunction associated with these processes, has given rise to human-specific reproductive complications, including recurrent early pregnancy loss and placental pathologies such as pre-eclampsia, in addi-tion to menstrual problems such as heavy or abnormal bleeding

Mechanisms of endometrial remodelling

Endometrial luminal epithelium The endometrium

undergoes substantial remodelling under the influence

of ovarian steroid hormones, and becomes receptive for only a few days in the mid-secretory phase of the

menstrual cycle (FIG. 1) The luminal epithelium is the first uterine point of contact for blastocysts, and dif-ferentiates considerably during the receptive phase to facilitate embryo attachment and subsequent implan-tation The transformation of the plasma membrane in cells of the luminal epithelium from a nonadhesive to an adhesive surface encompasses remodelling of elements that contribute to the endometrial barrier function, including the glycocalyx, epithelial polarity, epithe-lial–mesenchymal transition and the lateral junctional complexes (FIG. 1) 6 Importantly, in humans the placental trophoblasts invade between epithelial cells, without the epithelial destruction that is observed in other species with haemochorial placentation7 Defects in interac-tions between embryos and the endometrial epithelium contribute substantially to infertility and implantation failure8

The known molecular changes that occur in human endometrial luminal epithelium in relation to receptiv-ity affect the integrins, osteopontin, Notch signalling, heparin-binding EGF-like growth factor, cell-surface- associated mucins, glycodelin and ion channels, which have been reviewed elsewhere9 Some cytokines proba-bly also have important roles in endometrial epithelial receptivity For example, levels of IL-11 are lower in endometrial luminal epithelium in infertile women than in fertile women10 IL-11 regulates the

adhesive-ness of epithelial cells in vitro, probably by upregulating

expression of the plasma membrane proteins annexin A2 and flotillin-111, which are proposed to be essential for receptivity and embryo attachment11

The results of transcriptomic profiling studies have identified large numbers of genes that are upregulated

or downregulated in the induction of receptivity, but data sets vary considerably between studies (as has been reviewed elsewhere12), which suggests post-translational regulation of proteins at the endometrial epithelial sur-face is important (FIG. 1) Studies on the serine protease proprotein convertase subtilisin/kexin type 5 (PC6) have revealed that, in endometrial epithelium, PC6 is maximally expressed during the receptive phase, but its expression is lower in women with implantation failure than in reproductively healthy women13 PC6, via its proteolytic activity, post-translationally regulates anti- adhesion molecules and the organization of the plasma membrane in human endometrial epithelium, altering the apical architecture to provide a receptive surface13,14

Decidualization In human endometrial stromal cells

(ESCs), decidualization is the process of spontaneous, terminal differentiation that occurs in the mid-to-late secretory phase of each menstrual cycle, whereas in nonmenstruating species, this process is initiated dur-ing pregnancy (FIG. 1) In a menstrual cycle that does not result in conception, the terminally differentiated cells are shed during menses However, if pregnancy occurs, decidual cells promote the invasion of fetal extra villous trophoblasts that (along with uterine natural killer (uNK) cells) facilitate spiral-artery remodelling and protect the conceptus by conferring maternal immuno-tolerance of the fetal allograft15 The decidual cells also

Key points

• The human endometrium is a unique, dynamic tissue that is cyclically shed, repaired,

regenerated and remodelled, in preparation for embryo implantation

• Decidualization in women occurs spontaneously (regardless of the presence of an

embryo) during the mid‑to‑late luteal phase, necessitating endometrial shedding and

subsequent regeneration in the absence of conception

• Endometrial remodelling occurs primarily under the orchestration of oestrogen and

progesterone, but is influenced by many factors, including epigenetic signals and

stem/progenitor cells

• Abnormalities in endometrial remodelling lead to pathologies including infertility,

endometriosis and pregnancy disorders

• Understanding the processes that operate in the endometrium could provide

information that is applicable to nonreproductive pathologies such as cancer and

wound healing

Nature Reviews | Endocrinology

Blastocyst Trophectoderm

Inner cell mass

Luminal epithelium

Tight junction

Adherens junction

Ovulation

Glandular epithelium

Day

1

2

3

8 7

4

Post-translational regulation of the surface molecules

Pinopode Glycocalyx

EMT

Blood vessel

Perivascular cell

Mesenchymal stem cell

uNK

Macrophage

Decidual cell

Cell surface adhesion factors

Stromal fibroblast

MET Progesterone

Differentiation

shield the conceptus from environmental stress signals16, and ‘sense’ embryo quality to facilitate maternal rejection

of developmentally incompetent embryos17 Progesterone induces decidualization in stromal cells adjacent to spiral arterioles (FIG. 1) In vitro, decidualized

stem-cell-like perivascular stromal cells produce higher levels of cytokines and chemokines that are involved

in promoting decidualization and the recruitment

of trophoblasts than nonperivascular stromal cells16

Decidualization also requires cAMP18, and involves reprogramming of ESCs, which ensures that different genes are expressed at specific stages of differentiation19 After the initiation of decidualization, local par-acrine factors create a ‘wave’ of decidualization that spreads from spiral arterioles throughout the endome-trium (FIG. 1) Decidual regulation has been investigated predominantly in studies of individual molecules; more comprehensive studies of the proteome and secretome20

