Biology of Reproduction, 2019, 101(6), 1155–1166

doi:10.1093/biolre/ioy231
Precision Medicine in Assisted Reproductive Technologies Special
Issue
Advance Access Publication Date: 1 November 2018

Precision Medicine in Assisted Reproductive Technologies Special Issue

Biomarkers in abnormal uterine bleeding†

∗
Rohan Chodankar and Hilary O. D. Critchley
MRC Centre for Reproductive Health, Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh,
UK
∗
Correspondence: MRC Centre for Reproductive Health, Queen’s Medical Research Institute, The University of Edinburgh,
47 Little France Crescent, Edinburgh EH16 4TJ, UK. Tel: +441312426858, Fax: +441312426441,
E-mail: hilary.critchley@ed.ac.uk
†
Grant support: Medical Research Council (MRC Grants: G0000066, G0500047; G0600048; MR/J003611/1) for support of
several studies wherein data derived have been described in this review. This work was undertaken in the MRC Centre for
Reproductive Health which is funded by the MRC Centre grant MR/N022556/1.
Conference presentation: Presented in part at the 4th Biomarker Meeting: Personalized Reproductive Medicine, 12–13
April 2018, Valencia Spain.
Edited by Carlos Simón, PhD

Received 21 July 2018; Revised 23 October 2018; Accepted 31 October 2018

Abstract
Abnormal uterine bleeding (AUB) is an extremely common problem and represents a clinical area of
unmet need. It has clinical implications and a high cost for the healthcare system. The PALM-COEIN
acronym proposed by FIGO may be used as a foundation of care; it improves the understanding of
the causes of AUB, and in doing so facilitates effective history taking, examination, investigations,
and management.
Heavy menstrual bleeding, a subset of AUB, is a subjective diagnosis and should be managed in
the context of improving the woman’s quality of life. Available evidence suggests that there is poor
satisfaction with standard treatment options often resulting in women opting for major surgery
such as hysterectomy. Such women would benefit from a tailored approach, both for diagnosis
and treatment, highlighting the deficiency of biomarkers in this area.
This article focuses on the causes of AUB as per the PALM-COEIN acronym, the researched biomark-
ers in this area, and the potential pathogenetic mechanisms. In the future, these approaches may
improve our understanding of AUB, thereby enabling us to direct women to most suitable current
treatments and tailor investigative and treatment strategies to ensure best outcomes, in keeping
with the principles of personalized or precision medicine.
Summary Sentence
Abnormal uterine bleeding (AUB) is an extremely common problem and represents a clinical area
of unmet need. Identifying potential biomarkers in this area would allow for planning individualized
care and improving treatment outcomes.
Key words: angiogenesis, apoptosis, endometrium, estradiol/estradiol receptor, epigenetics, female reproductive
tract, menstrual cycle, proteomics, progesterone/progesterone receptor, uterus.

Introduction nomic, transcriptomic, proteomic, metabolomic, and imaging anal-

A biomarker is a characteristic that can be objectively measured and yses. Many commonly used tests in clinical practice can serve as
evaluated as an indicator of normal biological processes, pathogenic biomarkers, and the majority have been identified on the basis of
processes, or pharmacological responses to a therapeutic inter- insight or underlying physiology or biological mechanisms [1]. Ab-
vention. The ideal platforms for biomarker discovery include ge- normal uterine bleeding (AUB) is an extremely common problem


C The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction. This is an Open Access 1155
article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
1156 R. Chodankar and H. O. D. Critchley, 2019, Vol. 101, No. 6

Figure 1. FIGO classification of causes of AUB; “PALM-COEIN.”

and represents a clinical area of unmet need. Identifying potential Professor of the Practice of Physic at the University of Edinburgh.
biomarkers in this area would allow for planning individualized care. The term “metrorrhagia” probably came into use at the same time,
AUB was re-defined by Federation International de Gynecolo- with Cullen using the spelling “maetrorhagia” [10].
gie et d’Obstetrique (FIGO) in 2009 to introduce standardization of The introduction of the confusing modern term “dysfunctional
nomenclature and identify an etiological basis [2, 3]. Chronic AUB uterine bleeding” did not occur until the 1930s. Graves used the term
was defined as bleeding from the uterine corpus that is abnormal “dysfunctional uterine bleeding” to try and explain “impairment of
in volume, regularity, and or timing and has been present for the endocrine factors,” which normally controlled menstrual function
majority of the last 6 months. Acute AUB was defined as an episode [11].
of heavy bleeding that, in the opinion of the clinician, is of sufficient
quantity to require immediate intervention to prevent further blood
loss. Intermenstrual bleeding is defined as bleeding that occurs be- FIGO classification of AUB etiology
tween clearly defined cyclic and predictable menses and includes both Once a diagnosis of AUB has been established, the further classi-
randomly occurring episodes and those that manifest predictably at fication is based on the PALM-COEIN acronym. The system was
the same time in each cycle [2]. developed with contributions from an international group of both
Heavy menstrual bleeding (HMB) is a subcategory of AUB and clinical and nonclinical investigators from 17 countries on six con-
has a woman-centered approach to diagnosis. Rather than using tinents [3]. This system was created as a detailed assessment of the
objective measurements of volume or using PBAC (Pictorial Blood previously used menstrual terminology concluded that there is great
Assessment Chart) scores, NICE (National Institute for Care and confusion in the way these terminologies are used and there is an ur-
Excellence) define HMB as an excessive menstrual loss that interferes gent need for international agreement on the consistent use of terms
with the physical, social, emotional, and or material quality of life and definitions for symptoms, signs, and causes of abnormal uterine
[4]. This takes precedence over the previously used definitions of bleeding [12, 13].
menstrual blood loss of greater than 80 mL in both research and There are nine main categories: Polyp, Adenomyosis,
clinical settings [5–7]. Leiomyoma, Malignancy and Hyperplasia, Coagulopathy,
Ovulatory dysfunction, Endometrial, Iatrogenic, and Not oth-
erwise classified. In general, the components of the PALM group
History of menstrual terminology are discrete (structural) entities that may be identified visually with
From 430 BC until the early 1800s, the main menstrual symptom imaging techniques and or histopathology, whereas the COEIN
appearing in medical writings was excessively heavy bleeding [8]. group is related to entities that are not defined by imaging or
The description of periods in early writings also includes phrases histopathology (nonstructural) (Figure 1) [2].
such as “the flux is immoderate, either when the periods return The term “DUB,” which was previously used as a diagnosis when
too often, when they continue too long, or when too much blood there was no systemic or locally definable structural cause for AUB,
is discharged at one time.” Irregular and often light bleeding was should be abandoned [8]. These women generally have one or a
referred to as “the weeping of the womb” [9]. combination of coagulopathy, a disorder of ovulation, or primary
The term “menorrhagia” appears to have been used for the first endometrial disorder—the last of which is most often a primary or
time in the late 1700s in the lectures of Professor William Cullen, secondary disturbance in local endometrial hemostasis [2]. Similarly,
Biomarkers in abnormal uterine bleeding, 2019, Vol. 101, No. 6 1157

