Abstract
The endometrium is a multicellular tissue that is exquisitely responsive to the ovarian hormones. The local mechanisms of endometrial regulation to ensure optimal function are less well characterised. Transient physiological hypoxia has been proposed as a critical regulator of endometrial function. Herein, we review the literature on hypoxia in the non-pregnant endometrium. We discuss the pros and cons of animal models, human laboratory studies and novel in vivo imaging for the study of endometrial hypoxia. These research tools provide mounting evidence of a transient hypoxic episode in the menstrual endometrium and suggest that endometrial hypoxia may be present at the time of implantation. This local hypoxia may modify the inflammatory environment, influence vascular remodelling and modulate endometrial proliferation to optimise endometrial function. Finally, we review current knowledge of the impact of this hypoxia on endometrial pathologies, with a focus on abnormal uterine bleeding. Throughout the manuscript areas for future research are highlighted with the aim of concentrating research efforts to maximise future benefits for women and society.
Introduction
The human endometrium is a heterogeneous and dynamic tissue that undergoes cyclical breakdown and repair/regeneration more than 400 times during the female reproductive lifespan (Short 1976, Critchley et al. 2020). This occurs each month without scarring or loss of function. However, the regulation and local mechanisms of this endometrial breakdown and repair remain elusive. In particular, our knowledge of the contribution of local endometrial hypoxia to this process is in its infancy. The presence of hypoxia, usually defined as a partial oxygen pressure below 10 mmHg, is not an uncommon phenomenon in human physiology, for example, in bone marrow and intestinal mucosa (Suda et al. 2011, Zheng et al. 2015). Its presence in the menstrual endometrium has been proposed following progesterone withdrawal and intense vasoconstriction of the specialised spiral arterioles (Markee 1940). Unravelling the role of hypoxia in the endometrium has the potential to improve our understanding of menstrual and implantation disorders and reveal novel therapeutic strategies for those suffering from these common, devastating conditions.
Endometrial histology and ovarian hormone regulation
Histologically, the endometrium can be divided into the functional and basal layer (Noyes et al. 1950). The functional layer occupies the upper two-thirds of the endometrium and is composed of stroma and glands. This layer undergoes constant remodelling throughout the menstrual cycle and is shed during menstruation. The basal layer, adjacent to the myometrium, comprises the lower third of the endometrium.
Oestradiol is the dominant hormone in the first half of the menstrual cycle, during the proliferative phase. It acts via the oestrogen receptor (ER), which has two structurally related subtypes, ERα and ERβ (Lessey et al. 1988, Critchley et al. 2002). After ovulation, levels of oestradiol decline and the corpus luteum increases its progesterone production, prompting endometrial differentiation and decidualisation. This process, driven by cAMP signalling, reshapes the stromal compartment in order to keep the endometrium receptive for future implantation (Dunn et al. 2003). In contrast with non-menstruating species, where implantation of an embryo is required to trigger decidualisation (Brasted et al. 2003), the human endometrium spontaneously decidualises with endometrial stromal cells in close proximity to spiral arterioles initiating their own transformation (Gellersen & Brosens 2014). They morphologically transition from fibroblast-like cells to rounded epithelioid-like cells (Dunn et al. 2003).
Endometrial breakdown and regeneration
In the absence of implantation, the corpus luteum regresses, causing significant progesterone withdrawal (Corker et al. 1976, Maybin et al. 2011b). This decrease in progesterone levels triggers a cascade of local physiological inflammatory events that initiate menstruation.
Progesterone withdrawal leads to the induction of the transcription factor NFκB, which upregulates the expression of pro-inflammatory cytokines (IL6, TNF) and chemokines (CCL2, CXCL8) (King et al. 2001). In addition, this fall in progesterone levels increases endometrial cyclooxygenase 2 (COX-2), responsible for the synthesis of prostaglandins (PG) (Critchley et al. 1999). Increased levels of these inflammatory mediators drive the recruitment of myeloid leukocytes, activation of matrix metalloproteinases (MMPs) and the shedding of the upper endometrial layers (Critchley et al. 2001, Kelly et al. 2001). Hypoxia has been identified in the endometrium following progesterone withdrawal (Fan et al. 2008, Cousins et al. 2016b, Maybin et al. 2018) and may be due to vasoconstriction of the endometrial vessels. PGF2α and endothelin-1 (EDN1) are two endometrial factors with known vasoconstrictive properties that are present following progesterone withdrawal (Baird et al. 1996, Marsh et al. 1997). Vasoconstriction of specialised endometrial spiral arterioles may limit blood loss during menstruation. The subsequent tissue hypoxia does not appear to be necessary for endometrial breakdown but may have an important role in endometrial repair/regeneration (Maybin et al. 2018, Chen et al. 2020).
