Angiogenesis in abnormal uterine bleeding: a narrative review

Abnormal uterine bleeding (AUB) is defined by the International Federation of Gynecology and Obstetrics (FIGO) as a set of unusual menstrual symptoms caused by several uterine abnormalities, most often presenting as intermenstrual or heavy menstrual bleeding (IMB and HMB, respectively). While rarely life threatening, the impact of AUB is substantial as over one-third of all women will experience AUB at least once in their life, often negatively impacting work productivity and quality of life ( Munro et al. , 2018 ). AUB can be diagnosed if one or more of the following symptoms occur: the patients menstrual frequency, duration, regularity, and/or flow volumes are abnormal according to FIGO AUB definitions; the patient experiences IMB, defined as bleeding between cyclically regular onsets of menstruation; and the patient has unscheduled bleeding on medication with progestin, with or without oestrogens, such as birth control pills, intra-uterine devices, and injections ( Munro et al. , 2018 ). It is thought that AUB is initiated by a broad spectrum of potential causes, for example a deviant endocrine function, one or multiple uterine abnormalities (e.g. uterine fibroids, adenomyosis) or a combination of these ( Munro et al. , 2011a , b ). Patients with AUB can experience HMB, defined as uterine bleeding that occurs regularly and lasts >7 days and/or comprises blood loss >80 ml ( ACOG Committee on Practice Bulletins—Gynecology, American College of Obstetricians and Gynecologists, 2001 ). If AUB occurs in a cyclic context and no other causes are diagnosed, AUB could be caused by a primary endometrium disorder and can be triggered by changes in the concentrations of local vasoconstrictors or vasodilators ( Gleeson, 1994 ; Smith et al. , 1981a , b ). This type of AUB is classified as endometrial-AUB (AUB-E) by the FIGO AUB System ( Munro et al. , 2018 ). AUB-E is also frequently seen in perimenopausal women, owing to ovulatory dysfunction or anovulation caused by changes in endogenous hormone shifts ( Marnach and Laughlin-Tommaso, 2019 ; Khafaga and Goldstein, 2019 ). Unscheduled bleeding is frequently referred to as spotting or breakthrough bleeding. When spotting is caused by medical interventions or devices it is classified as iatrogenic-AUB (AUB-I) ( Munro et al. , 2011a ). Unfortunately, this is a recurrent reason for the discontinuation of effective and safe contraception and can lead to involuntary pregnancy ( Belsey, 1988 ). Human endometrium has the unique feature that it regenerates in a cyclic manner, which involves restoration and remodelling of the vascular morphology of the endometrium, as is shown in Fig. 1 ( Abberton et al. , 1999a ). Endometrium is composed of two layers of which the upper two-thirds is called the functional layer and is shed during menstruation. This process is characterized by rapid repair, without loss of function or residual scarring. The basal layer is the second layer, located adjacent to the myometrium and does not shed during menstruation. In the human menstrual cycle, three stages of neovascularization can be distinguished. The first stage is repair of damaged blood vessels during the menstrual phase, the second is rapid growth of the endometrial vessels during the proliferative phase, and the last is the development of spiral arterioles and the subepithelial capillary plexus during the secretory phase ( Jabbour et al. , 2006 ). Three factors are required to co-ordinate normal physiological menstruation and postmenstrual tissue repair. To regulate the cessation of the menstrual bleeding, the following changes occur: vasoconstriction of spiral arterioles to control blood flow; an effective haemostatic response that repairs the damaged vessels in the functional layer; and a properly timed re-epithelialization of the exposed basal endometrium ( Maybin et al. , 2018 ; Jain et al. , 2022 ). Withdrawal of progesterone at the end of the secretory phase and vasoconstriction of the spiral arterioles result in physiological transient hypoxia ( Martínez-Aguilar et al. , 2021 ; Jain et al. , 2022 ). Studies show that this transient hypoxia is specifically detected during tissue breakdown in the menstrual phase and is absent in the repair process of the endometrial surface. Subsequently, hypoxia stimulates hypoxia-inducible factor (HIF) that plays a pivotal role in the menstrual phase, since it induces angiogenesis and regulates energy metabolism and tissue remodelling ( Maybin et al. , 2018 ; Critchley et al. , 2020 ). After menstruation, the vascular and endometrial tissue undergo substantial proliferation under the influence of rising oestradiol levels ( Critchley et al. , 2020 ). During this proliferative phase, oestradiol and the previously induced hypoxia in the menstrual phase stimulate angiogenesis by triggering human endometrial stromal cells (HESCs). These HESCs produce vascular endothelial growth factor (VEGF) and simultaneously stimulate endothelial cells (ECs) to express angiopoietin 2 (Ang-2) ( Lockwood, 2011 ). During the proliferative phase, endometrial thickness increases significantly within a couple of days, without changing the microvessel density (MVD) ( Rogers and Gargett, 1998 ; Jabbour et al. , 2006 ). During the secretory phase, progesterone levels rise again, stimulating both the expression of Ang-1 by HESCS and a significant growth of the spiral arterioles ( Nayak et al. , 2005 ; Lockwood, 2011 ). Growth and coiling of these spiral arterioles is better described as arteriogenesis and is primarily stimulated by progesterone. During this arteriogenesis, capillaries become heavily coiled and coated with vascular smooth muscle cells (VSMCs), giving them the ability to change vessel diameter and regulate blood flow ( Carmeliet, 2000 ; Rogers and Abberton, 2003 ). In addition, both spiral and straight arterioles have been described ( Rogers and Abberton, 2003 ). Ultimately, when the corpus luteum diminishes in the late secretory phase, levels of progesterone and oestradiol decline, the functional layer is shed, and the menstrual breakdown begins ( Jain et al. , 2022 ). Derangement of these well-ordered and highly regulated processes during the menstrual cycle and haemostasis is thought to result in AUB-E. The human endometrial cycle during normal physiological menstruation. During the menstrual cycle, the human endometrium regenerates in a cyclic manner, which involves restoration and remodelling of the vascular morphology of the endometrium under the influence of oestradiol and progesterone. Ang-2: angiopoietin 2; ECs: endothelial cells; HESCs: human endometrial stromal cells; HIF: hypoxia-inducible factor; VEGF: vascular endothelial growth factor. Figure created with BioRender.com Munro et al. (2011) stated that aberrations in endometrial angiogenesis can lead to deficiencies in endometrial regeneration and, therefore, plays an important role in AUB ( Salamonsen et al. , 1999 ; Munro et al. , 2011a ). Angiogenesis is defined as the formation of new blood vessels from pre-existing ones and is triggered by the requirement of oxygen for tissue growth in endometrial proliferation ( Griffioen and Molema, 2000 ; Harmsen et al. , 2019 ). A schematic illustration of normal angiogenesis in the endometrium during the menstrual cycle is displayed in Fig. 2 . ECs of the arterial or venous vessels are surrounded by VSMCs and the capillaries covered by pericytes ( Kayisli et al. , 2015 ). They thereby support microvascular stability and play a central role in angiogenesis by supporting vascular development, maturation and differentiation ( Birbrair et al. , 2015 ). In response to angiogenic cues, such as hypoxia, ECs stimulate pericyte detachment by the release of Ang-2 ( Yetkin-Arik et al. , 2021 ). The most commonly described process to develop new capillaries, as for example in cancer-related angiogenesis, is called sprouting angiogenesis ( Jabbour et al. , 2006 ). For years it was assumed that sprouting angiogenesis was the main mechanism for the creation of new blood vessels. While this idea may still prevail, new vascularization can also emerge by alternative processes such as intussusception ( Pasut et al. , 2021 ) and vessel co-option or elongation ( Latacz et al. , 2020 ; Kuczynski and Reynolds, 2020 ). Intussusception involves splitting of the blood vessels and vessel elongation involves passive cell growth alongside existing vessels ( Rogers and Gargett, 1998 ; Yetkin-Arik et al. , 2021 ). Intussusception and vessel elongation both have structural advantages compared to sprouting angiogenesis, since the vessel wall remains intact during the creation of these new blood vessels, resulting in the continuation of blood flow through the original vessel ( Rogers and Gargett, 1998 ; Girling and Rogers, 2005 ). Recently, a new form of angiogenesis has been described where blood vessels coalesce to form a larger vessel ( Nitzsche et al. , 2022 ). This coalescent angiogenesis reduces the number of blood vessels and matures