the terms menorrhagia and metrorrhagia should also be abandoned gen exposure and development of adenomyosis. Increasing age with
[3]. increased duration of estrogen exposure, and tamoxifen use posi-
tively correlate with adenomyosis risk [26]; interestingly, cigarette
Demographics smoking may be protective [27]. Increasing age with cellular dam-
There is a significant clinical burden associated with AUB, affecting age and repair may be contributory. Other gynecological conditions
14–25% of women in the reproductive age group [14, 15]. About that may be associated with adenomyosis include uterine fibroids
20% of the 1.2 million referrals to specialist gynecologist services and endometriosis, although there is a debate if the latter is a sepa-
concern women with HMB [15]. Current NICE guidance clearly rate entity at all [28]. At a cellular level, matrix metalloproteinases
states that HMB should be managed in the context of improving the (MMPs) may initiate damage to the endo-myometrial junction via
woman’s quality of life, rather than treating a target blood loss vol- the basement membrane [29], and cyclo-oxygenase (COX) enzymes,
ume [4]. A recent HMB audit by the Royal College of Obstetricians vascular endothelial growth factor (VEGF), and stem cell progenitors
and Gynaecologists, assessing patient outcomes and experiences in may contribute to the development of adenomyosis [30, 31].
England and Wales, reported that 1-year post referral only 30% of In a study by Li et al, aberrations in angiogenesis were proposed
women (including those managed with surgery) were “satisfied” (or as mechanisms to explain histological changes in adenomyosis. A
better) at the prospect of current menstrual symptoms continuing, positive correlation was observed between VEGF expression and
as currently experienced, for the next 5 years [15]. MMP-2 and MMP-9 expression. A positive correlation was also
Thus, menstrual problems represent a clinical area of unmet need. found between mean vessel diameter (MVD) and MMP-2 or MMP-
Poor satisfaction with standard treatment options often results in 9 expression. It seems, therefore, that the elevation of MMP-2 or -9
women opting for major surgery such as hysterectomy. Such women expression may represent an important factor in the development of
would benefit from a tailored approach, both for diagnosis and treat- the disease, contributing to the invasion of endometrial tissues into
ment, highlighting the deficiency of biomarkers in this area. the myometrium and angiogenesis in adenomyotic implants [32].
Advances in genomics have ushered in a new era called “person- Recent evidence also points to epithelial to mesenchymal tran-
alized” or “precision” medicine, which takes into account individual sition (EMT) in the pathogenesis. Epithelial-to-mesenchymal transi-
genetic and other sources of variability in disease treatment and pre- tion is a process characterized by a loss of polarity of epithelial cells
vention. A strong rationale for the shift toward precision medicine and transition to a mesenchymal phenotype, which at a molecular
was laid by the National Research Council [16]. Genomic profiling level involves downregulation of epithelial markers (e.g. E-cadherin)
of endometrium may offer a significant step forward, in the health- and overexpression of mesenchymal markers (e.g. fibronectin and
care for women with AUB/HMB, both by directing patients to most vimentin), and activation of a number of transcription factors, in-
suitable current treatments and by informing new avenues for effec- cluding Snail, Slug, Twist, Zeb1, and SIP1 [33].
tive and personalized medical management. Reduced apoptosis and increased proliferation of the eutopic en-
In keeping with the PALM-COEIN classification system, AUB- dometrium could play a role in the pathophysiology of adenomyosis
E possibly represents a primary endometrial disorder, while AUB- [34]. This was further quantified using a Ki-67 (a nuclear and nucle-
L and AUB-A, which are still poorly understood, potentially, may olar protein that is strictly associated with cell proliferation) labeling
represent, a secondary endometrial disorder. index by Yang and colleagues [35]. The study involved analyzing eu-
topic endometrium in premenopausal women with and without ade-
nomyosis who underwent a hysterectomy. The endometrium was
Polyps and AUB (AUB-P) separated into endometrial stromal cells (ESCs). Markers for pro-
Endometrial polyps are epithelial proliferations arising from the en- liferation were investigated using nonradioactive assay kits, and im-
dometrial stroma and glands. The majority are asymptomatic The munohistochemistry (IHC) and those for apoptosis was analyzed us-
reported prevalence of endometrial polyps varies widely and ranges ing fluorescence-activated cell sorter. Sotnikova et al have suggested
from 7.8 to 34.9%, depending on the definition of a polyp, the diag- impaired cytokine production in the development of adenomyosis
nostic method used, and the population studied [17–20]. There are [36].
no identified biomarkers for polyps in clinical use, and their diag- Studies also propose an increased synthesis of local estrogen and
nosis relies on imaging, ultrasonography (USS), sonohysterography, possibly, progesterone resistance in women with adenomyosis. In-
and hysteroscopy [4]. creased aromatase and estrone sulfatase activity in glandular cells
of eutopic and ectopic endometrial tissues in women with adeno-
Adenomyosis and AUB (AUB-A) myosis can contribute to increased local uterine/endometrial estro-
Adenomyosis is defined as the presence of ectopic endometrial glands gen production, and reduced progesterone receptor B isoform (PR-B
and stroma in the myometrium, although it remains a poorly under- receptors) may contribute to local progesterone resistance [37, 38].
stood entity. The prevalence of adenomyosis is difficult to ascertain The concept of repeated tissue injury and repair in women with
because of a wide variation in diagnostic criteria both with imaging adenomyosis is supported in a study by Liu and colleagues. They pro-
modalities and with histology. It has been estimated that histological pose that platelet-induced activation of the TGF-β/Smad signaling
confirmation of adenomyosis ranges from 5 to 70% of patients who pathway may be a driving force in EMT, fibroblast-to-myofibroblast
undergo hysterectomy [21]. Adenomyosis is thought to cause HMB, transdifferentiation, and smooth muscle metaplasia in the develop-
dysmenorrhea and infertility [22] although not all studies agree that ment of adenomyosis leading to fibrosis. Platelets may also be in-
HMB is a causal association [23]. volved in uterine hyperactivity and myometrial hyperinnervation,
Risk factors for adenomyosis include increasing parity, termi- potentially contributing to adenomyosis symptoms [39].
nation of pregnancy, uterine curettage, and caesarean birth, all of Diagnosis of adenomyosis is based on histology (at hysterectomy)
which may disrupt the endo-myometrial junction and thereby al- and imaging. The common imaging modalities used for nonhisto-
low infolding of the endometrium with direct myometrial invasion logical diagnosis of adenomyosis include transvaginal ultrasound
[24, 25]. There also appears to be an association between estro- (TVUS) and magnetic resonance imaging (MRI), although a clear
1158 R. Chodankar and H. O. D. Critchley, 2019, Vol. 101, No. 6