Figure 1 | The pre-receptive, receptive and post-receptive endometrium The pre-receptive luminal epithelium (1) is

nonadhesive, owing to the presence of antiadhesive factors, including the glycocalyx, a polarized epithelium and lateral junctions that anchor cells tightly together During the pre-receptive phase, the glandular epithelium becomes highly secretory (2), uterine natural killer (uNK) cells proliferate and macrophages influx into the endometrium (3) To become

receptive, the luminal epithelium undergoes considerable changes (4): epithelial and blastocyst-secreted enzymes

post-translationally modify the glycocalyx; the epithelium undergoes epithelial–mesenchymal transition (EMT), becoming less polarized with fewer lateral junctions, and the adhesion-factor repertoire on the luminal epithelial surface changes Pinopodes (5) appear on the surface of the luminal epithelium at the initiation of receptivity, but their role in blastocyst–

epithelium adhesion is currently unclear Communication between blastocysts and uterine luminal epithelium further enhances receptivity (6) Decidualization (7) is initiated by progesterone in stromal cells adjacent to blood vessels, and in

vascular mesenchymal stem cells These cells undergo mesenchymal–epithelial transition (MET) to become rounded, secretory cells expressing the decidual markers prolactin and insulin-like growth factor-binding protein 1 Decidual cells secrete factors (such as hormones, cytokines, chemokines, lipids and noncoding RNAs) that act synergistically or additively to create a wave of decidualization (8) throughout the endometrium.

and microRNA (miRNA) signature21 of decidualization have not added substantially to the repertoire of pro-cesses that are known to be involved in decidualization

This repertoire has been reviewed elsewhere22 Although progesterone drives decidualization, other steroid recep-tors (specifically, oestrogen receptor (ER), glucocorti-coid receptor, mineralocortiglucocorti-coid receptor and androgen receptor) also have distinct roles22,23, and might confer specificity of hormone action

Few in vitro studies have investigated the role of

other cell types in the progress of decidualization The results of studies in mice that lack uterine glands show that these glands are essential for decidualization24, but whether they are similarly important in women is not known Human uterine glands secrete many factors that

are known to drive decidualization in vitro, but in vivo

secretion of these factors into the stroma has not been confirmed25 Leukocytes, including uNK cells, mast cells, T cells and dendritic cells, are essential for decidual angiogenesis during the initiation of pregnancy (FIG. 1) However, their function in decidualization is less clear

Murine models of dendritic-cell depletion indicate that these cells are required for decidual proliferation and dif-ferentiation26 Similarly, uNK cells in mice seem to main-tain decidual integrity27 However, the results of both

mouse and human in vitro co-culture experiments with

epithelial and stromal cells have not provided evidence that uNK cells initiate or promote decidualization28 Decidual leukocytes have specific phenotypes, and express distinct markers of differentiation and function compared with peripheral leukocytes Decidualized stro-mal cells secrete mediators that can act on, and influence the function and differentiation of, resident leukocytes29

Menstrual breakdown and repair Menstruation is

ini-tiated by the withdrawal of oestrogen and progesterone support in the absence of implantation and pregnancy, and is governed by a complex cascade of endocrine and paracrine signalling within the endometrium (FIG. 2)

In macaques, which are menstruating nonhuman pri-mates, the onset of menstruation can be blocked by progesterone replacement within 36 h of hormone withdrawal, but replacement after 36 h has no effect30 This result suggests a biphasic activation of menstrua-tion, in which endocrine signalling to cells expressing progesterone receptor initiates paracrine signalling to cells without progesterone receptor, which facilitates progesterone-independent effects that lead to menses

Intriguingly, endometrial tissue destruction and re- epithelialization occur simultaneously; re-epithelializa-tion is generally considered to start ~36 h after the onset

of menses, and is complete within a further 48 h The results of hysteroscopic analysis of the menstrual endo-metrium emphasize that menstrual shedding is a zonal event; areas undergoing breakdown can be observed adjacent to intact tissues from the previous cycle and areas that have already undergone re-epithelialization31 Decidualized stromal cells (FIG. 1) are essential for responding to endocrine cues and transmitting paracrine signals during menstruation, as they express the proges-terone receptor premenstrually32 and detect progesterone

withdrawal33 Hormone withdrawal from decidualized

stromal cells in vitro enhances inflammatory reactive

oxygen species via inhibition of superoxide dismutase activity, which upregulates nuclear factor κB (NF-κB) and prostaglandin G/H synthase 2 (PTGS2, also known

as COX-2) signalling relative to levels in the presence of progesterone and results in production of inflammatory factors, including prostaglandin F2α (REFS 33,34) (FIG. 2) Hormone withdrawal triggers the recruitment of inflammatory cells into the perimenstrual endometrium via alterations in chemokines derived from decidualized stromal cells33,35 (FIG. 2) Secretion of proteolytic enzymes

by leukocytes results in tissue breakdown at menses,

as reviewed elsewhere35, and local tissue lysis simulta-neously results in the production of cues for repair36 Expression of proteases and gene products involved

in extracellular matrix synthesis and repair is elevated specifically in stromal cells derived from areas of the endometrium that have undergone lysis36,37