them but increases blood flow. A separate mechanism of angiogenesis is mediated through incorporation of circulating ECs or endothelial progenitor cells in the existing vessel wall. This may contribute to sprouting angiogenesis, intussusception, co-option, and elongation ( Rogers and Gargett, 1998 ; Girling and Rogers, 2005 ). Angiogenesis in the endometrium of women with normal menstrual bleeding. This figure shows potential mechanisms of angiogenesis in the endometrium during the normal menstrual cycle. ANG1/2: angiopoietin 1/2; FGF: fibroblast growth factor; VEGF: vascular endothelial growth factor. The uterine endometrium is known to be one of the few tissues that undergoes significant angiogenesis in a cyclic matter. The understanding of when and what mechanism of angiogenesis occur during the menstrual cycle is poor ( Girling and Rogers, 2005 ). Although quantification of angiogenesis is difficult, as EC proliferation varies extensively at the same cycle stage between patients, studies suggest that angiogenesis may be highest in the proliferative phase ( Jabbour et al. , 2006 ). Moreover, Nayak and Brenner (2002) hypothesized that this heterogeneity in samples was caused by variation in hormone levels at the time of sampling. They subsequently showed in the artificially induced menstrual cycles of the rhesus macaque ( macaca mulatta ) that 8–10 days after progesterone withdrawal (mid-proliferative phase) angiogenic activity was the highest ( Nayak and Brenner, 2002 ). However, EC proliferation also continues during the secretory phase and a second peak in EC proliferation has been described during the mid-secretory phase (cycle day 19), which was shown in the human endometrium by analysing labelled nuclei ( Ferenczy et al. , 1979 ). Also measured in human endometrium, Girling and Rogers (2005) describe elongation as the main form of angiogenesis in the mid-late proliferative phase and suggest that there could be a role for sprouting or intussusception during the early mid-secretory phase ( Girling and Rogers, 2005 ). To date, no vascular sprouts were found in the human endometrium throughout the cycle ( Hii and Rogers, 1998 ; Rogers and Gargett, 1998 ; Girling and Rogers, 2005 ). To evaluate sprouting in endometrial angiogenesis, the distribution of integrin ανβ3 was studied in endometrial ECs. Integrin ανβ3 is a cell adhesion molecule and together with proliferating ECs are thought to be sprouting markers ( Hii and Rogers, 1998 ). Integrin ανβ3 and proliferating ECs were found within the existing vessel walls, and not on the outside, suggesting that sprouting is not the primary form of angiogenesis in the human endometrium ( Hii and Rogers, 1998 ; Girling and Rogers, 2005 ). Moreover, vessel elongation appears to be the major angiogenic mechanism during the mid-late proliferate phase of the menstrual cycle ( Maas et al. , 2001 ; Gambino et al. , 2002 ; Girling and Rogers, 2005 ). This was first demonstrated by Gambino et al. (2002) , as they found an increased mean vessel length per vessel branch in this phase, compared to all other phases of the menstrual cycle ( Gambino et al. , 2002 ). Previously, it has been hypothesized that sprouting angiogenesis may contribute to the rapid and fast outgrowth of endometrium tissue ( Girling and Rogers, 2005 ). However, so far this has not been proven and the exact angiogenic mechanisms that are responsible for the postmenstrual repair and secretory phase remodelling still need to be elucidated. This review hypothesizes that all the aforementioned mechanisms of angiogenesis could be involved in the complexity of human endometrial angiogenesis, as shown in Fig. 2 . Angiogenesis depends on a variety of proangiogenic factors in the tissue microenvironment surrounding the involved endothelium. Physiological angiogenesis differs from disease-related angiogenesis, as the latter is specifically regulated by local imbalances between pro- and antiangiogenic factors ( Ramjiawan et al. , 2017 ; Yetkin-Arik et al. , 2021 ). For example, hypoxia is an important environmental factor that is the major regulator of angiogenesis in tumour growth. Also, exogenous factors, such as endo- or exogenous hormones, play an important role in angiogenesis ( Reuwer et al. , 2012 ; Cioni et al. , 2020 ). Angiogenesis is extensively discussed in literature, mainly in the context of tumour growth but also in the pathophysiology of gynaecological conditions such as endometriosis and adenomyosis ( Rogers and Gargett, 1998 ; Nap et al. , 2004 , 2005 ; Harmsen et al. , 2019 ). Endometrial tissue of patients with endometriosis shows increased EC proliferation demonstrating a proangiogenic character of this condition ( Rogers and Gargett, 1998 ). Patients with AUB-E experience heavier and longer menstrual bleeding, probably triggered by decreased vasoconstriction and altered vessel maturation of predominantly spiral arterioles ( Jain et al. , 2022 ). Pathological hypoxia can be triggered by exogenous hormones that reduce endometrial blood flow, resulting in aberrant angiogenesis, causing AUB-I ( Lockwood et al. , 2004 ; Hickey et al. , 2006 ). Unlike HMB in AUB-E, it has been suggested that AUB-I originates from fragile capillaries and venules, as these vessels are superficial, irregularly distributed, and abnormally enlarged, which is caused by the lack of perivascular support from pericytes and VSMCs, eventually resulting in breakthrough bleeding ( Hanahan, 1997 ; Rogers et al. , 2005 ; Lockwood et al. , 2009 ). To study the pathophysiology of angiogenesis in the endometrium of patients with AUB, the different angiogenic pathways and separate steps of angiogenesis should be distinguished. An illustration of the possible mechanisms behind impaired angiogenesis in the endometrium of patients with AUB is displayed in Fig. 3 . In patients with AUB, angiogenesis could hypothetically be impaired by increased pericyte detachment, EC migration, and EC proliferation and, as a result of this, possibly increased and aberrant angiogenesis and/or impaired vessel maturation. Angiogenesis in the endometrium of women with abnormal uterine bleeding. This figure shows a hypothesis on how impaired angiogenesis in the endometrium could cause abnormal uterine bleeding via different angiogenic mechanisms, initiated by a significant imbalance between pro- and antiangiogenic factors. As a consequence of excessive proangiogenic factors, the combination of increased vessel formation and/or impaired vessel maturation could lead to endometrial and/or iatrogenic-AUB. ANG1/2: angiopoietin 1/2; FGF: fibroblast growth factor; VEGF: vascular endothelial growth factor. Angiogenesis is often induced by hypoxic conditions, as hypoxia stimulates the production of several factors and activity of their associated pathways ( Table I and Fig. 4 ). After hypoxia, HIF-1α ( Wang and Semenza, 1993 ; Griffioen and Bischoff, 2019 ) initiates several cellular responses in the HIF pathway, activating several proangiogenic factors such as VEGF, platelet-derived growth factor-β, and transforming growth factor-β1 (TGF-β1) ( Edeline et al. , 2012 ). Hypoxia also activates the CX-chemokine receptor 4 (CXCR4), which is involved in the tip cell proliferation of sprouting angiogenesis ( Yetkin-Arik et al. , 2021 ) and it induces transcription of the endothelial water channel protein, aquaporin-1 (AQP1) ( Mints et al. , 2007b ; Abreu-Rodriguez et al. , 2011 ). AQP1 was identified as a proangiogenic factor as it is linked to vascular permeability and facilitates EC migration during angiogenesis ( Tomita et al. , 2017 ). Before angiogenic sprouting occurs, quiescent ECs consist of a monolayer of cells surrounded by pericytes. Pericytes are microvascular cells that mostly cover capillary cells, thereby supporting microvascular stability. They also play a central role in angiogenesis by inducing vascular development, maturation and differentiation ( Birbrair et al. , 2015 ). Pericytes suppress EC activation by providing antiangiogenic factors, such as TGF-β1, thereby stabilizing the vessel ( Yetkin-Arik et al. , 2021 ). VEGF is thought to play a key role in angiogenesis, as it increases EC permeability and vasodilatation ( Griffioen and Molema, 2000 ). VEGF also regulates endothelial nitric oxide synthase (eNOS) downstream and has an important proangiogenic effect by producing nitric oxide (NO), which in turn increases vasodilatation, EC permeability and sprouting angiogenesis ( Valdes et al. , 2008 ). In addition, VEGF receptor 2 (VEGFR-2) activation leads to enhanced EC proliferation and migration, making the VEGF pathway an important pathway to explore in AUB-related angiogenesis ( Griffioen and Molema, 2000 ). The hypoxia-inducible factor pathway and its effect on several proangiogenic factors. Arrows indicate the direction of change. ADM: adrenomedullin; Ang-1/2: angiotensine-1/2; bFGF: basic fibroblast growth factor; BMP(R): bone morphogenetic protein (receptor); CLR: calcitonin receptor-like receptor; c-Src: cellular Src; EC: endothelial cell; eNOS: endothelial