consensus on imaging criteria remains lacking. A recent systematic a high risk of developing disease for prognosis and prediction of
review confirms that TVUS 2D is effective and should be consid- response, identification of individuals who are most likely to re-
ered as the first-line ultrasound imaging method for the diagnosis spond to specific therapeutic interventions, and prediction of which
of adenomyosis. Enhancing tools such as TVUS 3D improved upon patients will develop specific side effects. Proteomics analysis has
sensitivity when used with poor definition of the junctional zone, been used to compare the differential protein expression profile be-
while no improvement was noted in the overall sensitivity compared tween matched ectopic and eutopic endometrium of adenomyosis.
to TVUS 2D [40]. Newer imaging techniques such as elastography The study showed that a group of estrogen-responsive proteins were
in addition to conventional ultrasound may hold potential in the significantly altered and amongst them, Annexin 2 (ANXA2) was
future to assist with diagnosis of uterine focal lesions and may be identified as a key player in adenomyosis development by inducing
useful in preoperative planning [41]. A recent study has also pro- both metastasis and proangiogenesis of adenomyotic endometrial
posed that TV elastography can improve the diagnostic accuracy for cells. The invasive and metastatic potential involved in adenomyosis
adenomyosis, especially in differentiating it from uterine fibroids. was achieved by ANXA2-induced β-catenin/T-cell factor associated
The study also suggests a role for elastography in the assessment of EMT-like switch in endometrial cells, and the proangiogenic capacity
the developmental stage of adenomyotic lesions and a guide for the in local lesion was enhanced via ANXA2/HIF-1α/VEGF-A pathway
best treatment modality for the patient [42]. activation [48].
A meta-analysis comparing TV US and MRI in the adenomyosis Proteomic analysis using matrix-assisted laser desorption ioniza-
concluded that both techniques showed high levels of accuracy, al- tion time-of-flight mass spectrometry (MALDI-TOF-MS) has been
though a correct diagnosis was obtained more often with MRI [43]. studied in women with adenomyosis and endometriosis. The study
The Morphological Uterus Sonographic Assessment (MUSA) group compared protein peaks using the MALDI-TOF-MS system in the
has proposed uniform criteria for the diagnosis of both uterine fi- serum of women with endometriosis or adenomyosis to controls and
broids and adenomyosis to facilitate consistent reporting in both identified a possible biomarker for the conditions, but was unable to
daily clinical practice and for research purposes [44, 45]. differentiate between endometriosis and adenomyosis [49].
Several biomarkers have been explored in research settings for The nuclear factor kappa light chain enhancer of activated B cells
diagnosing adenomyosis, but none have been adapted for clinical (NF-κB) pathway has long been considered a proinflammatory sig-
use. Caveolin (CAV) proteins are the fundamental components of naling pathway, largely based on the role of NF-κB in the expression
caveolae that form different structural and functional microdomains of proinflammatory genes including cytokines, chemokines, and ad-
in a wide variety of cell types. A study evaluated the expression of hesion molecules. It also plays an important role in apoptosis and
CAV 1 in the ESCs in the human uterus affected by adenomyosis and cellular growth [50]. NF-κB activity is regulated by a family of pro-
concluded that loss of stromal CAV1 expression may play a critical teins known as IκBs. There are two pathways of (NF-kB) activation
role in the pathogenesis of adenomyosis. Loss of stromal CAV1 ex- that are known currently. The canonical pathway is triggered by mi-
pression enhanced metastasis of ESCs and enabled increased growth, crobial products and proinflammatory cytokines such as TNFα and
migration, and invasion of endometrial epithelial cells (EECs) that IL-1, while the alternative pathway is triggered by TNF-family cy-
might involve the release of RANTES in the stroma of the ectopic le- tokines but not TNFα [51, 52]. Immunoreactive proteins have been
sion. RANTES (also termed CCL5), a chemokine for monocytes and studied as potential biomarkers, including p65, p50, and p52. Nu-
activated T cells, significantly correlates with the severity of stages clear p65 immunoreactivity was positively associated with heavier
and dysmenorrhea in women with deep infiltrating endometriosis. menses and decreased PR-B, and increased nuclear p65 immunore-
The expression level of RANTES in the ectopic ESCs of adenomyosis activity in the ectopic endometrium was associated with the severity
patients was significantly higher than that of the eutopic counterpart. of dysmenorrhea in women with adenomyosis [34, 38].
Silencing of stromal CAV1 in ESCs could trigger nitric oxide (NO) Huang et al. proposed that an imbalance between apoptosis and
and prostaglandin E2 (PGE2) production in ESCs, potentially con- proliferation may contribute to the pathogenesis and progression of
tributing to the symptom of dysmenorrhea [46]. adenomyosis. The presence of ectopic endometrium in adenomyosis
Moesin, a protein encoded in human by the MSN gene, has been is likened to a tumor-like invasiveness, although poorly understood
proposed as a biomarker for adenomyosis. Using proteomic analy- [53]. Tyrosine kinase receptor B (TrkB) is a neurotrophic recep-
sis, a higher expression of moesin was noted in adenomyosis versus tor and contributes to tumor cells’ resistance to apoptosis, and the
normal endometrium. This was initially identified using IHC with acquisition of invasive and metastatic abilities [54]. Moreover, over-
a higher expression in ESC than in EEC and confirmed using RT- expression of TrkB in several types of human malignancy supports
PCR and western blot. An association between moesin as a marker this hypothesis. The study concluded that TrkB protein and TrkB
for EMT has been already proposed and may contribute to our un- mRNA in adenomyotic endometrium were elevated and positively
derstanding of the pathophysiology of adenomyosis. Adenomyosis correlated with the degree of dysmenorrhea. This may contribute
development mimics the process of tumor metastasis, which is char- to our understanding of the pathogenesis of adenomyosis and may
acterized by progressive transmyometrial invasion of endometrial represent a potential biomarker for disease progression in the future
cells and neovascularization in ectopic lesions. To explain the inva- [53].
siveness seen in adenomyosis, the authors propose a further review Tissue factor (TF) is a cell membrane-bound glycoprotein and a
of the phosphorylation of moesin in women with adenomyosis, as in member of the cytokine receptor family [55]. Tissue factor is mainly
certain tumors such as invasive gastric adenocarcinoma, the extent expressed in ESC and is regulated by progesterone. Tissue factor is
of invasiveness correlates with moesin expression [47]. involved in the pathogenesis of endometriosis, possibly in angiogenic
A common method employed for biomarker discovery is pro- and inflammatory signaling and has been evaluated as a biomarker
teomics, which is, in essence, a large-scale study of proteins. in women with endometriosis [56]. Tissue factor elevation in women
Proteomic-based approaches for biomarker investigation can be em- with endometriosis is thought to explain symptoms of dysmenorrhea
ployed in different aspects of medicine, such as elucidation of path- and HMB, and given the similarities between endometriosis and ade-
ways affected in disease, identification of individuals who are at nomyosis, the authors proposed to identify elevated TF expression
Biomarkers in abnormal uterine bleeding, 2019, Vol. 101, No. 6 1159