Oestrogen is not required for endometrial repair, as demonstrated by evidence from the study of ovariec-tomized women and women in natural menopause38

In vitro human studies have defined ‘wound-healing’

factors, including activin, vascular endothelial growth factor (VEGF), cysteine-rich secretory protein 3 and galectin-7, along with development-related path-ways, such as Wnt signalling pathways and mesen-chymal–epithelial transition (FIG. 2), which contribute

to re-epithelialization and endometrial wound repair independently of oestrogen37,39–42 However, once the endometrial surface is re-epithelialized, oestro-gen is required to stimulate glandular and stromal regeneration (FIG. 2)

Menstrual endometrium demonstrates the opposing processes of tissue destruction and repair simultaneously

in an inflammatory environment; both processes are ini-tiated by similar physiological cues Understanding how the menstrual endometrium limits inflammation, mod-ulates immune-cell activity, rapidly repairs and remains scar-free has implications for the development of treatments for a number of pathologies (BOX 1)

Stem/progenitor cells in regeneration Small

pop-ulations of adult stem/progenitor cells with classic stem-cell properties of clonogenicity, self-renewal and differentiation have been identified in human endome-trium43 (TABLE 1); these cells contribute to the ability of the endometrium to regenerate during each menstrual cycle (FIG. 2) Specific stem/progenitor cell types, includ-ing epithelial progenitors, mesenchymal stem cells and side-population cells (which are characterized by the efflux of DNA-binding dyes, a universal property of adult stem cells) might be involved in the regeneration

of different endometrial cellular compartments43 Epithelial progenitor cells have been identified in human endometrium as clonogenic cells that differen-tiate into large, gland-like structures44, and in mice as label-retaining cells that proliferate in response to oes-trogen, despite lacking ERs (TABLE 1) ERα-expressing niche cells that are closely associated with epithelial progenitor cells probably transmit the oestrogen signal

Nature Reviews | Endocrinology

25

1

2

3

8

4 5

6

Perivascular cell Mesenchymal stem cell

Macrophage

Neutrophil

Decidualized stromal cell

Stromal fibroblast

Blood vessel

Late secretory (days 24–28)

• No conception

• Corpus luteum demise

• Hormone withdrawal

Repair (days 2–5)

• Chemokines

• Growth factors

• Wnt signalling

• MET

Regeneration (days 5–14)

• Epithelial progenitor cells

• Mesenchymal stem cells

• Wnt signalling

• Notch signalling

Menstruation (days 1–5)

Eosinophil

NF-κB Vasoconstriction

Uterine bleeding

Functionalis Basalis

Epithelial progenitor cell

Luminal epithelium

• Growth factors

• Chemokines

Proteolytic enzymes

Tissue destruction

7

Growth factor activation COX-2

• PGE2

• PGF2α

9

to these ERα-negative cells Epithelial progenitors are thought to be located in the basalis region of the uterine glands (FIG. 1), where a high level of telomerase activ-ity (a feature of adult stem cells) has been detected43 Specific markers identifying epithelial progenitor cells are required to facilitate delineation of their role in endometrial proliferative disorders (BOX 2)

Human endometrium also contains a small population

of mesenchymal stem cells (eMSCs)43 (TABLE 1) Specific surface markers of clonogenic eMSCs demonstrate their perivascular localization in the endometrial functiona-lis and basafunctiona-lis45,46 (FIG. 1), as well as their presence within shed fragments in menstrual fluid43 eMSCs have been identified by the co-expression of CD146 and platelet- derived growth factor receptor β (PDGFRβ) markers as pericytes45 A single marker, sushi domain-containing

protein 2 (SUSD2, also known as W5C5)) identified 4% of endometrial stromal cells in 34 samples of stro-mal cells as eMSCs46 (TABLE 1) Gene profiling of fresh CD146+PDGFRβ+ cells47 and cultured SUSD2+ cells16 confirmed that eMSCs have a pericytic, perivascular signature, which suggests that eMSCs have an additional role in angiogenesis during stromal regeneration and placentation43 These endometrial perivascular cells are distinct from the stromal fibroblast (CD146−PDGFRβ+) and endothelial (CD146+PDGFRβ−) populations47 Side-population cells48 are also present within human endometrium; these populations are a mix of ERβ-expressing endothelial cells with some epithelial and stromal cells that do not express ERα or progesterone receptor49,50 (TABLE 1) In xenografts, the side-population cells regenerate human ‘endometrium’ consisting mainly

Figure 2 | Endometrial decidualization, menstruation, repair and regeneration Endometrial stromal cells (1) and

mesenchymal stem cells (2) undergo decidualization under the influence of oestrogen and progesterone In the absence

of conception and implantation (3), endometrial stromal cells ‘sense’ hormone withdrawal upon the demise of the corpus

luteum, and upregulate intracellular inflammatory signalling (4) and the release of inflammatory factors that contribute to

vasoconstriction of uterine blood vessels (5), recruitment of leukocytes (6) and propagation of the menstrual cascade