nitric oxide synthase; FGF-R: FGF-receptor; HIFα/β: hypoxia-inducible factor α/β; NO: nitric oxide; PLC-γ: phosphoinositide phospholipase C pathway; PI3K: phosphoinositide 3-kinase pathway; RAMP: receptor-activity modifying proteins; Smad: suppressor of mothers against decapentaplegic; TGF-β: transforming growth factor; TGF-βR1/2: TGF-β receptor 1 or 2; Tie-1/2: tyrosine kinase with immunoglobulin-like and endothelial growth factor-like domains-1/2; VEGF(R): vascular endothelial growth factor (receptor). Overview of several anti- and proangiogenic factors and the pathways they are involved in. General vasodilator Promotes EC growth by inducing cAMP Stabilization of the endothelial barrier Activates endothelial nitric oxide synthase (eNOS) ↑ Vascular network maturation Inhibits Ang-1 binding to Tie-2 ↑ Loss of pericytes Destabilizes vessel integrity ↑ Vascular permeability facilitates EC migration during angiogenesis ↑ Cell migration ↑ Tube formation by inducing endothelial progenitor cells (EPCs) Activates VEGFR-2, leading to EC proliferation and migration ↑ EC proliferation and migration Involved in the tip cell proliferation ↑ NO productions by eNOS ↑ cell growth and proliferation ↑ production of VEGF ↑ EC proliferation Upregulates VEGF Activates the transcription of several angiogenic growth factors, such as VEGF, Ang-1, Ang-2, and several others. VEGF ADM binding to the CLR ↑ Vasodilatation ↑ EC permeability ↑ Sprouting angiogenesis Binding to TGF-βR ↑ EC differentiation ↑ Vessel maturation Provided by pericytes Suppress EC activation by stabilizing the vessel ↑ EC proliferation and migration Thymidine phosphorylase, Tissue factor, STC-1 and CSPG4 are not in this table owing to limited literature on their angiogenic effect and influence on endometrium. The studies included in this review are some of the first to describe the endometrium related angiogenic processes. Ang-Tie: angiopoietin-tyrosine kinase with immunoglobulin-like and EGF-like domains; c-Src: cellular Src; MAPK: mitogen-activated protein kinases; MEK: a specific MAPK; PI3K: phosphatidylinositol-3 kinase; PLC-y: phospholipase C gamma. Furthermore, the need for oxygen activates the HIF pathway and production of other anti- and proangiogenic factors ( Yetkin-Arik et al. , 2021 ), such as the Ang-Tie pathway and adrenomedullin (ADM) ( Krock et al. , 2011 ), which is identified as a general vasodilator and a proangiogenic factor as it promotes EC growth in human ECs by inducing cAMP ( Nikitenko et al. , 2000 ). Literature indicates that ADM also plays a role in endothelial barrier stabilization and suggests that a decrease in ADM could potentially lead to increased blood loss caused by an increased barrier permeability ( Ha et al. , 2009 ). In addition, ADM binding to the calcitonin receptor-like receptor (CLR) stimulates NO production by downstream regulation, which in turn stimulates angiogenesis ( Wong et al. , 2012 ). The Ang-Tie pathway is involved in EC differentiation, as it stabilizes the blood vessel integrity by tyrosine kinase with immunoglobulin-like and endothelial growth factor-like domains type 1 (Tie-1). Binding of Ang-1 to Tie-2 induces maturation of the vascular network and is therefore proangiogenic. Ang-2 is antiangiogenic because it inhibits Ang-1 binding to Tie-2, antagonizing angiogenesis by causing pericyte-loss and destabilizing vessel integrity ( Griffioen and Molema, 2000 ; Yetkin-Arik et al. , 2021 ). The bone morphogenetic protein (BMP) family of proteins is part of the TGF-β superfamily and plays an important role in embryogenesis and implantation. It also seems to play a role in angiogenesis, as BMP-2 is reported to promote cell migration and tube formation ( Chen et al. , 2018 ). BMP-4 was shown to be crucial in VEGF signalling ( Rezzola et al. , 2019 ). The BMP ligands bind to BMP receptors (BMPR), activating the suppressor of mothers against decapentaplegic (Smad) proteins (among other factors), which are either receptor activators (Smad 1/5/8), receptor inhibitors (Smad 6/7), or common receptor mediators (Smad4) ( Pulkkinen et al. , 2021 ). In this review, we study the underlying pathophysiology of two types of AUB, namely AUB-E and AUB-I, involving patients without uterine malignancies. First, we review the literature regarding angiogenesis in the endometrium of patients with symptoms of HMB as part of AUB-E, secondly patients with AUB-E and HMB after treatment with exogenous hormones, and thirdly in AUB-I after use of exogenous hormones, either mentioned as spotting complaints or as breakthrough bleeding. We investigate whether AUB-E and AUB-I are caused by aberrant angiogenesis and discuss the differences in pro- and antiangiogenic factors involved.

The protocol of this narrative review was specified in advance, registered in the PROSPERO database in July 2020 (CRD42020169061) and conducted according to PRISMA guidelines ( Liberati et al. , 2009 ; Moher et al. , 2009 ). The systematic literature search included studies published up to September 2021 in the Cochrane Library, Embase, PubMed, and Web of Science databases and were independently assessed by two authors (E.E.D. and M.-A.M.). The following terms were used, including synonyms and closely related words: ‘angiogenesis’, ‘abnormal uterine bleeding’, and ‘endometrium’. The full search strategy and terms used can be found in Supplementary Data File S1 . An additional search was performed, which focussed specifically on exogenous hormone use in patients with AUB-E or AUB-I; however, this search did not result in new eligible articles compared to the original search. Eligibility criteria are shown in Table II . Search outcomes included original research papers only, with no limitations on publication year, randomized control trials (RCTs), and cohort studies or case–control studies that focused on an association between angiogenesis in the endometrium of patients with AUB. No language restrictions were applied. Articles had to be published as full papers in peer-reviewed journals. Papers that assessed the association of angiogenesis in the endometrium in patients with exogenous hormones were included and reported on separately. Papers that reported on AUB caused by other uterine abnormalities, described by Munro et al. (2011) as fibroids, adenomyosis, infections, or malignancies were excluded ( Munro et al. , 2011a ). In addition, case reports, letters, reviews or editorial articles and papers, animal studies and studies on non-human cells maintained in vitro were excluded. Papers that used menstrual effluent or serum levels of angiogenic factors only, without biopsies of the endometrium, were also excluded. Finally, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were not included in the review. Although MMPs and TIMPs play a very important function in the extracellular matrix (ECM), and these pathways are important in the interplay for angiogenesis, we did not include these regulators in this review because intervention was shown to be very complex and resulted in significant adverse events ( Rani et al. , 2021 ). Two authors (E.E.D. and M.-A.M.) independently screened title and abstracts and, if eligibility was expected, the full article was acquired and reviewed. Any disagreements were resolved by discussion. In addition, references were checked for remaining eligible studies. Inclusion and exclusion criteria of the literature search. Premenopausal women with AUB With and without exogenous hormones Uterine abnormalities, like fibroids or adenomyosis In vitro and in vivo experiments with non-human cells Angiogenic factors in endometrium Vessel characteristics Menstrual effluent Serum levels Randomized controlled trial Case–control Cohort study Meta-analysis Case reports Reviews AUB: abnormal uterine bleeding. Using a standardized form, data from published studies was extracted independently by the authors (E.E.D. and M.-A.M.), including but not limited to characteristics such as study design, the number of included patients and outcomes. The included studies were divided into papers reporting on the comparisons of three groups of patients: AUB-E, AUB-E with exogenous hormone supplementation, and AUB-I. The results were subdivided into outcomes linked to angiogenesis-related vessel morphology and angiogenic parameters, including factors and their receptors. Two authors (E.E.D. and M.-A.M.) independently performed a quality assessment of the included studies. To estimate the quality of the RCTs, the case–control, and cohorts studies, the Cochrane Risk of Bias tool version 2.0 (RoB 2) and the Newcastle–Ottawa Scale (NOS-RoB) were used respectively ( Supplementary Data Files S2 and S3 ) ( Stang, 2010 ; Sterne et al. , 2019 ; Jørgensen et al. , 2016 ; Wells et al. , 2021 ). For comparability in the NOS-RoB, two population characteristics were chosen as important potential confounders: if groups were comparable for age, defined as reproductive age (15–49 years) by the World Health Organization (WHO) and history of hormone use ( World Health Organization, 2006 ).