Table 1. Researched biomarkers and potential pathogenesis in adenomyosis.

r Increased MMP expression [32]
r Epithelial to mesenchymal transition (EMT) [33]
r Increased proliferation (KI-67) and reduced apoptosis in the eutopic endometrium [35]
r Increased local estrogen production—increased aromatase and estrone sulfatase activity [35]
r Increased local progesterone resistance mediated by progesterone receptor B isoform (PR-B receptors) [38]
r Repeated tissue injury and repair—platelet-induced activation of the TGF-β/Smad signaling pathway [39]
r Reduced expression caveolin (CAV) proteins [44]
r Increased moesin expression [47]
r Increased Annexin 2 (ANXA2) expression [48]
r Activation of NF-κB [51, 52]
r Increased expression of TrkB [51,52]
r Increased SLIT/ROBO expression [60, 61]
r Imaging—elastography [41, 42]

in women with adenomyosis [57]. The study showed increased TF biomarkers have been identified (discussed below), none are in clin-
immunoreactivity in the adenomyotic endometrium (eutopic and ec- ical use. It is well established that fibroids are monoclonal tumors
topic) versus controls (no adenomyosis) and had a strong association arising from the smooth muscle cells of the myometrium. Fibroids
with HMB and dysmenorrhea [57]. contain three different cell populations: fully differentiated smooth
SLIT is a secretory glycoprotein that acts via its receptor ROBO, muscle cells, intermediate cells, and fibroid stem cells, which in turn
a transmembrane protein. SLIT-ROBO system is reported to func- are crucial to fibroid growth. A genetic hit in the myometrial stem
tion as a chemoattractant to recruit vascular endothelial cells to cell can produce fibroid stem cells. These genetic hits include mu-
sites for vasculogenesis [58]. Increased SLIT expression correlates tations in the mediator complex (MED) 12 gene and chromosomal
with increased MVD and is a marker for tumor angiogenesis [59]. rearrangements on the high mobility group A (HMG2A) gene on the
SLIT immunoreactivity is increased in endometriosis. Its elevation long arm of chromosome 12 [68–71].
may be a constitutive biomarker for recurrence of endometriosis, Endocrine-disrupting chemicals (EDCs) are substances in our en-
and given the similarities between adenomyosis and endometriosis, vironment, food, and consumer products that interfere with hor-
SLIT has been explored as a potential biomarker in women with mone biosynthesis, metabolism, or action resulting in a deviation
adenomyosis [60]. In comparison to the normal endometrium, Nie from normal homeostatic control or reproduction and there is evi-
and colleagues demonstrated that SLIT expression was higher in the dence to suggest that exposure to EDCs, especially in critical phases
ectopic endometrium of women with adenomyosis, while ROBO1 of uterine development such as in utero and early childhood, may
immunoreactivity and MVD were higher in both eutopic and ectopic result in genetic mutations influencing fibroid growth [72, 73].
endometria of women with adenomyosis and that these biomarkers Fibroids are steroid hormone-dependent tumors; however, un-
positively correlated with the severity of dysmenorrhea [61]. Table like differentiated fibroid cells, fibroid stem cells have a very low
1 summarizes the researched biomarkers and potential pathogenetic expression of estrogen and progesterone receptors, indicating that
mechanisms of adenomyosis. these hormones exert their tropic effects on fibroid stem cells via a
paracrine mechanism. Fibroids also secrete increased transforming
Leiomyomas and AUB (AUB-L) growth factor-beta 3 (TGF-β3) in response to steroids (see below).
Uterine fibroids (myomas, leiomyomas) are the most common be- TGF-β3 is a cytokine that is involved in cell differentiation, em-
nign tumors in women of reproductive age present in almost 80% bryogenesis, and development and is believed to regulate molecules
of all women by the age of 50 [62]. Fibroids tend to be twice or even involved in cellular adhesion and extracellular matrix (ECM) forma-
three times more common in black women as compared to other tion. TGF-β has a direct effect on fibroid ECM production, stimulat-
racial or ethnic groups [63]. The association between AUB and fi- ing collagen expression as well as plasminogen activator inhibitor-1
broids is complex and poorly understood, as women with fibroids expression [74]. The role of TGF-β in fibrotic processes such as liver
may be asymptomatic; however, a strong association exists between cirrhosis and pulmonary fibrosis is well established, and further re-
submucous myomas and AUB, demonstrated as early as 1956 [64]. search may improve our understating of fibrotic pathways associated
The proposed mechanisms of how fibroids may cause AUB in- with fibroids [75].
clude an increase in the endometrial surface, an increase in uterine The role of the WNT/β-catenin pathway is also of importance in
vascularization, changes in patterns of myometrial contractility, ul- fibroid growth. Mutations in MED 12 genes are believed to lead to
ceration of the surface of a myoma, myoma degeneration, and uterine alterations in the WNT/B-catenin pathway expression and signaling.
venous ectasia by compression effect from the myoma(s) [65]. These This results in degradation of cytoplasmic β-catenin and increased
proposed mechanisms often relate to fibroid size, but cannot explain nuclear β-catenin, which is associated with increased fibroid bur-
completely the relationship between AUB and fibroids. There is a den in murine models [76, 77]. In human fibroid cells, silencing
correlation between AUB and the degree of distortion and penetra- the MED 12 gene results in decreased WNT/β-catenin pathway sig-
tion of the uterine cavity associated with the fibroid(s). Submucous naling, thereby slowing fibroid growth [70]. The WNT/β-catenin
myomas (FIGO 0, 1, 2, and 3) are thought to be most symptomatic pathway also results in increased expression of TGF-β3. A recent
[66]. Distortion of the uterine cavity by fibroids is also proposed to study by Sinclair et al. suggested that leiomyoma-secreted TGF-β3
explain other symptoms such as infertility [67]. induces BMP-2 resistance in the endometrium by downregulation
In recent years, our understanding of fibroids at a molecular level of BMPR-2, likely causing defective endometrial decidualization.
and cellular has significantly improved. Although several potential TGF-β3 also reduces expression of plasminogen activator inhibitor-1
1160 R. Chodankar and H. O. D. Critchley, 2019, Vol. 101, No. 6

Table 2. Researched biomarkers and potential pathogenesis—fibroids.

r Chromosomal rearrangements on the high mobility group A (HMG2A) gene [71]
r Mutations in the mediator complex (MED) 12 gene [68–70]
r Exposure to endocrine-disrupting chemicals (EDCs) [72, 73]
r Increased production of transforming growth factor-beta 3 (TGF-β3) with increased BMP-2 resistance [74]
r WNT/β-catenin pathway [70, 76–78]
r Altered cytokines such as interleukins (IL) -10,13,17 [84]
r Imaging—elastography [41]

Table 3. Detection of coagulopathies (adapted from Kouides et al [87]).

Structured history—positive screen if

a. Excessive menstrual bleeding since menarche, or

b. History of one of the following—postpartum hemorrhage, surgery-related bleeding, or bleeding associated with dental work, or
c. History of two or more of the following—bruising greater than 5 cm once or twice/month, epistaxis once or twice/month, frequent gum bleeding,
family history of bleeding symptoms.