However, these inflammatory and growth factors (4), proteolytic enzymes (7) and recruited immune cells (8) also

contribute to repair after menstruation, in concert with processes such as mesenchymal–epithelial transition (MET) (9)

and Wnt signalling, to restore endometrial homeostasis (10) Activation of endometrial epithelial progenitor cells and

perivascular mesenchymal stem cells (11), possibly involving Wnt signalling or Notch signalling, drives cellular

replacement in the glands and stroma respectively, to mediate regeneration of the endometrium COX-2, prostaglandin G/H synthase 2 (PTGS2); NF-κB, nuclear factor κB

of stromal and vascular tissue, with occasional epithelial gland-like structures49–51 Similarly, SUSD2+ eMSCs gen-erate stromal tissue in xenografts46 However, in human

endometrium in vivo, whether one or more stem/

progenitor cell type regenerates endometrial tissue, or

a stem/progenitor cell hierarchy exists, is not known

In an experimental model of wound repair, eMSCs modulated chronic inflammation outside the uterus, which suggests these cells have a role in communica-tion and regulacommunica-tion of macrophages52 Determination

of the function of these cells in endometrial physiology has the potential to identify their roles in endometrial disorders (BOX 2)

Endometrium–embryo crosstalk

The pre-implantation microenvironment Uterine fluid

provides the natural environment for sperm transport and blastocyst hatching together with pre-implantation development, as well as peri-implantation embryonic–

maternal interactions The fluid contains not only the nutrients necessary for blastocyst growth, but also important regulatory molecules and microvesicles53 Specific proteins secreted from the endometrium inter-act with the blastocyst to facilitate implantation25,54,55 (FIG. 1) mi RNAs in uterine fluid are taken up by pre- implantation mouse embryos and alter embryonic

mRNA expression in vitro56 Uterine fluid must also contain factors to protect the mother and embryo from bacteria and other pathogens57

Many classes of molecules, from simple salts and amino acids through to proteins, steroids and lipids are contained in uterine fluid These molecules are derived from multiple sources, including endometrial epithelial secretions, selective transudation from blood, leukocyte activation and possibly Fallopian tubal secretions and peritoneal fluid

Glucose, lactate and pyruvate are required for human blastocyst development58 Alterations in the levels of these factors might also alter the pH of the local environment

Proteins in uterine fluid include leukaemia inhibitory factor (LIF), VEGF, IL-11 and other chemokines and cytokines that are probably synthesized in the endome-trium and secreted into the uterine cavity54, establishing a complex milieu to facilitate implantation The amino acid profile of uterine fluid has been determined, but the full molecular composition is not yet known59 The mech-anisms involved in the regulation of levels of nutrients and ions, and the relationships between these compo-nents and their relative importance in the establishment

of pregnancy, are still to be determined

Blastocysts and endometrial epithelium Successful

implantation and pregnancy outcome require both a receptive endometrium and an appropriately developed blastocyst Blastocysts enter the uterine cavity during the receptive phase and remain for up to 72 h before implan-tation (FIG. 1) After blastocyst hatching from the zona pellucida, the trophectoderm comes into close contact with, and firmly adheres to, the receptive endometrial luminal epithelium, which initiates implantation (FIG. 1) The influence of blastocysts on receptivity and implan-tation is poorly defined in humans, although hormonal, epigenetic and metabolomic cues have been identified

Blastocysts communicate with the endometrium via cell-surface proteins and secreted factors60 (FIG. 1) Human chorionic gonadotropin (hCG) is secreted

by hatched human blastocysts in close apposition to the endometrial epithelium61 Treatment of primary human endometrial epithelial cells (EECs) with hCG, as well as infusion of hCG into the uterine cavity of humans and baboons, mediates the production of factors that are associated with endometrial receptivity, including LIF, VEGF, IL-11 and prokineticin-1 (REFS 62–65)

Human blastocysts require glucose metabolism, but exhibit an idiosyncratic metabolic mechanism that pro-duces high levels of lactate in close proximity to the uter-ine epithelium, creating a low pH environment66 This process is thought to promote local endometrial tissue disaggregation, facilitating trophectoderm cell invasion into the endometrium via modulation of epithelial VEGF production54

Human blastocysts regulate EEC adhesion and gene expression via secreted regulators67 Culture media

derived from blastocysts generated by in vitro

fertiliza-tion (IVF) that subsequently implant (resulting in a live birth) enhance primary human EEC adhesion, unlike IVF blastocysts that do not successfully implant68 Human blastocysts that are determined by morphology

to be of high quality during IVF culture, but that do not subsequently implant after transfer, secrete mi RNAs that are not secreted by blastocysts that implant68 mi RNAs secreted by IVF blastocysts during culture might reflect their quality and implantation potential.miR-661, bound

to the RNA-binding protein argonaute-1, is secreted spe-cifically by human IVF blastocysts that do not implant68 miR-661 is also taken up by primary human EECs in culture and blocks their adhesive capacity68,69 This anti- adhesion effect of miR-661 is mediated, at least in part,