Shedding of the functional endometrial layer necessitates repair of the denuded endometrial surface and regeneration of endometrial tissue. This takes place when oestradiol and progesterone levels are low but local glucocorticoid action may be increased (McDonald et al. 2006, Kaitu’u-Lino et al. 2007a, Rae et al. 2009). Evidence from mouse models and human tissue studies suggest that hypoxia is required for physiological endometrial repair (Fan et al. 2008, Maybin et al. 2018). The processes involved are likely to be similar to those of wound healing, involving haemostasis, inflammation, proliferation and remodelling (Velnar et al. 2009, Mutsaers et al. 2015).
Detection of Hypoxia throughout the menstrual cycle
The first suggestion that hypoxia was present at menses derived from findings in a primate model in 1940 (Markee 1940). Transplantation of endometrial explants to the Rhesus macaque eye allowed direct observation of intense vasoconstriction of spiral arterioles and focal bleeding following progesterone withdrawal. Since then, the use and refinement of animal models for the study of menstrual physiology and endometrial hypoxia has become more common.
In vivo animal models
Menstruation is restricted to humans and few other species. These include higher order primates (baboons, Rhesus macaques), the elephant shrew (Van der Horst & Gillman 1940), certain bats (Hamlett 1934, Rasweiler & de Bonilla 1992, Zhang et al. 2007) and the spiny mouse (Bellofiore et al. 2017). The majority of menstrual studies have been carried out in rodents and non-human primates, including the Rhesus macaque (Brenner & Slayden 2012).
Rodent models
Despite physiological differences between mice and humans (e.g. a shorter length of cycle and lack of spontaneous decidualisation) mouse models replicate the events of human menstruation and decidualisation well (Wang et al. 2013, Cousins et al. 2016a, Armstrong et al. 2017). The feasible management of large experimental groups, short breeding times and availability of laboratory antibodies/reagents provide advantages over macaque models. Mouse models also offer the possibility of genetic, environmental and pharmacological manipulation of hypoxia (see ‘Role of hypoxia throughout the menstrual cycle’ section). Technically, culling by carbon dioxide (CO2) inhalation can impact tissue hypoxia and may distort results. Hence, cervical dislocation is the recommended culling method for these studies. Great care must be taken to handle, process and fix tissue rapidly to capture the physiological events of menstruation.
The mouse model of simulated menstruation
The menses-like model was first described in 1984 (Finn & Pope 1984) and further optimised in the 2000s (Brasted et al. 2003). Since then, it has been the most popular model to investigate the dynamics of endometrial repair (Fan et al. 2008, Evans et al. 2011, Cousins et al. 2014, Maybin et al. 2018, Chen et al. 2020) (Fig. 1). Mice are ovariectomised and supplemented with exogenous oestradiol and progesterone to mimic the human hormonal endometrial environment. They require artificial induction of decidualisation, via a transcervical or surgical intrauterine injection of oil. Once decidualisation has taken place, progesterone withdrawal leads to active bleeding in the mouse uterus and subsequent repair (Fig. 1A). Alternatively, the simulation of menses can be achieved by inducing pseudopregnancy (Fig. 1B). In this model, female mice are mated with vasectomised males to mimic fertilisation events. Progesterone withdrawal occurs naturally or is induced by ovariectomy or administration of a progesterone antagonist (Rudolph et al. 2012).
Rodent models of simulated menstruation. (A) An exogenous hormone mouse model of simulated menstruation. Female mice are ovariectomised (ovex) and allowed to recover for 7–14 days before being given subcutaneous injections of oestradiol (E2). A progesterone (P4) implant is subcutaneously inserted and lower dose E2 injections administered. The decidualisation stimulus (oil) is transcervically administered. In order to induce a menstrual-like event, the P4 implant is subsequently removed (T0). This triggers a menstrual-like bleed (8 h after P4 withdrawal-T8) and subsequent endometrial regeneration (24 h after P4 withdrawal-T24). (B) A pseudopregnancy mouse model of simulated menstruation. Female mice are mated with vasectomised males to induce pseudopregnancy. Three to four days after the formation of the vaginal plug, decidualisation is externally induced via uterine oil injection. Two days after the decidualisation stimulus, mice are ovariectomised (ovex) to trigger P4 withdrawal (T0). Using this approach, the endometrial breakdown is apparent 12–16 h after P4 withdrawal (T12) and reepithelialisation can be detected 24 h after P4 withdrawal. Optionally, mice can receive daily subcutaneous injections of E2 to prevent atrophy of the uterus following ovariectomy. (C) Xenograft mouse model. Female immunodeficient mice are ovariectomised (ovex) and allowed to recover for 7–14 days before the implantation of the endometrial patches/dissociated endometrial cells. At the time of implantation or shortly after the formation of the xenografts, mice are treated with E2 and P4 for 21–28 days to induce the menstrual cycle. When the P4 pellet is removed, menstruation and successive regeneration take place in the xenograft for the next 4–8 days.