(PAI-1), ATIII, and thrombomodulin in the endometrium, likely con- did so positively, whereas 43 did so negatively. Among the genes
tributing to AUB/HMB. In the past, TGF-β3 has been shown to be that were upregulated with the size of the intramural fibroid, there
involved in ECM remodeling and proliferation which could modu- was an association with larger blood vessel size, a feature that corre-
late fibroid growth [78]. lates well with the angiogenesis involved in fibroid vascular supply
AUB/HMB associated with fibroids may be explained by a com- and growth. Similarly, immune response and response to wounding
plex interplay between coagulation, neo-angiogenesis, and vaso- were underrepresented when a fibroid was present. An impairment
constriction. As described above, fibroids secrete TGF-β3, which of maturation and differentiation of lymphocytes in women with
in turn may alter normal hemostatic and fibrinolytic pathways large leiomyomas suggested a decrease in the local immune response
through PAI-1, ATIII, and thrombomodulin in endometrium [78]. [85].
Evidence also suggests an increased expression of fibroblast growth Despite all the advances discussed above, a biomarker for identi-
factor and fibroblast growth factor receptor in the endometrium fying causal factors underlying AUB in women with fibroids remains
of women with fibroids [79]. Other angiogenic factors such as elusive currently, and reliance for identifying the presence of uter-
heparin-binding epidermal growth factor, platelet-derived growth ine fibroids is placed on imaging modalities (USS, MRI, sonohys-
factor, VEGF, parathyroid hormone-related protein, and prolactin terography) and hysteroscopy. Table 2 summarizes the researched
are also altered in women with fibroids [80]. This could explain al- biomarkers and potential pathogenetic mechanisms of leiomyomas.
tered neo-angiogenesis and HMB with fibroids. Endothelin-1 (ET)
and prostaglandin F2 alpha (PGF2 α) are potent vasoconstrictors that
regulate menstruation [81]. Vasoconstrictors that regulate myome- Malignancy and AUB (AUB-M)
trial contractility (ET-1, PGF2 α) and spiral arteriole vasoconstriction It is beyond the scope of this article to discuss malignancy-related
(ET-1) are altered in women with fibroids [82, 83]. PGF2 α produc- biomarkers as it is primarily focused on benign pathology.
tion is increased in women with uterine fibroids [82]. Endothelin-
1 acts via the ETA -R and ETB -R receptors and higher levels of
endothelin-1 have identified in the endometrium as compared to the
Coagulopathy and AUB (AUB-C)
fibroid tissue and myometrium. In addition, higher levels of ETA -R
Coagulopathies are reported to affect 13% of the women present-
are identified in the myometrium compared to fibroid tissue and vice
ing with HMB [86]. The systemic disorders of hemostasis may be
versa for ETB -R. These alterations suggest disordered ET function
identified in 90% of women using a structured history (see Table 3)
in women with uterine fibroids [83]. The consequence of perturbed
[87].
expression of these vasoconstrictors results in alerted myometrial
There are clearly defined biomarkers for this cause of AUB, so
contractility and dilatation of endometrial stromal venous spaces
long as health professionals are meticulous at screening women at
and may explain HMB associated with fibroids.
risk and offering onward referral to appropriate specialists. These
Changes in circulating levels of cytokines such as interleukins
biomarkers include a full blood count, measurement of individual co-
(IL)-10, 13, and 17 have been identified in women with fibroids [84].
agulation factor quantity and/or activity, D-Dimer, fibrinogen, inter-
In general, an infection is accompanied by an inflammatory process;
national normalized ratio, partial thromboplastin time, prothrombin
however, an inflammatory response evidenced by altered cytokine
time, thrombin time, platelet function test, ristocetin cofactor, von
levels in the endometrium (out with infection), as a mechanism for
Willebrand factor antigen, and several more. It is beyond the scope
HMB associated with fibroids, remains to be established.
of this article to discuss individual biomarkers for AUB-C.
A clinical and functional genomics analysis in women with fi-
In the original FIGO PALM-COEIN system, women with
broids was undertaken and concluded that that intramural leiomy-
AUB associated with the use of anticoagulants were categorized
omas not affecting the endometrial cavity alters the expression pat-
with coagulopathies (AUB-C). In the updated classification (2018),
tern of some endometrial genes, but the genes involved in implanta-
they are considered iatrogenic and classified as AUB-I. This in-
tion are not affected. The study identified that the expression of 69
cludes the modern, nonvitamin-K antagonists such as rivaroxa-
genes strongly correlated with the size of the myoma, and 26 genes
ban that appears to have a greater impact on the volume of
Biomarkers in abnormal uterine bleeding, 2019, Vol. 101, No. 6 1161