by downregulation of the production of nectin-1 in primary human EECs68

Box 1 | Translating endometrial biology to other pathologies

Skin wounds

Chronic skin wounds commonly exhibit deficient re‑epithelialization Understanding

how the endometrium undergoes rapid repair after menstruation could lead to novel

insights into the development of treatments to promote repair of chronic wounds

Chronic inflammatory diseases

The endometrium limits inflammation during menstruation to prevent excessive tissue

destruction Translating the mechanism by which inflammation is restricted could aid

the resolution of chronic inflammation

Stem-cell dysfunction

Cyclic activation of stem cells is required for endometrial regeneration after

menstruation This process occurs monthly for an average of 450 menstrual cycles,

but stem‑cell senescence occurs in women with recurrent pregnancy loss

Delineation of the factors and mechanisms involved in cyclic activation could aid the

treatment of recurrent pregnancy loss and other diseases associated with stem‑cell

dysfunction

Fibrotic diseases

Repair of the endometrium following menstrual shedding is scar‑free Understanding

how the endometrium remains scar‑free despite inflammation and tissue destruction

each month could lead to novel therapies for fibrosis

Human blastocysts, via their secreted mediators, alter endometrial receptivity Secretion of specific signalling molecules that influence receptivity is probably affected

by the quality of blastocysts As lactate and noncoding RNAs have roles in cancer development and invasion, determining how they function and are regulated in the highly controlled process of embryo invasion has implications for our understanding of cancer biology

Endometrial pathologies and treatments

Endometriosis Endometriosis is characterized by the

growth of ectopic endometrial tissue outside the uterus;

this tissue cycles similarly to eutopic endometrium, undergoing inflammation, shedding and regeneration in response to hormonal changes across the menstrual cycle

Endometriosis affects ~10% of women of reproductive age; affected women commonly present with pelvic pain and dysfunctional menstrual bleeding

Sampson’s theory of retrograde menstruation70 is the most widely accepted cause of endometriosis This theory posits that fragments of shed endometrium reflux through the Fallopian tubes into the peritoneal cavity

As most women experience retrograde menstruation, alterations in the function of tissue fragments or an ina-bility of the immune system to clear the fragments could exist in women who develop endometriosis Altered ER function has been proposed to modulate apoptosis and inflammasome activation, possibly enabling fragments

of refluxed endometrial tissue to survive71 and establish endometriosis Alternatively, women with endometriosis might have variants of susceptibility genes that have been identified in genome-wide association studies (GWAS),

or they could have epigenetic alterations72

In support of Sampson’s theory, endometrial stem/

progenitor cells together with niche cells in shed tissue fragments can establish endometriotic lesions (BOX 2)

Clonogenic, self-renewing endometrial cells are pres-ent in endometriotic lesions in adult women73, and in a subset who had a neonatal ‘menstrual bleed’, these cells might have been present from birth74, remaining viable and dormant until rising oestrogen levels at menarche initiated the growth of ectopic endometrium

Establishment of ectopic endometriotic lesions prob-ably alters gene expression and cellular function within the eutopic endometrium A proliferative transcriptomic fingerprint is maintained within the early-secretory eutopic endometriotic endometrium75; eutopic ESCs from women with endometriosis do not undergo decidualization, and they have alterations in gene expression suggestive of resistance to progesterone-mediated differentiation76 However, even in the absence of progesterone stimulation

in vitro, endometriotic eutopic stromal cell gene expression

is altered compared with endometrial stromal cells isolated from women without endometriosis, and this underlying difference could contribute to the altered responses to

progesterone stimulation in vitro76 The phenomenon of progesterone resistance might not be restricted to endome-triosis5, as similar genetic alterations have been observed in ESCs isolated from women with recurrent pregnancy loss and polycystic ovary syndrome (PCOS)77,78 Evidence from

a transcriptomic analysis suggests that eutopic endometrial gene expression is more dysregulated in severe endometri-osis than in mild endometriendometri-osis79 By contrast, pilot data with the endometrial receptivity array suggest that eutopic endometrial gene expression is not altered in different stages of endometriosis80 However, levels of noncoding RNA and protein production, as well as post-translational modifications within the eutopic endometrium, might be altered, mediating functional cellular changes81 Despite the association of endometriosis with infer-tility, embryo implantation proceeds normally in women with endometriosis who receive supplementary steroid

Table 1 | Properties of endometrial stem/progenitor cells Stem/progenitor

cell Stem-cell property Cell types and markers Frequency among endometrial cells Refs

Clonogenic cells (human) Ability of a single cell to form a colony when seeded

at low density in culture

Epithelial progenitor cells <1% 44

Mesenchymal stem cells (human)

Differentiate into multiple mesodermal cell types (fibroblasts, adipocytes, chondrocytes, osteocytes and smooth-muscle cells)

Side-population cells (human) Efflux DNA-binding dyes (Hoechst) because

of high expression of plasma-membrane transporter molecules

Endothelial cells (ERβ+) 51%* 50 Epithelial cells (ERα–PR–) 27% 50

Label-retaining cells (mouse) Quiescent, proliferate rarely and retain DNA-synthesis

label for a long time

Epithelial (ERα–); proliferate in response to oestradiol 3% 172,173 Stromal (84% ERα–, 16% ERα+);