Citation: Reproduction 161, 1; 10.1530/REP-20-0268
The first work to describe the presence of hypoxia during endometrial breakdown and repair in the mouse utilised the ‘pseudopregnancy’ model variant (Fan et al. 2008). Pimonidazole is a hypoxic marker that, when oxygen partial pressures are below 10 mmHg, forms protein adducts which can be visualised using specific monoclonal antibodies. Due to its chemical stability, pimonidazole is considered one of the most reliable means of tissue oxygen level detection, even when it is temporally and spatially transient. Fan et al. found the endometrial area undergoing regeneration to be hypoxic and that this hypoxia decreased and eventually disappeared with endometrial reepithelialisation (Fan et al. 2008). Subsequent confirmation of the presence of menstrual hypoxia was found in the ‘exogenous hormone’ model of simulated menses (Cousins et al. 2016b, Maybin et al. 2018, Chen et al. 2020). Using pimonidazole, hypoxia was detected during bleeding and later confined to areas undergoing active repair. Hypoxia may also be present in the endometrium at the time of implantation. As the uterine epithelium contains no blood vessels during initial embryo contact, it has been suggested that the onset of implantation occurs in a hypoxic environment (Daikoku et al. 2003). The detection of pimonidazole adducts in the area of implantation in mice reinforces this hypothesis (Pringle et al. 2007).
Another method to determine tissue hypoxia is the detection of the oxygen-sensing transcription factor hypoxia-inducible factor (HIF). HIFs have a key role in the cellular response to oxygen and are heterodimers composed of two subunits: a constitutively expressed beta subunit (HIF1B) and an O2-sensitive alpha subunit (Semenza 2000). There are three known α subunits: HIF1A, HIF2A, and HIF3A. HIF1A and HIF2A are the most common alpha isoforms and present overlapping but distinct target gene specificities (Mole et al. 2009). HIF3A is structurally different from the other isoforms and is the least characterised (Pasanen et al. 2010). Along with promoting genes related to nitrogen metabolism and immune response, HIF3A has the ability to inhibit HIF1A/2A action (Zhang et al. 2014).
The regulation of HIF takes place predominantly at the protein level. In normoxia, prolyl hydroxylase domain enzymes (PHDs) hydroxylate specific residues within the alpha subunit, leading to its ubiquitination and subsequent degradation via the proteasome (Salceda & Caro 1997). In hypoxia these PHDs are inhibited, resulting in HIFA stabilisation. HIFA translocates to the nucleus, dimerises with HIF1A and binds to hypoxia-response elements (HREs) to enhance transcription of a plethora of genes involved in energy metabolism, angiogenesis, tissue remodelling and inflammatory responses (Semenza 2012).
The presence of nuclear HIF1A protein is, therefore, indicative of active HIF1 and consistent with tissue hypoxia. Using this approach, HIF1A has been detected during menstruation in both the exogenous hormone (Maybin et al. 2018, Chen et al. 2020) and pseudopregnancy menstruation models (Chen et al. 2015), decreasing during endometrial regeneration. Examination of HIF1A and HIF2A in the mouse uterus during pre-implantation (day 4) and decidualisation (days 5–8) of pregnancy, revealed HIF1A was present in the luminal epithelium prior to implantation and throughout the epithelium and stroma during decidualisation and implantation (Daikoku et al. 2003). HIF2A was seen in the stroma on day 4 and limited to cells surrounding the blastocyst on day 5. The authors suggested that HIF1 was involved in maintaining oxygen homeostasis and that HIF2 was driving the angiogenesis necessary for successful implantation.
Various concerns have been raised about using HIF as a hypoxic surrogate marker. Transient hypoxic events can be too brief to stabilise HIF for immunohistochemical detection (Wang et al. 1995). Antibody unreliability is an added factor, which is compounded by the fact that tissue collection and fixation can also affect HIF detection (Zhang & Salamonsen 2002). Furthermore, HIF stabilisation can be induced by NF-κB-driven cytokine production in a non-hypoxic dependent manner, and hypoxia can exert downstream effects independently of HIF signalling (Lin & Simon 2016).