The search resulted in 2158 references. After removal of duplicates and title/abstract screening, 84 full-text articles were assessed for eligibility. Three records were added by additional sources, resulting in 36 included articles. The selection process is shown in Fig. 5 . PRISMA 2009 flow diagram for the search carried out for this narrative review . The characteristics of the included studies are presented in Tables III , IV , and V . In accordance with the NOS-RoB assessment, patient populations were considered to be comparable for a history of hormone use and reproductive age, defined as 15–49 years according to WHO guidelines ( WHO, 2006 ). With the exception of one study, which included patients with AUB defined as either HMB or irregular uterine bleeding ( Zhang et al. , 2010 ), all studies involved patients with AUB complaints defined only as HMB. AUB was defined objectively in nine studies ( Kooy et al. , 1996 ; Abberton et al. , 1999a and 1999b ; Mints et al. , 2007c , 2010 ; Ha et al. , 2009 ; Andersson et al. , 2015 ; Maybin et al. , 2017 , 2018 ), according to the alkaline haematin method (AHM) or the pictorial blood assessment chart (PBAC). The AHM requires collection of all menstrual sanitary products for laboratory analysis, and by calculation >80 ml defines HMB ( Hallberg and Nilsson, 1964 ). The PBAC score is calculated by the degree of bloodstaining of sanitary products, in which a score of >100 points defines HMB ( Janssen et al. , 1995 ). AUB was subjectively defined from patients’ medical history or based on the UK National Institute for Health and Care Excellence (NICE) guideline no. 44, in four studies ( Hewett et al. , 2002 ; Biswas Shivhare et al. , 2014 , 2018 ; Lu et al. , 2021 ). This guideline defines HMB based on subjective complaints of excessive menstrual blood loss that interferes with a woman's physical, social, emotional, and/or material quality of life ( NICE, 2018 ). In 12 studies, the diagnosis of AUB was not specified ( Sangha et al. , 1997 ; Blumenthal et al. , 2002 ; Hewett et al. , 2002 ; Mints et al. , 2005 , 2007a , b , c , 2010 ; Zhang et al. , 2010 ; Biswas Shivhare et al. , 2014 , 2018 ; Richards et al. , 2017 ). In all papers that studied exogenous hormones, AUB at baseline was either not described, based on subjective complaints or defined by the WHO criteria, which is based on subjective registration ( Rogers et al. , 1993 ). In general, AUB during exogenous hormone exposure was registered by menstrual calendar and based on the amount of spotting days. Characteristics of studies on angiogenesis in the endometrium of patients with endometrial abnormal uterine bleeding, compared with normal menstruation controls. Matching for age and/or hormonal use. Phases described: 2 phases : proliferative (P) and secretory (S); 3 phases : menstrual (M), P, and S; 4 phases : P, early (ES), mid (MS), and late secretory (LS); 5 phases : M, P, ES, MS, and LS; 6 phases: M, early proliferative (EP), mid proliferative (MP), ES, MS, and LS; 7 phases : M, EP, MP, late proliferative (LP); ES, MS, and LS. Methods described: BrdU assay: 5-bromo-2-deoxyuridine incorporation assay; CCT: corrosion casting technique; ELISA: enzyme-linked immunosorbent assay; HUVEC: human umbilical vein endothelial cells; IHC: immunohistochemistry; QIA: Quantitative image analysis; RPA: ribonuclease protection assay; RT-PCR: real-time PCR; WB: western blot. AUB classification: AHM : alkaline haematin method; requires collection of all menstrual sanitary products for laboratory analysis. By calculation >80 ml defines heavy menstrual bleeding (HMB) ( Hallberg and Nilsson, 1964 ); NICE guidelines: National Institute for Health and Care Excellence. HMB (NICE clinical guideline 44) is defined as excessive menstrual blood loss which interferes with a woman's physical, social, emotional, and/or material quality of life. It can occur alone or in combination with other symptoms ( NICE, 2018 ); PBAC: pictorial blood assessment chart; the degree of bloodstaining of these sanitary products corresponds to a score, with >100 points defines HMB ( Janssen et al. , 1995 ); World Health Organization terminology: bleeding: any bloody vaginal discharge that required the use of such protection as pads or tampons; spotting: any bloody vaginal discharge that was not sufficient to require protection; bleeding/spotting episode: one or more consecutive days during which blood loss (bleeding or spotting) had been entered on the calendar record, each episode being bounded by two or more bleeding/spotting-free days, a single bleeding/spotting-free day within a bleeding/spotting episode was counted as part of the episode surrounding it; bleeding/spotting-free interval: two or more consecutive days during which blood loss (bleeding or spotting) had not been entered on the calendar record; each interval being bounded by bleeding/spotting days; reference period: the period of time (length of diary) measured in number of days on which analysis was to be based ( Rogers et al. , 1993 ). AUB: breakthrough bleeding, spotting or menstrual bleeding according to definition mentioned at footnote d; HMB: menorrhagia; no MBL: amenorrhoea, no bleeding/spotting days during the reference period; normal menstrual bleeding (NMB): eumenorrhoea, normal blood loss. Only used the outcomes of the case–control study, not the mouse model. AUB-E: endometrial abnormal uterine bleeding. Characteristics of studies on the effect of exogenous hormones on angiogenesis in patients with endometrial abnormal uterine bleeding. Matching for age and/or hormone use. LNG-IUS: levonorgestrel-releasing intra-uterine system. Methods described: IHC: immunohistochemistry; QIA: quantitative image analysis; WB: western blot. AUB classification: AHM: Alkaline haematin method; requires collection of all menstrual sanitary products for laboratory analysis. By calculation >80 ml defines HMB ( Hallberg and Nilsson, 1964 ); NICE guidelines: National Institute for Health and Care Excellence. Heavy menstrual bleeding (HMB) (NICE clinical guideline 44) is defined as excessive menstrual blood loss which interferes with a woman's physical, social, emotional and/or material quality of life. It can occur alone or in combination with other symptoms ( NICE, 2018 ). PBAC: pictorial blood assessment chart; the degree of bloodstaining of these sanitary products corresponds to a score, with >100 points defines HMB ( Janssen et al. , 1995 ); World Health Organization terminology: bleeding: any bloody vaginal discharge that required the use of such protection as pads or tampons; spotting: any bloody vaginal discharge that was not sufficient to require protection; bleeding/spotting episode: one or more consecutive days during which blood loss (bleeding or spotting) had been entered on the calendar record, each episode being bounded by two or more bleeding/spotting-free days, a single bleeding/spotting-free day within a bleeding/spotting episode was counted as part of the episode surrounding it; bleeding/spotting-free interval: two or more consecutive days during which blood loss (bleeding or spotting) had not been entered on the calendar record; each interval being bounded by bleeding/spotting days; reference period: the period of time (length of diary) measured in number of days on which analysis was to be based. Only used the outcomes of the case–control study, not the mouse model. HMB: menorrhagia. AUB: abnormal uterine bleeding. Characteristics of studies on angiogenesis in the endometrium of patients with (hormonal induced) iatrogenic abnormal uterine bleeding. Matching for age and/or hormone use; NA: not applicable. DMPA: depo-medroxyprogesterone acetate; LNG-IUS: levonorgestrel-releasing intra-uterine system; Norplant: subdermal levonorgestrel implant; Progestasert: progesterone-releasing IUS; Oral continuous HT (hormone therapy): oestrogen and progestin taken daily; Oral cyclic combined HT (hormonal therapy): daily oestrogen with progestin for 14 days each cycle; Oral or IM (intramuscular) Progestin-only: Primolut (norethisterone, 5 mg/day taken orally) or Provera (depo-medroxyprogesterone acetate, 150 mg injected intramuscularly every 3 months). Methods described: ELISA: enzyme-linked immunosorbent assay; HUVEC: human umbilical vein endothelial cells; IHC: immunohistochemistry; QIA: quantitative image analysis; RT-PCR: real-time PCR; WB: western blot. AUB: breakthrough bleeding, spotting or menstrual bleeding according to definition mentioned at ‘ footnote d’ ; HMB: menorrhagia; no MBL: amenorrhoea, no bleeding/spotting days during the reference period; normal menstrual bleeding (NMB): eumenorrhoea, normal blood loss. AUB classification: World Health Organization (WHO) terminology: bleeding: any bloody vaginal discharge that required the use of such protection as pads or tampons; spotting: any bloody vaginal discharge that was not sufficient to require protection; bleeding/spotting episode: one or more consecutive days during which blood loss (bleeding or spotting) had been entered on the calendar record, each episode being bounded by two or more bleeding/spotting-free days, a single bleeding/spotting-free day within a bleeding/spotting episode was counted as part of the episode surrounding it; bleeding/spotting-free interval: two or more consecutive days during which blood loss (bleeding or spotting) had not been entered on the calendar record; each interval being bounded by bleeding/spotting days; reference period: the period of time (length of diary) measured in number of days on which analysis was to be based. Only presented the outcomes of the case–control study, not of the HESC (human endometrial stromal cells) model. Only presented the outcomes of the second experiment, not the first experiment with Norplant and with or without vitamin E. AUB: abnormal uterine bleeding. The included studies staged the phase of the menstrual cycle of the endometrial biopsy based on histology according to the Noyes or Fox and Buckley criteria, patients’ medical history/cycle day, or it was not specifically