menstrual bleeding than the traditional, vitamin K antagonists Instead, changes in the microenvironment may reprogram the few
such as warfarin [13, 88]. functional cells remaining after menstruation to regenerate a new
functional layer [91].
It is well established that progesterone withdrawal secondary to
Ovulatory disorders and AUB (AUB-O)
the demise of the corpus luteum in the absence of pregnancy is the sig-
Anovulation is observed at extremes of age, in association with en-
naling event for the onset of menstruation. A key role is played by de-
docrine disorders such as hypothyroidism, polycystic ovarian syn-
cidualized ESCs, as they remain responsive to progesterone through
drome, hyperprolactinemia, and with factors such as mental stress,
the secretory phase. They retain progesterone receptor (PR) expres-
extremes of weight, excess exercise, and even drugs that interfere
sion, thereby allowing the endometrium to respond to progesterone
with the hypothalamic–pituitary–ovarian axis such as dopamine ag-
withdrawal. Progesterone withdrawal is proposed to have two major
onists. Anovulatory cycles tend to present as an alteration in cycle
effects: (a) increased levels of cytokines and prostaglandins into the
length (often > 38 days) and AUB/HMB due to the effect of unop-
endometrium and consequently (b) influx of leukocytes, activation
posed estrogen on the endometrium.
of MMPs, and destruction of the ECM [92]. The action of MMPs
Although a well-structured history and examination may iden-
is thought to be independent to progesterone withdrawal after an
tify many cases, specific tests may be ordered to rule out en-
initial inflammatory response.
docrinopathies. Thus, there are clinically relevant biomarkers for
Neutrophil type leukocytes predominantly increase in the en-
this cause of AUB, such as serum thyroid stimulating hormone and
dometrium and contain high levels of MMPs and can activate
thyroxine levels, prolactin levels, gonadotropin levels (FSH/LH), sex
local MMPs, initiating endometrial breakdown. Increased B-cell
hormone binding globulin, free androgen index, and so on. It is be-
lymphoma 2 (BCL 2– an apoptosis regulator) levels secondary
yond the scope of this article to discuss individual biomarkers for
to progesterone withdrawal, limit neutrophil activity in the en-
endocrinopathies contributing to AUB-O.
dometrium and prevent a damaging chronic inflammatory response
Bao et al. have presented data to identify serum amyloid protein
[93]. Macrophages also increase perimenstrually and produce cy-
A (SAA) as a potential biomarker to differentiate between ovula-
tokines and proteases and are involved in tissue remodeling and
tory and anovulatory AUB. Using surface-enhanced laser desorption
debris removal [94, 95].
ionization TOF-MS, they identified three protein peaks correspond-
Inflammatory responses in the endometrium are mediated via
ing to SAA, VEGF, and anti-vitamin K epoxide reductase (VKOR).
the NF-κB pathway, secondary to steroid hormone withdrawal. NF-
Given that SAA is highly expressed in individuals with nonimmune
kB increases the transcription of a wide variety of genes, includ-
inflammation, the authors hypothesized a similar response in women
ing cytokines (IL-1, IL-6), chemokines (CXCL8/IL-8, chemokine
with AUB, that SAA was highly expressed in the menses (sera and
ligand 2/CCL-2), and adhesion molecules [96]. Increased IL-8
supernates) of anovulatory and ovulatory women with AUB versus
mRNA expression in premenstrual endometrium and localiza-
controls. The role of VEGF is well established in menstruation and
tion to perivascular cells by the withdrawal of progesterone has
endometrial repair [89]. In this study, VEGF was highly expressed
been shown by Milne et al. [97]. A role for cyclo-oxygenase
in ovulatory women with AUB but poorly expressed in anovulatory
(COX)-2 following progesterone withdrawal has also been demon-
patients suggesting a possible aberration in angiogenesis in anovula-
strated [98]. Inhibition of the COX enzymes or NF-kB at the
tory AUB. Vitamin K is essential in the clotting cascade and requires
time of progesterone withdrawal significantly decreased the amount
VKOR for this process. The authors noted a poor expression of
of bleeding and endometrial breakdown and leukocyte influx in
VKOR in the menses of women with ovulatory AUB possibly sug-
a murine model [99].
gesting a defect in clotting [90].
Menstruation has been proposed to occur also as a result of a
physiological process of ischemia and reperfusion. Ischemia has not
Endometrial disorders and AUB (AUB-E) been detected in the human endometrium during menstruation to
AUB that occurs in the absence of an identifiable histological or date; however, evidence supports the occurrence of hypoxia in the
structural cause (AUB-L, AUB-A, AUB-P), in the context of regular endometrium. Markers for hypoxia (CAIX and hypoxia inducible
menstrual cycles (ovulatory) and with coagulopathy ruled out, in factor-1α [HIF-1α]) have been detected in the human endometrium
the absence of iatrogenic causes (AUB-I) usually represents a pri- during menstruation [100]. The current evidence supports the
mary endometrial disorder (AUB-E). AUB-E is thought to be caused role of hypoxia and HIF-1α in the process of endometrial repair
by a local disturbance(s) in endometrial function—deficiencies or ex- during menstruation [101].
cesses of proteins or other entities that have an adverse impact on Vasoconstriction of the uterine spiral arterioles mediated by
hemostasis, normal angiogenesis, vascular integrity, or endometrial PGF2α and ET-1 is considered to play a role in determining blood
repair. AUB-E is a diagnosis of exclusion; a well-structured history loss during menstruation. PGE2 is known to have a vasodilatory
and examination often help, but there is no commercially available effect [102]. Evidence supports the theory of HMB secondary to a
testing. Hence, a clear role for developing biomarkers exists for this reduced endometrial expression of ET-1 and an altered PGF2α/ PGE2
cause of AUB. ratio. The reduced maturity of the uterine spiral arteriole vessel wall,
The endometrium is a complex multicellular tissue that lines the increased gaps in the endothelial cell lining, and reduced vascular
inside of the endometrial cavity and involves interactions of immune, smooth muscle proliferation may all contribute to HMB [103–106].
endocrine, and vascular systems. It is morphologically divided into Cessation of menstruation relies on an intact endometrial coag-
functional and basal layers. The functional layer occupies the upper ulation system. Endometrial endothelial injury initiates immediate
two-thirds of the endometrium. During endometrial repair and pro- activation and aggregation of platelets to form a plug. The subse-
liferation, mitosis occurs in the functional layer of the endometrium, quent stage of hemostasis involves the formation of fibrin via the
a highly active layer consisting of glands supported by stroma. Stud- coagulation cascade. Tissue plasminogen activator and urokinase
ies now demonstrate that the basal layer may not serve as a source of plasminogen activator drive the production of plasmin, and PAI in-
stem cells for endometrial regeneration after normal menstruation. hibits fibrinolytic activity [107]. There is evidence that an overactive
1162 R. Chodankar and H. O. D. Critchley, 2019, Vol. 101, No. 6