Alongside detection of pimonidazole and HIF, hypoxia-inducible factor downstream targets may indicate a hypoxic response in the mouse menstrual endometrium. HIF1A-mediated induction of the angiogenic factors vascular endothelial growth factor (VEGF) and the chemokine receptor CXCR4 was increased during menstruation and endometrial repair (Fan et al. 2008, Chen et al. 2015, Cousins et al. 2016b, Maybin et al. 2018).
Xenograft mouse model
The xenograft mouse model provides an alternative model for the study of menstrual physiology and pathology (extensively reviewed in Kuokkanen et al. 2017). Human functional endometrium is transplanted into immunodeficient mice (Fig. 1C). This is usually collected during the proliferative phase and can be transplanted as (1) small fragments (1–2 mm3) of endometrial tissue (Guo et al. 2011, Coudyzer et al. 2013) or (2) dissociated endometrial cells from epithelial and stromal fractions that are mixed before implantation (Masuda et al. 2007, Polotsky et al. 2009).
The recipient mice are selected to limit xenograft tissue rejection, but the immunodeficient strain used can vary. The most commonly used in xenograft menstruation models is the severe combined immunodeficiency (SCID) mouse, which has T and B cell deficiencies (Gaide Chevronnay et al. 2009, Guo et al. 2011, Coudyzer et al. 2013). The best engraftment results are achieved with the non-obese diabetic (NOD)/SCID/γcnull mice (NOG), which also have defective NK cell activity (Matsuura-Sawada et al. 2005, Masuda et al. 2007).
Generally, the patches of endometrial tissue are placed subcutaneously in mice (Guo et al. 2011, Coudyzer et al. 2013) with a survival time of 4 weeks, whereas the dissociated endometrial cells are implanted below the kidney capsule and survive up to 10 weeks (Masuda et al. 2007). This latter mode of implantation allows extension of the duration of experiments, making this the method of choice for studies of the proliferation kinetics of the endometrium after pharmacological treatments (Polotsky et al. 2009).
Xenograft menstruation studies mainly focus on endometrial regeneration and the role of ovarian steroids in orchestrating the process (Gaide Chevronnay et al. 2009, Guo et al. 2011, Coudyzer et al. 2015) and use the endometrial fragments model variant. To date, this mouse model has only been employed once to study the presence of hypoxia during menstruation (Coudyzer et al. 2013). In 2013, Coudyzer et al. subcutaneously implanted endometrial patches on SCID female mice and tested for signs of hypoxia in the resulting xenograft using several methods. First, they directly measured the local partial oxygen pressure (pO2) using electron paramagnetic resonance and OxyLite fluorescent probes. They also studied the presence of pimonidazole staining and HIF1A using immunohistochemistry (IHC). The authors did not detect hypoxia during endometrial breakdown or repair using any of these methods. These results contrast with findings in the mouse model of simulated menses and may be partially explained by the xenograft model itself. Endometrial tissue architecture and vasculature is severely compromised following transplantation and may impair vasoconstriction and prevent endometrial hypoxia. Moreover, endometrial breakdown and repair are considered inflammatory events, as they involve pro-inflammatory cytokine production and myeloid leukocyte recruitment (Finn 1986). Therefore, the necessary immunosuppressed state of the recipient mice may alter physiological menstrual endometrial events. The SCID model aims to suppress T and B-cell mediated transplant/xenograft rejection without substantially affecting the innate immune response and may be more relevant than other immunocompromised recipient mice (Guo et al. 2011, Donoghue et al. 2012).
Spiny mouse
The common spiny mouse (Acomys cahirinus) is, to date, the only known rodent to display spontaneous decidualisation and natural menstruation (Bellofiore et al. 2017, 2018).
Although anatomically different, the spiny mouse uterus has physiological similarities to the human endometrium. For example, the spiny mouse displays spiral arteriole remodelling in the perimenstrual phase (Bellofiore et al. 2018). In addition, endometrial decidualisation is tightly controlled, not compromising the structural integrity of the endometrial glands or the myometrium, as observed in other mouse models (Bellofiore et al. 2018). Hypoxia has not been examined in this rodent to date and these studies are awaited with interest.