described ( Fox and Buckley, 1983 ; Noyes et al. , 2019 ). Results were given for two (proliferative and secretory) or up to seven phases (menstrual, early—mid—late proliferative and early—mid—late secretory phases). The results in Table VI are presented only for the proliferative and secretory phases, and if results were significantly different between, for example, the mid and late secretory phases this was specified per study. If hormones were continuously used, endometrium does not show different histological phases and the different phases were not distinguished. Hormone type and length of use is specified, as shown in Table V . Presented outcomes are defined as vascular morphology outcomes or as angiogenic parameters. Outcomes regarding angiogenesis in the endometrium of patients with endometrial abnormal uterine bleeding. bFGF: basic fibroblast growth factor; FGF-R1: fibroblast growth factors receptor 1; VEGF(-R): vascular endothelial growth factor (-receptor); TGF-β1: transforming growth factor-β1; Ang: angiopoietin; ADM: adrenomedullin; CLR: calcitonin receptor-like receptor; BMP: bone morphogenetic protein; eNOS: endothelial nitric oxide synthase; AQP: aquaporin; HIF: hypoxia-inducible factor; CXCR: CX-chemokine receptor; MVD: microvascular density. Lu et al. (2021) distinguishes between outcomes in the endometrial stromal cells (ESC), glandular epithelium and bloods vessels. Abberton et al. (1999a) distinguishes between outcomes in spiral and straight arteries. Biswas Shivhare et al . (2018) distinguishes between the early secretory phase (ESP) and the late secretory phase (LSP). The search included 31 case–control studies, three cohort studies and two RCTs. The RoB assessment of all included studies is shown in Supplementary Data File S4 . According to the NOS-RoB, one cohort study was of ‘good’, one of ‘fair’ quality, and one of ‘poor’ quality: the latter assessment was based on the difference in age of the compared populations, the outcome assessments and follow-up adequacy. Of the 31 case–control studies, 8 were of ‘good’, 17 of ‘fair’, and 6 of ‘poor’ quality. In general, the non-response rate and the selection process of included groups was poorly described in all studies that were defined as ‘fair’. If, additionally, comparability of the included groups was lacking, for example if they were not matching for important confounders, this resulted in a ‘poor’ overall quality assessment. However, overall, the case definition of the included patient populations and (methods of) ascertainment of exposure were accurate in all included studies. The RoB assessment for the RCTs was assessed as ‘high’ and ‘some concerns’ as the randomization process was poorly described in both studies and selection bias of the reported results could not be excluded. This review covers the statistically significant results of the original publications, unless mentioned otherwise. The results of 35 identified parameters, from 20 studies that compared endometrium tissue of patients with and without AUB-E, are presented in Table VI . The VEGF pathway was investigated in several studies. One study showed an increase in VEGF in the endometrial glands during the secretory phase but not in the proliferative phase ( Zhang et al. , 2010 ). VEGF-A and its receptor subtypes were studied by Mints et al. (2005 , 2007a , b , c ) and published in four different articles. They found an increased VEGF-A and VEGFR-1/2/3 protein levels in both the proliferative and secretory phase in patients with AUB-E, with one exception. Mints et al. (2007c ) showed an increase of VEGFR-2 in the proliferative phase, but this difference was not significantly different in the secretory phase ( Mints et al. , 2005 , 2007a , b , c ). In contrast, Maybin et al. (2018) found no difference in VEGF levels in both the proliferative and secretory phase, but HIF-1α and VEGF did decrease peri-menstrually in patients with AUB-E compared to normal controls ( Maybin et al. , 2018 ). This could be suggestive of delayed endometrial repair during menstruation in patients with AUB-E. The downstream regulation of eNOS by VEGF was studied in one trial and showed an increase of eNOS in both phases in patients with AUB-E ( Blumenthal et al. , 2002 ). Three studies compared parameters from the Ang-Tie pathway and found conflicting outcomes. As Ang-1 binding to Tie-2 has a proangiogenic effect and Ang-2 antagonizes the binding of Ang-1 to Tie-2, the Ang-1/Ang-2 ratio is thought to be the most relevant outcome for this pathway. Two studies found an increased Ang-1/Ang-2 ratio in both cycle phases in the AUB-E group ( Blumenthal et al. , 2002 ; Hewett et al. , 2002 ). However, only one study included patients with objective AUB-E and their results showed an increased Ang-1 expression in patients with AUB-E. The proangiogenic factor ADM was decreased in the secretory phase according to Ha et al. (2009) , while the CLR expression was increased. This may indicate that the EC is more sensitive to ADM binding and leads to an increase of the NO pathway, stimulating angiogenesis ( Fig. 4 ) ( Ha et al. , 2009 ). Three studies compared TGF-β1 and found no difference between the proliferative and secretory phase ( Abberton et al. , 1999b ; Maybin et al. , 2017 ; Lu et al. , 2021 ), and a decreased expression peri-menstrually in patients with AUB-E ( Maybin et al. , 2017 ). Two studies also analysed the TGF-β receptors, which show a decreased expression ( Lu et al. , 2021 ) or no difference ( Maybin et al. , 2017 ). In addition, Abberton et al. (1999b ) studied endothelin-1, a proangiogenic factor that stimulates vessel maturation by mediating vascular permeability and vasoconstriction ( Krock et al. , 2011 ). Endothelin-1, as well as basic fibroblast growth factor (bFGF) and the FGF-receptor 1, was found to be decreased in patients with AUB-E, compared to normal controls ( Sangha et al. , 1997 ; Abberton et al. , 1999b ). Though FGF is supposedly a proangiogenic factor, it is also involved in vessel maturation and the development of spiral arterioles in the endometrium. Consequently, a reduction of FGF and endothelin-1 disturbs development of the endometrium and could lead to abnormal bleeding patterns ( Sangha et al. , 1997 ; Abberton et al. , 1999b ). One study demonstrated that AQP1 expression is decreased in both phases of the menstrual cycle in patients with AUB-E. Despite the fact that AQP1 is an proangiogenic factor, impaired expression of AQP1 could lead to abnormal vessel formation by aberrant endothelial permeability or EC proliferation and migration ( Mints et al. , 2007b ). No differences were found between patients with and without AUB-E in one study evaluating the expression of BMP 2/4/6, BMPR1A/1B, and Smad 4/6/7 ( Richards et al. , 2017 ). In addition, Smad2 and -7 were found to be unchanged in both phases and, overall, Smad3 and -4 were decreased in the secretory phase in patients with AUB-E ( Lu et al. , 2021 ). In line with these results, Maybin et al. (2017) found lower expressions of Smad2 and-3 peri-menstrually and decreased phosphorylated Smad2/3 during the late secretory phase ( Maybin et al. , 2017 ). However, they also found an increase of Smad2 in the secretory phase, possibly explained by the fact that HMB was defined differently in both studies, knowingly subjectively according to NICE guidelines by Lu et al. (2021) and objectively with AMH by Maybin et al. (2017) . Only BMP7 was increased in patients with AUB-E; therefore, they conclude that BMP7 is possibly involved in endometrial differentiation and tissue integrity, both crucial for embryo implantation ( Richards et al. , 2017 ). Ten studies report on vessel morphology in patients with and without AUB-E ( Table IV ). Increased angiogenesis can lead to an increased MVD, and an imbalance in pro- and antiangiogenic factors can increase the number of wall gaps, vessel diameter and perimeter, and decreased pericyte coverage or wall thickness. This could indicate that vessel maturation is impaired, resulting in more fragile bloods vessels. MVD was investigated in six studies, five of which showed no significant changes in both the proliferative and secretory phases ( Mints et al. , 2005 , 2007b , 2010 ; Makhija et al. , 2008 ; Andersson et al. , 2015 ), while one study that found an increase in MVD in the secretory phase ( Zhang et al. , 2010 ). One study found that pericyte coverage of >50% was decreased in the proliferative phase and also in the secretory phase in patients with AUB-E compared to the control group ( Andersson et al. , 2015 ). One study reported on vessel diameter, and perimeter and wall gaps, which were all increased in the secretory phase of patients with AUB-E, compared to normal controls. Wall gaps also were increased in the proliferative phase, although the other characteristics were similar ( Mints et al. , 2007c ). In addition, Andersson et al. (2015) found an increased wall thickness in patients with AUB-E, independent of the cycle phase ( Andersson et al. , 2015 ) whereas Abberton et al. (1999a ) found no difference in vessel wall thickness. Proliferation and attraction of VSMCs occurs with vessel wall remodelling, for example during the maturation process of newly formed vessels, and is profoundly co-regulated by VEGF ( Darden et al. , 2019 ). It is also involved in the next step after angiogenesis, namely arteriogenesis ( Jeremy et al. , 1999 ), in which endometrial spiral arterioles are surrounded by VSMC ( Abberton et al. , 1999b ). Total VSCM was identified in four different studies and was generally found to be unchanged in patients with AUB-E, with two exceptions. One study found a decrease of VSMC in the proliferative phase and one in the secretory phase in patients with AUB-E ( Abberton et al. , 1999a and 1999b ; Biswas Shivhare et al. , 2014 ; Andersson et al. , 2015 ). Another study found no differences in VSMC thickness and additionally studied VSMC differentiation via two proteins: calponin and smoothelin. Both proteins were unchanged in the proliferative phase, but calponin was