fibrinolytic system in the endometrium interferes with hemostasis role of Chlamydia trachomatis and AUB has also been described,
and contributes to HMB [108]. and its prevalence is thought to be underestimated as a cause of
Angiogenesis and spiral arteriole maturation are essential compo- AUB [118].
nents of repair during menstruation, a process that is usually com-
pleted by cycle day 5. Vascular endothelial growth factor, a key
mediator of vascular function, is increased in women at menses, and Future directions
is regulated by hypoxia [109]. This process is independent of steroid Although to date, there is a limited success in the clinical use of
hormones. Recently, interest has increased in the role of immune cell biomarkers for women with AUB, this remains an area of unmet
influx at the time of menstruation the role they may play in the regu- need. In a medical context, the word “phenotype” is used to refer to
lation of endometrial bleeding. The uterine natural killer (uNK) cells some deviation from normal morphology, physiology, or behavior.
may play an important role in spiral arteriole maturation, which in Deep phenotyping can be defined as the precise and comprehensive
turn will impact upon vasocontraction and potentially reduced men- analysis of phenotypic abnormalities in which the individual com-
strual blood loss [110]. Dysregulation of uNK cells in HMB has also ponents of the phenotype are observed and described. The emerging
been demonstrated, which may have an impact on endometrial vas- field of precision medicine aims to provide the best available care
cular development and or endometrial preparation for menstruation. for each patient based on stratification into disease subclasses with
[111]. a common biological basis of disease [119]. There is a clear need for
Newer research has focused on the role of hypoxia and the HIF- developing a system of deep phenotyping for women with AUB, such
1α in endometrial repair using a murine model of simulated menstru- that individualized and personalized care can be offered to ensure
ation. Validation of the mouse model of menstruation has already best results with treatment strategies.
been performed [95]. The study by Maybin et al. demonstrated re- A role for the endometrial microbiome has been proposed, and
duced endometrial HIF-1α in women with AUB/HMB. The study a study by Pelzer et al. using microbial community profiling re-
suggests that HIF-1α regulates response within cells to low oxygen vealed differences in the endometrial microbial community profiles
levels (hypoxia), increasing the production of a number of repair fac- for (1) the endocervix compared to the endometrium, and (2) women
tors and therefore playing an important role in repair of the denuded with HMB versus dysmenorrhea [120]. This allows for further ex-
endometrial surface [101]. ploration in this field to try and understand the pathogenesis and
Despite the vast improvements in the understanding of the cel- develop management strategies for women with AUB.
lular and molecular basis of menstrual physiology, clinically usable The role of exosomes using proteomic analysis in predicting ad-
biomarkers remain lacking in women with AUB-E. verse pregnancy outcomes and suggesting pathophysiologic mecha-
nisms has been explored in the context of preterm birth [121]. Exo-
somes act as proxies for cells and therefore serve as better biomarkers
Iatrogenic AUB (AUB-I)
than secreted biochemicals from cells. Exosomes may hold a role in
AUB may be associated with the use of exogenous steroids, usually as
the future as tools for “noninvasive” tissue sampling in women with
continuous estrogen and or progesterone therapy results in unsched-
AUB. Exosomes derived from menstrual blood have already been
uled bleeding (BTB) [112]. Drugs that interfere with ovarian steroid
used for other applications [122]. This remains an unexplored field
release may have a similar effect (GnRH agonists and antagonists)
with the potential to discover new biomarkers. Uterine fluid obtained
and aromatase inhibitors. The use of intrauterine contraceptive de-
by lavage or as aspirates, menstrual loss supernatants, and sera may
vices may contribute to chronic endometritis (CE) and AUB [113].
all have a future role to play [90, 123].
A structured history and examination and exclusion of other causes