Macaque models
Macaques have morphologically similar uteri to humans, a similar length of menstrual cycle and they display spontaneous decidualisation (Brenner & Slayden 2012). Macaques also experience menstrual abnormalities (e.g. heavy menstrual bleeding (HMB)) and can be fitted with tampons, hence they are exceptional candidates for evaluating therapies for menstrual disorders (reviewed in Brenner & Slayden 2012). Despite menstruating naturally, macaques are routinely ovariectomised and treated with oestradiol and progesterone to create artificial menstrual cycles and enable accurate timing of endometrial sampling. However, the need for larger experimental groups, longer experimental times and the increased cost of these experiments has meant many researchers are now preferentially using rodent models to study menstrual physiology.
As previously mentioned, the first indication of endometrial tissue hypoxia was observed in endometrial explants transplanted to the eye of rhesus macaques in the 1940s (Markee 1940). Rather than hypoxia, Markee observed pulses of intense vasoconstriction in the spiral arterioles that he associated with localised hypoxic ischemia. This hypothesis was later supported by the detection and increased expression of HIF1A in the functional layer of the macaque endometrium during menstruation (Brenner & Slayden 2012), consistent with the presence of endometrial hypoxia.
Ex vivo human endometrial studies
HIF1A protein has been identified, both by Western blot and IHC, in human endometrial biopsies collected during the late secretory and menstrual phases (Critchley et al. 2006, Maybin et al. 2018). HIF1A staining was localised in the glandular and stromal cells in the functional endometrium, whereas in the basal layer HIF1A staining was restricted to the glands.
In contrast, HIF2A is present exclusively during the early-mid secretory phase (Maybin et al. 2018). Downstream targets of HIF, such as VEGF and carbonic anhydrase IX (CA9), have also been shown to be increased during the menstrual and proliferative phases (Charnock-Jones et al. 1993, Sharkey et al. 2000, Punyadeera et al. 2006, Maybin et al. 2011b).
In vivo human endometrial studies
Detection of human endometrial hypoxia in vivo has been largely via measurements of perfusion, initially investigated using thermal heat dissipation (Prill & Götz 1961) and later by a Xenon-133 clearance technique (Fraser et al. 1987) (Fig. 2). Both methods are invasive, and results were conflicting as suffering from variable calibration and poor spatial and temporal resolution, respectively. The introduction of Doppler ultrasound allowed perfusion measurements in individual spiral arterioles (Kupesic & Kurjak 1993), but this showed an increase in flow the day before ovulation, in contrast with the 133Xe clearance study which found a fall at this time. Laser Doppler fluxmetry was able to assess endometrial perfusion using a fibre optic probe (Gannon et al. 1997), finding blood flow peaks in the early proliferative and early secretory phase, but spatial resolution was limited. The more sensitive three-dimensional power Doppler angiography (3D-PDA) was also used in spiral arterioles (Raine-Fenning et al. 2004) and revealed a significant pre-ovulatory peak in perfusion, followed by a post-ovulatory fall and gradual increase through early to mid-secretory phases. In general, there has been little consensus regarding changes in endometrial blood flow over the menstrual cycle and how to measure such changes. MRI methods may now offer a better alternative, although there has been little work on the application of these techniques to detect endometrial hypoxia.
In vivo methods with the potential to detect markers of endometrial hypoxia in women. Left: previous in vivo work to assess human endometrial hypoxia, shown with structural MRI of the uterus and surrounding tissues. Right: potential non-invasive imaging methods for translation from other body areas. [+] Advantages of each technique, [−] Disadvantages of each technique. Relevant references shown for each. DCE-MRI, dynamic contrast-enhanced MRI.
Citation: Reproduction 161, 1; 10.1530/REP-20-0268
To our knowledge, functional investigation of the normal endometrium has been limited to MR spectroscopy (Sarac et al. 2004, Celik et al. 2005). This technique detects the presence of specific metabolites in the body by examining the resonant frequencies of the hydrogen protons within them. In particular, lactate is a product of anaerobic respiration (and therefore a marker of hypoxia) and has been detected in normal secretory and proliferative endometrium (Sarac et al. 2004, Celik et al. 2005). Although lactate is arguably a more direct marker of hypoxia than measurement of perfusion, analysis and acquisition of spectroscopy data is technically challenging (Lange et al. 2006) and spatial resolution tends to be poor.
Dynamic contrast-enhanced (DCE) MRI is a technique that can detect hypoxia indirectly by measuring perfusion using an exogenous gadolinium-based contrast agent (CA) (Sourbron 2010). Passage of the CA through the tissue can be modelled to allow perfusion to be estimated as part of a model-fitting process (Sourbron & Buckley 2012). The technique has been applied in the normal endometrium (Majd et al. 2017) but showed no differences between the secretory and proliferative phases. The advantage of DCE-MRI for hypoxia imaging is its good spatial resolution, but imaging and analysis can be complex (Brix et al. 2004, 2009, Michaely et al. 2008) and there is no gold standard for validation of the technique. Use of DCE-MRI to detect a reduction in perfusion related to hypoxia in the menstrual cycle would require a specialised imaging protocol and robust data analysis using a complex model, including estimation of parameter uncertainties.