increased and smoothelin was decreased in the secretory phase ( Biswas Shivhare et al. , 2014 ). Increased EC proliferation was seen in patients with AUB during both phases compared to control samples ( Kooy et al. , 1996 ). EC density was compared by several parameters, with three of the four markers showing no difference in the proliferative phase and an increase in the secretory phase ( Biswas Shivhare et al. , 2018 ). In addition, this study showed that several ECM proteins (fibronectin, osteopontin, and collagen-IV) were decreased in the secretory phase in patients with AUB-E, with one exception: osteopontin was decreased in the early secretory phase and increased in the late secretory phase. These differences in protein levels could indicate altered maturation of the endometrial vessels in AUB-E ( Biswas Shivhare et al. , 2018 ). The effect of exogenous hormones on angiogenesis in the endometrium of patients with AUB-E was studied in three studies and patients with AUB-I in 13 studies ( Tables VII and VIII , respectively). All studies that examined AUB-E used the levonorgestrel-releasing intra-uterine system (LNG-IUS), thus progestin-only hormone therapy. Also, all the selected literature that examined AUB-I used progestin-only medication, with the exception of Rogers et al. (2005) who assessed the effect of combined oestrogen and progestin hormonal therapy. None of the included literature studied the effect of GnRH agonists or antagonists. Outcomes regarding the effect of exogenous hormones on angiogenesis n the endometrium of patients with endometrial abnormal uterine bleeding. VEGF: vascular endothelial growth factor (a- or b-: acidic- or basic-). AUB: abnormal uterine bleeding. FGF: fibroblast growth factor, ADM: adrenomedullin; MVD: microvascular density; αSMA: α-smooth muscle actin; MHC: myosin heavy chain; vWF: von Willebrand factor. Outcomes regarding the effect of exogenous hormones on angiogenesis in the endometrium of patients with iatrogenic abnormal uterine bleeding. VEGF: vascular endothelial growth (-R: receptor) factor (a- or b-: acidic- or basic-); FGF: fibroblast growth factors; TGF-ß1: transforming growth factor-beta1; EGF: epidermal growth factor; STC-1: stanniocalcin-1; CSPG4: cleaved chondroitin sulphate proteoglycan 4; MVD: microvascular density; αSMA: α-smooth muscle actin; MHC: myosin heavy chain; vWF: von Willebrand factor; PCNA: proliferating cell nuclear antigen. LNG-IUS: levonorgestrel-releasing intra-uterine system; Progestasert-IUS: progesterone-releasing uterine contraceptive device; Norplant: subdermal levonorgestrel implant; DMPA: depo-medroxyprogesterone acetate; Oral continuous HT (hormone therapy): oestrogen and progestin taken daily; Oral cyclic combined HT (hormonal therapy): daily oestrogen with progestin for 14 days each cycle; Oral or IM (intramuscular) Progestin-only: Primolut (norethisterone, 5 mg/day taken orally) or Provera (depo-medroxyprogesterone acetate, 150 mg injected intramuscularly every 3 months). ND: length of use not described. BL: bleeding site during hysteroscopy in endometrium; NBL: non bleeding during hysteroscopy in endometrium. First outcomes in both lamina functionalis and lamina basalis, second outcome only in the subepithelial plexus. Table VII shows three studies that compared the endometrium of patients with AUB-E, with and without LNG-IUS exposure. After short-term exposure (<4 months) an increase was found in VEGF-D expression in LNG-IUS exposed endometrium, compared to controls ( Donoghue et al. , 2012 ). Conversely, no differences were seen in VEGF, or in acidic FGF or bFGF in LNG-IUS patients after long-term (3 years) exposure, compared with control samples ( Hague et al. , 2002 ). Also, the angiogenic factors ADM and thymidine phosphorylase, of which the latter is associated with pathophysiological angiogenesis ( Sengupta et al. , 2003 ), showed no differences in samples after long-term (3 years) LNG-IUS exposure, compared to control samples ( Hague et al. , 2002 ). The MVD of blood vessels was increased after short-term and long-term LNG-IUS treatment in comparison to patients without LNG-IUS treatment ( Hague et al. , 2002 ; McGavigan et al. , 2003 ). In contrast, Donoghue et al. (2012) did not find a difference in MVD of blood vessels after short-term LNG-IUS exposure compared to AUB-E patients without treatment. Furthermore, an increase in diameter and perimeter of the blood vessel was seen after short-term LNG-IUS treatment compared to controls, and the maximal width of the largest luminal diameter for each sample was also increased ( McGavigan et al. , 2003 ; Donoghue et al. , 2012 ). Literature shows that the increased diameter in both blood and lymphatic vessels could be caused by increased VEGF-D expression ( Rissanen et al. , 2003 ) and, therefore, this could indicate vascular remodelling. Donoghue et al. (2012) performed immunostaining for lymphatic vessels and found no difference in lymphatic MVD but did find an increased lymphatic vessel diameter ( Donoghue et al. , 2012 ). After long-term exposure, EC proliferation was reduced in patients with LNG-IUS compared to AUB-E controls ( Hague et al. , 2002 ). As presented in Table VIII , two studies examined the effect of LNG-IUS treatment ( Roopa et al. , 2003 ; Rogers et al. , 2005 ), two studies observed the effect of Norplant treatment ( Lau et al. , 1999 ; Runic et al. , 2000 ), two studies looked at the effect of depo-medroxyprogesterone acetate (DMPA) ( Shapiro et al. , 2015 , 2017 ), and one study examined the effect of oral cyclic or continuous hormonal therapy on angiogenic factors or receptors in patients with AUB-I compared to controls with normal menstrual bleeding (NMB) ( Rogers et al. , 2005 ). In patients with short- and long-term use of Norplant (1 year, respectively), the VEGF staining index (correlation of VEGF and microvascular density) was higher, compared to endometrium of patients with NMB in glandular and stromal tissue ( Lau et al. , 1999 ). One study showed an increase in VEGF, 3 and 6 months after LNG-IUS insertion, although VEGF mRNA levels were not elevated ( Roopa et al. , 2003 ). In addition, after LNG-IUS, progestin-only or oral combined hormonal therapy, a different study found no changes in VEGF immunohistochemistry (IHC) compared to controls ( Rogers et al. , 2005 ). An increase was found in bFGF and TGF-ß1 protein immunostaining, and their corresponding mRNAs, after 1 month, with an increasing staining pattern 6 months after LNG-IUS insertion in patients with AUB-I compared to controls with NMB ( Roopa et al. , 2003 ). Another angiogenic polypeptide involved in EC proliferation is endometrial epidermal growth factor (EGF). EGF and EGF mRNA were not different after 3–12 months of LNG-IUS exposure ( Roopa et al. , 2003 ). However, an increase in immunostaining of EGF-receptor (EGFR) was seen after 3 and 12 months of Norplant insertion compared to proliferative-phase control specimens. This increased in EGF was also seen by Western blot analysis of the non-bleeding tissue sites after 12 months of Norplant exposure, when compared to secretory-phase control specimens ( Runic et al. , 2000 ). Tissue factor (TF) is an important initiator for haemostasis and could potentially promote aberrant angiogenesis and produce fragile vessels if overexpressed ( Runic et al. , 2000 ; Lockwood, 2011 ). Unexpectedly, TF was decreased in the endometrium in Norplant users after 3 months, compared to controls. However, within this Norplant-exposed group, endometrium samples of bleeding sites showed an increase in TF compared with non-bleeding sites after 12 months ( Runic et al. , 2000 ). The progesterone receptor (PR) is likely to regulate TF expression, but no difference was found in the PR protein levels between Norplant users and controls ( Runic et al. , 2000 ). Again, when bleeding and non-bleeding sites were compared in Norplant users, the PR was increased after 12 months. Two studies analysed the effect of 3 months of DMPA exposure on angiogenesis in the endometrium of patients with AUB-I compared to controls with NMB ( Shapiro et al. , 2015 , 2017 ). They found an overexpression of stanniocalcin-1 and stronger immunostaining for cleaved chondroitin sulphate proteoglycan 4, both mediators of angiogenesis and linked to malignant tumour formation ( Law and Wong, 2013 ; Shapiro et al. , 2015 , 2017 ). In summary, when patients with AUB-I were compared to control groups with NMB, VEGF staining was increased in two studies and not affected in one study. FGF, TGF-β, and EGFR were increased in patients with AUB-I, and EGF showed no difference between groups. When bleeding sites in Norplant users were compared with non-bleeding sites, TF and PR expression were increased in the group with bleeding complaints. Besides stimulation of vessel maturation, FGF and TGF-β are both also proangiogenic factors, acting by stimulating EC proliferation and differentiation and have a synergistic effect on the induction of angiogenesis, if combined with VEGF. TF is an important haemostasis initiator, so it seems logical that TF and its receptor are increased in bleeding sites of patients with AUB-I. Eight studies examined the effect of exogenous hormone exposure on morphological characteristics of vessels ( Table VII ). An increase in MVD was observed in patients with AUB-I and LNG-IUS compared to controls with NMB. This was found both in short- and long-term treatments, 1–3 months and 12–60 months, respectively ( Stephanie et al. , 2007 ). In addition, an increase in MVD was seen in patients exposed to Norplant, after 3–12 months ( Rogers et al. , 1993 ; Runic et al. , 2000 ). In contrast to this increase in MVD, two studies in patients who used Progestasert-IUS for 54 months and LNG-IUS for 5–48 months (average 12.3 months) found a decrease in MVD ( Shaw et al. , 1981 ; Rogers et al. , 2005 ). No difference in MVD was found after oral combined cyclic and continuous hormonal therapy (oestrogen/progestin), or after oral or intramuscular progestin-only therapy, when compared to