12% proliferate in response to oestradiol

ER, oestrogen receptor; PDGFRβ, platelet-derived growth factor receptor β; PR, progesterone receptor; SUSD2, sushi domain-containing protein 2 *Frequency of cell types in the heterogeneous side-population cell population.

hormones82 Oocyte-donation studies have not demon-strated any association between endometriosis, embryo implantation and pregnancy outcome82,83 Endometriosis does not seem to negatively affect pregnancy outcome when standard endometrial-priming protocols are fol-lowed84,85, although it should be noted that these studies did not examine different stages of endometriosis In two meta-analyses86,87, severe endometriosis was found

to negatively affect the probability of pregnancy success

in women undergoing IVF

Diagnosis of endometriosis currently requires the direct surgical visualization of lesions at laparoscopy, so the identification of appropriate biomarkers is the sub-ject of considerable interest Noninvasive or minimally invasive biomarkers are urgently required, particularly for adolescent endometriosis The existence of a herit-able component to endometriosis is well supported, as the risk of disease is elevated in first-degree relatives

of women with severe endometriosis, and concordance

is high for disease incidence and stage in monozygotic twins88 However, despite extensive GWAS, no diag-nostic genetic signature is available yet, which suggests the involvement of epigenetic, rather than genetic, regulation89

Single and combined biomarkers, along with global techniques such as miRNA analysis, transcriptomics and proteomics, have been investigated in relation to endo-metriosis, as reviewed elsewhere90 However, no single biomarker or biomarker panel has been independently confirmed and tested for sensitivity and specificity A lack

of concordance between the results of different studies has prompted the establishment of guidelines for standardiza-tion of surgical and clinical data collecstandardiza-tion and biological sample collection and storage in endometriosis research91

Assisted reproductive technology Ovarian-stimulation

protocols and embryo-culture techniques have improved dramatically since the birth of the first ‘test-tube’ baby

in 1978, but pregnancy success rates with assisted reproductive technology (ART) have not significantly altered ART success in stimulated cycles remains around 25–30%92

Endometrial receptivity is abnormal in women undergoing ART93 The altered hormonal milieu that results from controlled ovarian hyperstimulation (COH) affects the development and timing of endometrial receptivity94 Women treated by ART with COH who do not become pregnant after fresh-embryo transfers tend

to have prematurely advanced endometrial histology

on day 2 after hCG treatment for oocyte maturation95 This abnormal histology correlates with the presence

of markers of decidualization, and elevated leukocyte numbers and activation; these alterations are not nor-mally seen until later in the menstrual cycle95 Similarly, implantation that occurs late in natural cycles, when the endo metrium is highly differentiated, is more likely to result in miscarriage than earlier implantation96 A study

of endometrial gene expression during IVF demon-strated that ovarian stimulation with follicle-stimulating hormone (FSH) and gonadotropin-releasing hormone (GnRH) antagonist leads to the occurrence of gene expression characteristic of a decidualized, late-secretory endometrium earlier in the cycle than in the absence of ovarian stimulation97

A promising approach to maximize reproductive success in ovulatory infertile couples is embryo freez-ing, with transfer of a thawed embryo into a natural cycle98 This strategy bypasses the detrimental effects of COH and supraphysiological hormone levels, enabling

Box 2 | Potential roles of endometrial stem/progenitor cells in endometrial disorders 43

Endometriosis

Normal stem/progenitor cells are shed into the peritoneal cavity by retrograde menstruation and probably establish ectopic clonal endometriotic growths170 In early‑onset endometriosis, retrograde neonatal uterine bleeding resulting from withdrawal of maternal hormones might seed the pelvic cavity with endometrial stem/progenitor cells that survive and become activated as oestrogen levels begin to rise at puberty74

Adenomyosis

The presence of normal endometrial stem/progenitor cells in an abnormal niche might enable ectopic endometrium to grow in the myometrium Inappropriate differentiation of endometrial mesenchymal stem cells into smooth‑muscle cells could account for the associated smooth‑muscle hyperplasia

Endometrial cancer

Mutations in the genome or epigenome of endometrial epithelial stem/progenitor cells might generate cancer stem cells that are responsible for tumour initiation, progression, metastasis and recurrence171

Thin, dysfunctional endometrium

Diminished activity of normal endometrial stem/progenitor cells, with an inability to respond to oestrogen stimulation, results in an atrophic endometrium (<7 mm thick) that is insufficient for embryo implantation and subsequent establishment of pregnancy43