Other existing MRI techniques could be applied to measure endometrial hypoxia (Fig. 2). T2* is a characteristic tissue relaxation time that depends on inhomogeneities in the main magnetic field produced by the scanner as well as rapidly changing inhomogeneities induced by the presence of other nearby molecules. Detection of a reduction in T2* is commonly assumed to be due to the presence of deoxyhaemoglobin and therefore, tissue hypoxia. This technique has been used in the myometrium (Kido et al. 2007, Imaoka et al. 2012) and has a high spatial resolution necessary to investigate the endometrium. T2* can change for a number of other reasons (e.g. local haematocrit, hemosiderin, calcification and tissue iron deposition) therefore changes should be interpreted with caution. Similarly, a non-invasive perfusion technique known as arterial spin labelling (ASL) (Ferré et al. 2013) could be extended from existing work in the myometrium (Takahashi et al. 2016) to the endometrium, though it can be technically challenging. Finally, the extensive work on hypoxia measurements in cancer (Horsman et al. 2012) could be applied in the endometrium. Oxygen-enhanced (OE) MRI (O’Connor et al. 2019) allows a change in the tissue relaxation time T1 as a result of the patient breathing 100% oxygen through a mask to be related to the oxygen status of the tissue (O’Connor et al. 2016). These minimally invasive MRI techniques may provide key information on the presence of human endometrial hypoxia throughout the menstrual cycle, with potential diagnostic and therapeutic benefits for women.
Role of hypoxia throughout the menstrual cycle
Mice have the experimental advantage of genetic or pharmacological alteration to assess the role of hypoxia in endometrial function. Hif1a heterozygote mice have revealed that HIF1A is required for normal menstruation, and decreased HIF1A delays endometrial repair (Maybin et al. 2018). Pharmacological stabilisation and inhibition of HIF1A in mice have confirmed this role (Chen et al. 2015, Maybin et al. 2018). Mice placed in hyperoxic chambers (75% O2) during menses had reduced local endometrial hypoxia at menstruation and delayed endometrial repair (Maybin et al. 2018). Hif2a deficiency restricted to uterine stromal cells in a mouse implantation model revealed a key role in decidualisation, endometrial receptivity, embryonic implantation and survival (Matsumoto et al. 2018).
This emerging evidence for the presence and important role of hypoxia and HIF in endometrial function presents an exciting and developing research area (Fig. 3). The effects of hypoxia on the important menstrual processes of inflammation, proliferation and tissue remodelling remain to be elucidated.
Overview of the presence and role of hypoxia in endometrial physiology. (A) Hypoxia during implantation. Endometrial stromal cells undergo decidualisation under the influence of progesterone. Hypoxia-inducible factor (HIF)2A in these uterine stromal cells supports decidualisation, embryo invasion and survival. Endometrial blood vessels undergo dynamic remodelling that may be influenced by hypoxia/HIF. (B) Hypoxia during endometrial breakdown. Vasoconstriction of the endometrial vessels may limit blood loss during menstruation and cause transient endometrial hypoxia to stabilise HIF1A. The endometrial leukocyte population may be altered in number and/or function by hypoxia/HIF. (C) Role of hypoxia during endometrial repair. Hypoxia is not detected in endometrial areas that have reepithelialised, while those areas undergoing active regeneration remain hypoxic. This hypoxia is thought to promote endometrial VEGF (alongside other factors), which is responsible for reepithelialisation and new blood vessel formation. P4, progesterone; G, glands; BV, blood vessel; VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factor.
Citation: Reproduction 161, 1; 10.1530/REP-20-0268
Impact of hypoxia on inflammation
Inflammation is a key event during implantation, at menstruation and the subsequent endometrial repair. There is a peri-menstrual influx of leukocytes into the endometrium, in particular, neutrophils and macrophages (Armstrong et al. 2017). Interactions between the inflammatory response and hypoxia are well described at other tissue sites (Cramer et al. 2003, Taylor 2008, Taylor et al. 2016) but the impact of hypoxia on the endometrial inflammatory response is less well characterised.