controls. However, if patients with progestin-only therapy were compared to patients with combined therapy, the patients with progestin-only therapy showed a decrease in MVD ( Rogers et al. , 2005 ). An increased number of blood vessel defects was reported in samples taken from patients with Progestasert-IUS compared to normal controls after 54 months of follow-up ( Shaw et al. , 1981 ). Studies that investigated the effect of exogenous progestin on blood vessel maturation found an increased number of immature and partially mature vessels in patients with 1–3 months of LNG-IUS treatment, compared to unexposed NMB endometrium, while the number of mature blood vessels was decreased. This effect was still significantly increased after 12 months of LNG-IUS treatment, but the contrast was smaller after 12 months, in comparison to 3 months of LNG-IUS treatment ( Stephanie et al. , 2007 ). After 3–12 months of Norplant exposure, the endometrium of patients with AUB-I showed an increased number of immature blood vessels, while the number of partially mature and mature blood vessels was decreased, compared to the endometrium of the proliferative phase of patients with HMB ( Rogers et al. , 2000 ). They also assessed vessel maturation in patients with amenorrhoea after 3–12 months of Norplant exposure. This effect appeared only moderate because no difference was seen in the number of immature or partially mature blood vessels; however, a decrease was found in the number of mature vessels. In addition, an increase in immature vessels was found after short- and long-term LNG-IUS exposures, and a decrease in mature vessels after short-term exposure compared to both controls and long-term exposure ( Stephanie et al. , 2007 ). In addition, endometrium samples showed a decrease in VSMC number and proliferation after DMPA exposure, compared to samples of the same patients before exposure, again indicating impaired vessel maturation after progestin exposure ( Kayisli et al. , 2015 ). These results indicate disruption in the maturation of blood vessels after exogenous progestin use in a time-dependent matter, especially in patients with AUB complaints. After 12 months of Norplant treatment, Runic et al. (2000) compared endometrium samples of bleeding sites with non-bleeding sites and found an increased blood vessel perimeter, but no difference in MVD and wall thickness ( Runic et al. , 2000 ). In addition, EC migration was reduced in patients after 3–12 months of Norplant and DMPA, compared to controls with NMB ( Subakir et al. , 1995 , 2000 ).

Both anti- and proangiogenic factors are altered in patients with AUB-E and AUB-I, which could be a pathophysiological explanation for how HMB and spotting complaints emerge, supporting our hypothesis that angiogenesis is altered in these patients. However, many different angiogenic factors and receptors have only been investigated in single studies; therefore, the results have to be interpreted with caution. This review indicates that HMB in patients with AUB-E is caused by a change in angiogenic factors, which probably leads to immature vessels or less stable vessel walls, rather than an increase in MVD. Spotting in patients with AUB-I does seems to be related to the combination of an increased MVD and impaired vessel maturation after short-term hormone exposure. Our findings provide points of interest for future research to identify key targets for antiangiogenic therapy to treat patients with AUB.

This review provides a summary of the available literature on angiogenesis in the endometrium of women with AUB-E, and in patients with AUB-E and AUB-I after use of exogenous hormones. Figure 6 provides an overview of the interactions between the angiogenic parameters reported on in this review. Schematic overview of pro- and antiangiogenic parameters assessed in this review. Blue: proangiogenic parameter; red: antiangiogenic parameter. ↑ increase, = no change, ↓ decrease. a/bFGF: acidic/basic fibroblast growth factor; ADM: adrenomedullin; Ang: angiopoietin; AQP: aquaporin; BMP(-R): bone morphogenetic protein (-receptor); CLR: calcitonin receptor-like receptor; CSPG4: cleaved chondroitin sulphate proteoglycan; CXCR4: CX-chemokine receptor4; HIF: hypoxia-inducible factor; EGF(-R): epidermal growth factor (-receptor); eNOS: endothelial nitric oxide synthase; FGF-R1: fibroblast growth factors receptor 1; NO: nitric oxide; PR: progesterone receptor; Smad: Smad proteins; STC-1: stanniocalcin-1; TF: tissue factor; TGF-β1: transforming growth factor-β1; Tie-1/2: tyrosine kinase with immunoglobulin-like and endothelial growth factor-like domains-1/2; TP: thymidine phosphorylase; VEGF(-R): vascular endothelial growth factor (-receptor). The HIF-1α initiated VEGF and Tie-Ang pathway characteristics were the most studied angiogenic parameters. In studies with objectively defined AUB, increased VEGF, VEGF-A, VEGFR-1, and VEGFR-2 were reported, while no differences in these factors were found in studies with subjectively reported AUB. In patients with subjectively reported AUB-E, the Ang-1:Ang-2 ratio, and proangiogenic Tie-1 were increased. However, one study found no difference in Ang-2 levels and only a decrease in Ang-1 in the secretory phase. As this is the only study that included patients with objectively measured AUB, this may indicate a possible relation between a decrease of Ang-1 in patients with AUB-E and decreased vessel maturation, considering that Ang-1 supports vessel maturation. However, caution is required in drawing this conclusion as this is based on one study only. In patients with AUB-E and AUB-I caused by exogenous hormones, VEGF expression was increased after short-term exposure and unchanged after long-term exposure, compared to controls. This may suggest a role of the VEGF pathway in stimulating angiogenesis in hormone-induced spotting or break-through-bleeding, in a time-dependent manner. In addition, other proangiogenic factors, such as bFGF, its receptor FGF-R1, ADM, and endothelin-1, were decreased in patients with AUB-E compared to normal controls. Conversely, in patients with AUB-I, bFGF was increased after short-term hormone exposure. These factors are in involved in EC proliferation and differentiation, as well as vessel maturation. This could suggest two somewhat contradictory outcomes: an increase in these factors could result in increased angiogenesis by mobilizing ECs, leading to AUB, and a decrease in these factors may lead to an increase in poor vascular maturation and, thus, to vascular dysfunction. Although unchanged during the proliferative phase, TGF-β1 and its downstream Smad2/3/4 proteins were decreased peri-menstrually. The Smad family is involved in wound healing and repair, and a suboptimal TGF-β response could thereby decrease postmenstrual repair, leading to HMB ( Maybin et al. , 2017 ; Lu et al. , 2021 ). Moreover, Maybin et al. (2018) showed lower levels of HIF-1α and VEGF peri-menstrually, potentially suggesting delayed endometrial repair during menstruation in women with HMB. Conceivably, the genesis of AUB in patients with AUB-E and AUB-I is caused by a different combination of abnormalities in angiogenic factors. In general, VEGF expression increased and Ang-I decreased in patients with objectively defined AUB-E: this could suggest decreased vessel maturation in these patients. In patients with AUB-I, angiogenic factors were increased after short-term exposure and unchanged after long-term exposure to exogenous hormones. This could be in agreement with the fact that spotting complaints with exogenous hormone use gradually disappear over time ( Hillard, 2014 ) and, hypothetically, angiogenesis in the endometrium will normalize. However, these findings should be interpreted with caution and do not imply causation. In addition, decreasing complaints of spotting may be linked to selection bias, with patients who experience severe or persisting AUB complaints being more likely to stop hormone therapy after short-term use or switch to another (surgical) treatment ( National Institute for Health and Clinical Excellence, 2005 ; Daud and Ewies, 2008 ). Vasoconstriction of spiral arterioles is essential to limit menstrual blood flow, as a small increase in diameter will lead to an extensive increase in blood flow. For instance, if the diameter is increased 2-fold this will lead to a 16-fold decrease in flow resistance ( Jain et al. , 2022 ). Blumenthal et al. (2002) found an increase in eNOS in both the secretory and proliferative phase in patients with AUB-E ( Blumenthal et al. , 2002 ). As eNOS produces NO and NO increases vasodilatation, HMB could hypothetically be caused by an increase in spiral arteriole diameter under the influence of increased NO levels ( Jeremy et al. , 1999 ; Valdes et al. , 2008 ). This could indicate that, apart from angiogenesis, several other mechanisms are involved in AUB. The MVD was generally not increased in patients with AUB-E. Although other morphological parameters of vessels did show changes, such as diameter, perimeter, congestion, wall gaps were increased and pericyte vessel wall coverage was decreased in the endometrium of the AUB-E group. An increase in vessel diameter and perimeter could be a sign of maturation, though when pericyte coverage is absent and wall gaps and defects are increased, this can lead to fragile and leaking microvessels, thereby causing AUB ( Runic et al. , 2000 ). These results could therefore indicate impaired angiogenesis and disturbed vessel maturation in the patient group with solely AUB-E, similar to the effects found in short-term exogenous hormone exposure in patients with AUB-E and AUB-I. This was supported by two studies that showed disturbed vessel maturation in patients with AUB-I ( Rogers et al. , 2000 ; Stephanie et al. , 2007 ) and undisturbed vessel maturation in Norplant users with NMB ( Rogers et al. , 2000 ). In contrast to patients with AUB-E, in patients with AUB-I, the MVD outcomes were very different for different types of hormones and their length of exposure. These findings could