Asherman syndrome

Damage or loss of normal endometrial stem/progenitor cells from injury to the endometrial basalis layer or postpartum infection in a setting of low oestrogen levels results in complete obliteration of the endometrium

by fibrous tissue43

Endometrial ablation

Heat‑induced damage to normal endometrial stem/progenitor cells prevents future growth of endometrial tissue

natural endometrial development The combination

of this approach with endometrial-receptivity testing could optimize reproductive success Biomarkers for receptivity have been reviewed elsewhere99,100 Both endometrial tissue and uterine fluid have been inves-tigated by genomic, proteomic and metabolomic methods in attempts to find a marker, or a cohort of markers, to use as a fingerprint to define a receptive endometrium Current effort is being directed to the validation of an RNA-based endometrial receptivity array101, which detected a nonreceptive endometrium

in ~25% of women with implantation failure during the expected time of endometrial receptivity, indicating a displacement of the implantation window The remain-ing women with implantation failure were classified as having a receptive endometrium during the expected window However, although such a test is useful, it can-not be performed in the implantation cycle because it requires an endometrial biopsy specimen, as well as time to complete the analysis Development of a rapid, noninvasive test that can be performed before embryo transfer, to facilitate clinical decision-making, is required

to overcome these limitations

PCOS Up to 20% of women of reproductive age have

PCOS102 The syndrome presents as hyperandrogen-ism, either clinical (hirsutism) or biochemical, with oligo-ovulation or anovulation, as well as polycystic ova-ries, and is often associated with hyperinsulinaemia and obesity Infertility affects 40% of women with PCOS103, mainly as a result of ovulation failure, with progesterone deficiency resulting from the lack of corpus luteum for-mation, which subsequently affects endometrial devel-opment However, even when ovulation is restored, women with PCOS have low pregnancy rates and high miscarriage rates (~73%)104, which are attributed

to the elevation of levels of oestrogen and androgens, and the presence of the metabolic syndrome (including hyperinsulinaemia and obesity), all of which affects the endometrium

The endometrium in women with PCOS is progester-one-resistant, exhibiting defective decidualization78,105,106 and altered levels of inflammatory mediators, which are likely to contribute to pregnancy complications and reductions in fertility107,108 In a gene-array analysis,

466 genes were differentially regulated in the mid- secretory endometrium in women with PCOS com-pared with unaffected women, and the expression pat-tern was indicative of progesterone resistance in PCOS78 Endometrial receptivity factors, such as glycodelin and LIF, are also dysregulated in the endometrium in women with PCOS78,104 Pregnancy success in IVF cycles

is affected, because of endometrial insulin resistance109 Specifically, the insulin-regulated facilitated glucose transporters GLUT1 and GLUT4 and the insulin receptor substrate (IRS) 1 are downregulated in the endometrium

in women with PCOS110 Interventions, including lifestyle changes and treat-ment with the insulin-sensitizing drug metformin, have been tested to determine their potential to restore menstrual cyclicity and fertility111 Results indicate that

restoration of ovulation and improvement in men-strual function, along with normalization of hormo-nal parameters (free testosterone, FSH and luteinizing hormone) can be achieved in women with PCOS by treatment with metformin112–114 or by intervention with hypocaloric diets and physical activity110,115,116 These interventions, which ranged from 6 weeks to 6 months

in duration, also improved endometrial function and expression of receptivity markers Endometrial blood flow112,113, GLUT4, GLUT1 and IRS1 (REFS 110,117) and the ERα:ERβ ratio116 were all improved from the levels

in untreated women with PCOS Evidence also indi-cates that metformin treatment throughout pregnancy continues to affect endometrial function, which results

in a decreased risk of miscarriage and an increased rate

of pregnancy and live birth compared with untreated women118–120

Receptivity is characterized by low androgen levels, and the elevation of androgens in women with PCOS

is likely to alter receptivity121 The endometrium in women with PCOS does not exhibit the expected downregulation of the androgen receptor during the receptive phase121 However, compared with untreated PCOS, metformin treatment and lifestyle interventions can reduce levels of free testosterone and endometrial expression of the androgen receptor, normalizing the endometrial androgen environment114,116,117

Thin endometrium and Asherman syndrome Repair

mechanisms and endometrial stem/progenitor cell function are potentially compromised in women with thin (<7–8 mm), dysfunctional endometrium that fails

to regenerate sufficiently for embryo implantation, and

in Asherman syndrome (intrauterine scarring), even with long-term use of oestrogen to stimulate endo-metrial growth43 (BOX 2) Endometrial stem/progenitor cells might be lacking, or unable to respond to oestro-gen via niche cells expressing ER122 In two case studies

of Asherman syndrome, autologous bone-marrow cells were administered into the sub-endometrial zone via a needle123, or infused into the uterine arterioles under ultrasonographic guidance These cell-based treatments were followed by oestrogen replacement for several months, but the results were modest in terms of endo-metrial receptivity, and the lack of controls necessitates caution in interpretation124

Endometrial ‘scratch’ Local endometrial injury (for

example, by biopsy or a scratch) is receiving attention for its potential effect on pregnancy success in IVF cycles This procedure is now recommended by up to 83% of IVF clinicians125, and the administration of an injury during the cycle preceding embryo transfer is proposed to double live-birth rates126

An important issue in the application of this technique

is to define the clinical population for whom endometrial injury could prove beneficial The initial results127, pub-lished in 2003, demonstrated a beneficial effect of endo-metrial injury on pregnancy outcome in IVF cycles in women with recurrent implantation failure who were clas-sified as good responders to hormonal stimulation Since

then, a large number of randomized, controlled trials (RCTs) and observational studies have been conducted, with results that either dispute128–130 or corroborate131–133 those of the original study A Cochrane meta-analysis126 of