Impact on neutrophils
Neutrophils comprise up to 15% of the total endometrial cell numbers during menstruation (Poropatich et al. 1987, Salamonsen & Lathbury 2000). Their influx is tightly regulated, displaying a rapid, short-lasting induction, which coincides with the upregulation of chemokines and cytokines. This temporal dynamic has been observed in both the mouse model of simulated menses and in human endometrial samples (Armstrong et al. 2017). Neutrophils are important mediators of endometrial breakdown, which has been confirmed by their depletion in the mouse model of menstruation (Kaitu’u-Lino et al. 2007b). However, the depleting agent used in this study also affects the monocytic cell lineage. Activated neutrophils release enzymes, such as neutrophil elastase and cathepsin G. These enzymes activate MMPs produced by endometrial stromal cells and cause degradation of the extracellular matrix (Salamonsen & Lathbury 2000). In airway inflammation, hypoxia boosts neutrophil degranulation and protease release (Hoenderdos et al. 2016). It would be informative to determine whether hypoxia has similar effects in the endometrial environment during menses.
Neutrophils also produce reactive oxygen species (ROS) that might participate in the endometrial breakdown. The potential role of ROS in menstruation has been reported (Sugino et al. 1996), suggesting that free oxygen radicals may contribute to endometrial shedding by causing tissue damage. Indeed, the inhibition of ROS generation in the mouse model of simulated menstruation has been shown to abrogate endometrial breakdown (Wu et al. 2014).
Neutrophil depletion in mouse models also affected endometrial regeneration (Kaitu’u-Lino et al. 2007b). Little is known about the impact of hypoxia on neutrophils during the endometrial repair. The concept that hypoxia has an effect on neutrophil number and function is derived from studies of tumour biology. In a mouse model of endometrial carcinoma, there was spatiotemporal correlation between hypoxia and neutrophil infiltration within the tumour (Blaisdell et al. 2015). Accumulation of pimonidazole and nuclear staining of HIF1A was detected slightly prior to neutrophil infiltration. These results are consistent with those observed in the mouse model of simulated menses, where pharmacological inhibition of HIF1A decreased the number of endometrial neutrophils present during active bleeding (Maybin et al. 2018). The role of hypoxia in promoting neutrophil recruitment in endometrial carcinoma was confirmed by placing mice in hyperoxic chambers (60% O2) (Mahiddine et al. 2020). This resulted in a dramatic reduction in neutrophil influx within the tumour and also improved the ability of these cells to oppose tumour growth through increased activation and expression of several MMPs and ROS production. This is consistent with hypoxia not only affecting the recruitment of neutrophils, but also their function. Determining the effects of hypoxia on neutrophil number and phenotype in the normal endometrium would be of great interest to advance our understanding of menstrual physiology.
Effects of hypoxia on neutrophils have also been observed in benign tissues. Airway inflammation studies have revealed that hypoxia, via HIF1A and HIF2A, prolonged neutrophil lifespan by inhibiting apoptosis (Walmsley et al. 2005, Thompson et al. 2014). Glucocorticoids have also been shown to delay neutrophil apoptosis in vitro, but this did not occur in the presence of hypoxia (Marwick et al. 2013). Neutrophil apoptosis has been identified in the menstrual endometrium of mice, when hypoxia is present (Armstrong et al. 2017). In addition, glucocorticoids have been identified as having an important role in the human menstrual endometrium (McDonald et al. 2006, Rae et al. 2009). The impact of hypoxia on endometrial myeloid apoptosis has not been examined to date.
Impact on macrophages
Macrophages have been detected in the endometrium throughout the menstrual cycle, both close to the endometrial glands and in the stromal compartment (Bonatz et al. 1992). They show a peri-menstrual peak in number, reaching up to 15% of the cell total number at the time of menses (Salamonsen & Woolley 1999). Like neutrophils, it is proposed that macrophages play a critical role in the onset of endometrial breakdown via production and release of MMPs (reviewed in Critchley et al. 2001, Thiruchelvam et al. 2013). There are also indications of their involvement in glandular remodelling (Garry et al. 2010) and endometrial regeneration (Maybin et al. 2012, Cousins et al. 2016a), including the regulation of angiogenesis (Thiruchelvam et al. 2016).
Macrophages are remarkably plastic cells, capable of shifting towards different phenotypes by sensing the surrounding microenvironment (Martinez et al. 2008). Thus, their microenvironment may affect their recruitment and function. Historically, macrophage polarisation has been categorised as classical (M1) or alternative (M2). M1 phenotype is associated with microbicidal properties and M2 reflects a more regulatory, anti-inflammatory phenotype. More recently, macrophage polarisation is understood to be a dynamic spectrum of macrophage transition in response to environmental cues (Martinez & Gordon 2014). As there is mounting evidence for hypoxia in the local endometrial environment at menstruation (Cousins et al. 2016b, Maybin et al. 2018), it is important to determine its effect on endometrial macrophages.