be related to the fact that quantification of MVD varied, sometimes reported as MVD/mm 2 or MVD/unit area. Moreover, the mechanism involved in angiogenesis might differ between groups. In general, MVD was increased after short-term exogenous hormone use and decreased, or was unchanged, after long-term use in patients with AUB-E and AUB-I. For LNG-IUS specifically, the MVD after long-term exposure was decreased, compared to short-term exposure ( Stephanie et al. , 2007 ). Unexpectedly, EC proliferation was decreased in patients with AUB-I after LNG-IUS exposure, which may be explained by the fact that 3 years of treatment led to a faded angiogenic effect ( Hague et al. , 2002 ) or that the control group suffered from AUB-E, as Kooy et al. (1996) showed increased EC proliferation in patients with AUB-E, compared to controls with NMB ( Kooy et al. , 1996 ). This result is in line with the findings of Kayisli et al. (2015) , who showed that endometrial VSMC number and proliferation were decreased in patients exposed to long-acting progestin-only contraceptives, resulting in thin-walled, hyperdilated fragile microvessels, potentially causing AUB-I ( Kayisli et al. , 2015 ). However, these results are in contrast with a recent review that points out that, in patients with NMB, both EC proliferation and MVD do not change during the menstrual cycle. This review suggests that normal angiogenesis involves the faster process of vessel elongation and intussuscepted angiogenesis (splitting of existing blood vessels) ( Yetkin-Arik et al. , 2021 ). One could hypothesize that in patients with AUB-E, especially those suffering from HMB, a combination of angiogenic mechanisms could be responsible for the formation of new vessels ( Fig. 3 ). Moreover, if coalescent angiogenesis contributes to new blood vessel formation, a decrease in the number of vessels and therefore a decrease in MVD are expected ( Nitzsche et al. , 2022 ). It is likely that in patients with AUB-E and AUB-I, a combination of different angiogenic mechanisms may play a role. This could be one of the explanations for why increased angiogenesis is not always associated with a change in MVD but could lead to vessels of insufficient quality or unstable vessels with an impaired function. To compare angiogenesis between patients, the MVD may not always be the most appropriate parameter. As the MVD is also dependent on other endometrial elements, such as glandular and stromal cells, changes in MVD could also mean changes in these structures since a relative proportion of vessels is measured ( Shaw et al. , 1979 ; Makhija et al. , 2008 ). Furthermore, in patients with short-term exposure to exogenous hormone, an increase in MVD of mostly immature vessels could potentially be based on more vessel elongation, intussuscepting, or sprouting angiogenesis as compared to the extent of coalescent angiogenesis, thereby causing spotting complaints. More research is needed to elucidate the mechanisms responsible for angiogenesis in women with NMB, AUB-E, and AUB-I. Unexpectedly, EC migration was found to decrease in patients with AUB-I after short-term Norplant or DMPA exposure. This seems contradictory to the fact that, in patients with AUB-I, a higher MVD was found. This paradox might be explained by a lower blood vessel regression rate in Norplant and DMPA users, compared to other tissue in the endometrium. Thus, after 3–9 months of progestin exposure, the endometrial microvasculature is in a relative steady state, with a high vascular density but an overall low angiogenic activity. An alternative explanation could be that the authors used umbilical vein EC, which are characterized as macrovascular EC. It is possible that responses in microvascular EC, as present in endometrium tissues, differ from those in cells originating from macrovessels ( Subakir et al. , 1995 , 2000 ). This is the first narrative review that discusses impaired angiogenesis in the endometrium of patients with AUB, and AUB initiated by exogenous hormones. Strengths of this review include registration in the PROSPERO register of systematic reviews, the systematic approach according to PRISMA guidelines, the search of several databases, and the use of internationally accepted checklists to assess the RoB for included publications. In addition, this review systematically presents the broad range of angiogenic parameters and morphological characteristics of vessels. Nonetheless, there are some limitations. First, RoB assessment showed that only 8 of the 35 included studies were of good quality. Second, most studies lack objective scoring of AUB and include patients with subjective AUB; it could be questioned if the included patients with subjective blood loss actually suffer from objective AUB. Third, when interpreting the long-term effects of exogenous hormones, it should be considered that results can be influenced by selection bias, because patients with severe AUB-I are more prone to stop their treatment compared to patients with less severe symptoms. It is reported that 15% of the women who had an LNG-IUS placement for HMB had it removed within 6 months after insertion because of bleeding complaints ( Daud and Ewies, 2008 ). Also, up to 60% of women remove the LNG-IUD within 5 years because of bleeding complaints and adverse progestogenic effects ( National Institute for Health and Clinical Excellence, 2005 ). Moreover, it is possible that different doses and exposure times of exogenous hormones show different effects but because of the large heterogeneity in hormone studies it was not possible to address this further. Fourth, although the diagnostic techniques mostly involved IHC, quantification of these results differs among studies and is dependent on the subjective interpretation of the researchers. The magnification power of the fields analysed ranged from 40 to 400× and the unit of measurement was sometimes presented as measurements per power field, per unit area or per mm 2 . To minimize the differences in quantification, some studies compared the IHC outcomes with another analysis method, used scoring systems and/or automated computerized analysis. Studies show that if quantitative analysis of IHC is carried out by experienced researchers, different scoring systems do show significant correlation ( Walker, 2006 ). In addition, a large proportion of the included literature was published over 10 years ago. Exogenous hormones have developed over time and thus could be outdated, such the Progestasert-IUS. In addition, two out of the eight studies that objectified AUB by using the PBAC or AHM were published <10 years ago ( Andersson et al. , 2015 ; Maybin et al. , 2018 ), which potentially could be the reason for the varying outcomes. Furthermore, as MMPs and their inhibitor TIMPs are considered to be involved mainly in ECM degradation, they were not included in this review. The literature shows that these factors play a role in regulating HESCs, by stabilizing vascular ECM and endometrial stromal cells, and are regulated by progesterone. Dissolution of ECM or unstable stroma could therefore contribute to the severity of complaints of AUB ( Lockwood, 2011 ; Schatz et al. , 2016 ). Finally, and perhaps most importantly, this review includes many different markers, angiogenic factors and receptors, treatment types, treatment durations, and comparisons with different control groups (HMB or NMB). These factors were also measured at different phases of the menstrual cycle, which makes it difficult to compare the outcomes presented. Some factors are only analysed in one study, and if factors were analysed in different studies but measured in different phases of the menstrual cycle, the results are not comparable because of rapid changes in endometrial gene expression. Despite the heterogeneous literature about this very complex process, the authors are confident that the outcomes of studies support the hypothesis that disturbed angiogenesis is important in provoking AUB and emphasizes the need for further research. AUB accounts for one-third of all outpatient gynaecological visits and >70% of all gynaecological consults in the perimenopausal years ( Khafaga and Goldstein, 2019 ). New insight into the genesis of this disease is essential to develop new therapeutic strategies. This review supports our hypothesis that in patients with subjectively and objectively defined AUB-E and AUB-I, both anti- and proangiogenic factors are altered, likely leading to changes in vessel morphology. It is also the first review that shows a systematic presentation of several anti- and proangiogenic factors in patients with AUB-E and AUB-I. Nevertheless, the fact that one angiogenic factor can induce angiogenesis but simultaneously also play an essential role in vessel maturation makes it difficult to identify the best point of interest to intervene in this process. Future research should focus on identifying key angiogenic factors in a patient group with objectively defined AUB, to gather direct evidence to support the hypothesis that AUB symptoms are related to aberrant angiogenesis. In addition, it is important to distinguish between short- and long-term exogenous hormone exposure and to study the dose–effect relationship. Our results show that the most interesting systems involve factors from the HIF-, VEGF-, FGF-, and Ang-Tie pathways. Aberrant angiogenesis is known to play a major role in cancer, making this process a major point of focus in the development of antiangiogenesis therapies ( Ramjiawan et al. , 2017 ; Yetkin-Arik et al. , 2021 ). As this includes antiangiogenesis therapy in gynaecological cancers, this could also be interesting for the treatment of AUB. Naturally, antiangiogenesis therapy should be combined with birth control methods to avoid teratogenesis. To support our hypothesis that altered angiogenesis is related to AUB or infertility, additional research is the necessary first step to identify specific targets that can be used to intervene in the angiogenic pathways.

Click here for additional data file.