14 RCTs provided evidence that endometrial injury dou-bles live-birth rates in IVF cycles in women with recurrent implantation failure However, this analysis could have underestimated the overall effect of endometrial injury, because women in the control groups could also have undergone some degree of endometrial manipulation126 The number and quality of the studies that were included

in the meta-analysis has been criticized, and the number

of participants in individual studies was generally low134 Overall, results suggest that endometrial damage might only be effective in women undergoing a fresh- embryo transfer cycle who have experienced recurrent implantation failure (two or more failures), which suggests that these women have abnormal receptivity135 The posi-tive effect of endometrial injury on embryo implantation was not replicated in oocyte recipients129, or in the results

of a well-designed RCT of women without recurrent implantation failure130 However, subgroup analysis in oocyte recipients suggested that endometrial injury

is beneficial as the number of previous failed embryo transfers increases129 In a study of women with recurrent implantation failure who underwent endometrial injury132, increased maternal age, elevated FSH during the proliferative phase of previous cycles and dimin-ished ovarian reserve were negatively associated with pregnancy outcomes

The mechanisms underlying the effects of local endo-metrial injury are unknown One possibility is that injury induces an inflammatory reaction within the uterus, improving synchronicity between the endometrium and the embryo126 However, as the endometrial damage is caused in the cycle preceding ovarian stimulation and embryo transfer, how it affects the subsequent cycle is unclear

Well-controlled studies are now required, focusing on women with recurrent implantation failure, determining the optimal timing of injuries and number of injuries per

menstrual cycle These studies present ethical challenges, but are necessary to prevent the withholding of a ben-eficial procedure, or the provision of an unproven one

Future avenues of endometrial research

The results of research in several areas relating to endo-metrial biology have suggested the potential benefits of future investigations (BOX 3)

The endometrial microbiome The concept of the sterile

uterus is no longer considered valid Indeed, the uterus and other tissues (including lung and bladder) widely cited as being free of bacteria are now known to harbour unique microbiota In the endometrium, deep sequenc-ing of a hypervariable region of the 16S ribosomal RNA gene identified 15 phylotypes that were present in each

of 19 samples from nonpregnant women136 In 90% of

these samples, Bacteroides spp were dominant, and Proteobacteria spp and Firmicutes spp were also

com-mon, presenting a unique uterine core microbiome136 that is quite different from that of the vagina Notably,

Bacteroides spp regulate certain mechanisms in the gut

that are relevant to the endometrium, including epithe-lial-cell maturation and maintenance, mucosal-barrier reinforcement and interactions with the host immune system to control other bacteria However, the low level microbial presence in the uterus is not associated with inflammation137

The endometrial epithelial surface and uterine fluid contain hormonally-regulated immunomodulatory molecules that are important to control infection57 and

to maintain the uterine microenvironment in a non-inflammatory state to enable its functions, including sperm chemotaxis, embryo development and implanta-tion Among the immunomodulatory molecules within uterine fluid, the oestrogen-regulated antileukoprotein-ase, a whey-acidic-protein-motif protein138 and human β-defensin-2 (REF. 139) (one of four β-defensins with dif-ferent cyclical expression profiles in the endometrium140) have antibacterial activity against both Gram-positive and Gram-negative bacteria141 Interferon-ε, the only oestrogen-regulated interferon in the endometrium,

is secreted from human uterine epithelial cells142, and might provide essential antiviral activity Neutrophils are also a source of antimicrobials, and are abundant during menstruation, when the epithelial layer is not intact140

Epigenetics and noncoding RNA Evidence suggests

that human endometrial remodelling, receptivity and the development of epigenetic pathologies are epigenet-ically regulated Epigenetics describes heritable changes that do not alter the genomic DNA sequence, but involve stable modifications of chromatin, DNA, pro-tein or noncoding RNA143 Emerging evidence indicates that hormonal and local paracrine responses within the endometrium are, in part, epigenetically regulated

Endometrial global histone acetylation varies across the menstrual cycle, which suggests epigenetic regulation

of gene expression Abnormal epigenetic modifica-tions might be associated with impaired receptivity and implantation failure144

Box 3 | Future directions in reproductive research

• Understanding epigenetic mechanisms that alter intergenerational inheritance and

contribute to endometrial pathologies

• Developing personalized medicine

• Developing platforms for timely, noninvasive assessment of endometrial receptivity,

endometriosis and early pre‑eclampsia

• Developing models to study environmental toxins and endocrine disrupters,

implantation and early post‑implantation pregnancy disorders

• Refining the use of endometrial stem/progenitor cells to enhance receptivity and

treat endometrial disorders

• Defining the dialogue between the blastocyst and the endometrium, and using this

information to enhance receptivity and treat infertility

• Developing models to recapitulate human endometrial disorders

• Establishing a biobank of material from patient cohorts with standardized collection

procedures

• Altering the microbiome to improve reproductive health

• Developing targeted treatment‑delivery strategies