Current pathology practice utilizes chromogenic IHC, and im-
help to secure the diagnosis. Often cessation of the drug or removal
proving the technology available may allow us to identify clinically
of the device (implant, intrauterine device) helps resolve the problem.
usable biomarkers. Multiplexed IHC (mIHC) approaches are now
The role of biomarkers in this category is limited.
available, offering greater insights into disease heterogeneity and the
characterization of systems biology mechanisms driving disease, as
Not otherwise classified (AUB-N) well as helping to conserve limited tissues. Multiplexed IHC offers
Entities such as CE (not secondary to IUD use), arteriovenous mal- greater insight into molecular cascades, preserves tissue context, and
formations (AVMs) [114], endometrial pseudoaneurysms, and my- allows for improved accuracy through the application of image anal-
ometrial hypertrophy have been associated with or contribute to ysis, with the use of landmark markers specifically to indicate tissue
AUB/HMB. Cesarean sections scar defects such as “isthmoceles” architecture [124]. Application of such technology has already been
may also contribute to AUB [115, 116]. In addition, there may be used in the field of cancer. Kim et al. have demonstrated quantitative
other disorders that would be defined only by biochemical or molecu- proteomic profiling of breast cancers using mIHC, so that individu-
lar biology assays that should be placed in this category. Considering alized cancer therapy can be offered [125].
these entities are extremely rare, the search for a viable endometrial In keeping with advances in technology, elastography (MRI and
biomarker is only of academic value. USS) is being increasingly used in the assessment of women with
Most women with AVMs, endometrial pseudoaneurysms, and fibroids and adenomyosis serving as noninvasive imaging biomark-
myometrial hypertrophy will be diagnosed using imaging techniques. ers. More importantly, there are being used to tailor therapy and
Chronic endometritis is poorly understood but plays an important predict treatment response for allowing individualized care [126]. A
role in AUB and poor reproductive outcomes and is currently di- recent study showed that fractional change in stiffness value of uter-
agnosed by histology. A study by Tortorella et al, proposed the ine fibroids measured by magnetic resonance elastography would be
use of biomarkers in the menstrual effluent for diagnosing CE. related to the treatment outcomes after magnetic resonance guided
They identified that proinflammatory cytokines are increased in focused ultrasound [127].
menstrual effluents of women with CE with IL-6 and TNF-alpha Identifying differential gene expression in women with
having a high screening capacity for the condition [117]. The AUB/HMB, i.e. a gene signature, may advance our understanding
Biomarkers in abnormal uterine bleeding, 2019, Vol. 101, No. 6 1163

of the mechanisms responsible for HMB and allowed tailored treat- nongravid women of reproductive age. Int J Gynecol Obstet 2011;
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ration and Mr Ronnie Grant for graphical assistance.
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Conflict of interest statement
Fernandez E. Comparison of ultrasonography and hysteroscopy in the
RC is supported as a Clinical Research Fellow by Bayer AG and has diagnosis of intrauterine lesions in infertile women. J Am Assoc Gynecol
no other conflict of interest. HODC has clinical research support for Laparosc 1998; 5(4):375–378.
laboratory consumables and staff from Bayer AG and provides con- 20. Anastasiadis P, Koutlaki N, Skaphida P, Galazios GC, Tsikouras P,
sultancy advice (but with no personal remuneration) for Bayer AG, Liberis V. Endometrial polyps: prevalence, detection, and malignant po-
tential in women with abnormal uterine bleeding. Eur J Gynaecol Oncol
PregLem SA, Gedeon Richter, Vifor Pharma UK Ltd, AbbVie Inc.,
2000; 21:180–183.
Myovant Sciences GmbH. HODC receives royalties from UpToDate
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for an article on abnormal uterine bleeding.
pathogenesis, diagnosis, and management. Best Pract Res Clin Obstet
Gynaecol 2017; 40:68–81.
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