Under physiological conditions, M2 macrophages are involved in angiogenesis and cellular clearance, hence promote wound healing. However, tumour-infiltrating macrophages (TAMs) are often correlated with poor cancer prognosis (Kawanaka et al. 2008). TAMs have been shown to be retained in hypoxic regions of tumours through the Sema3A/Neuropilin-1signaling axis, which is regulated by HIF2A (Casazza et al. 2013). The influence of hypoxia on TAMs is not only limited to the macrophage number but also influences their phenotype. Indeed, specific TAM phenotypical subsets have been reported depending on intra-tumoral oxygen levels (Laoui et al. 2014).
Non-tumoral studies have also linked HIF to changes in macrophage phenotype. In a model of endotoxemia, HIF1A and HIF2A were differentially expressed in M1 and M2-macrophages, respectively (Takeda et al. 2010). In addition, in the context of obesity and adipose tissue inflammation, HIF1A has been proven to promote inflammation and insulin resistance through M1 macrophage polarisation whereas HIF2A ameliorated the effects via M2-macrophage induction (reviewed in Lin & Simon 2016). Interestingly, HIF1A was found to be decreased in mouse adipose tissue when glucocorticoid activation was suppressed, suggesting a crucial role of glucocorticoids in HIF-dependent macrophage polarisation (Chapman et al. 2013). Thus, different research fields converge around the concept that HIF1A may be required for M1 polarisation of macrophages, while HIF2A might promote M2 polarisation.
The menstrual endometrium presents a unique model of transient, physiological hypoxia in which to study macrophage number and phenotype. HIF2A may have a role in the recruitment and function of macrophages during implantation, when endometrial HIF2A was found to be present (Maybin et al. 2018). However, a recent study of mice with a targeted deletion of Hif1a in myeloid cells resulted in decreased pregnancy rates and increased miscarriage rates, suggesting that HIF1A dependent pathways in myeloid cells are also important for the maintenance of pregnancy (Köstlin-Gille et al. 2019). It would be informative to establish if the balance between HIF1A/HIF2A determines the pro-inflammatory or anti-inflammatory fate of the endometrium.
Impact of hypoxia on proliferation
After ‘injury’, fibroblasts must migrate and proliferate in the damaged area, where they produce extracellular matrix (ECM) components that contribute to repair (Gonzalez et al. 2016). This production must be tightly regulated to prevent excessive ECM growth, scar formation and fibrosis (Ruthenborg et al. 2014). In dermal tissue, hypoxia has been shown to stimulate macrophage growth factors that may contribute to fibroblast proliferation and tissue repair (Murdoch et al. 2005). Macrophage production of platelet-derived growth factor (PDGF) enhances fibroblast mitosis, while transforming growth factor β (TGFB) promotes the formation of the ECM (Ruthenborg et al. 2014). In addition, hypoxia has been proven to induce the transcription of VEGF, connective tissue growth factor and adrenomedullin in endometrial stromal tissue (Maybin et al. 2011a, Maybin et al. 2012). Hence, hypoxia may induce a pro-repair environment by modifying the secretome of endometrial cell populations.
To complete tissue restoration, reepithelialisation of the affected area must take place. In the skin, this is achieved through the migration and proliferation of keratinocytes towards the injury site (Ruthenborg et al. 2014). Stabilisation of HIF1A in a mouse model of skin wound healing revealed its role in promoting keratinocyte proliferation and migration to the injured area, accelerating wound closure (Kalucka et al. 2013). This is consistent with the findings of delayed endometrial repair with decreased HIF1A (Maybin et al. 2018).
Impact of hypoxia on vascular remodelling and angiogenesis
Angiogenesis and vascular remodelling are crucial events in the endometrium throughout the menstrual cycle. Optimal vascular function is necessary to support the repair of the functional endometrial layer and to supply the thickened endometrium required for successful implantation and placentation.
VEGF is a key mediator of both physiological and tumoral angiogenesis and may be induced by hypoxia (Carmeliet 2005). VEGF mRNA and protein have been detected during all phases of the menstrual cycle, both in the stromal compartment and the glandular epithelium ( Charnock-Jones et al. 1993, Shifren et al. 1996, Punyadeera et al. 2006) but was maximal during menses (Sharkey et al. 2000, Graubert et al. 2001, Maybin et al. 2011b). Studies in mouse and macaque models of menstruation have shown that blocking VEGF dramatically decreases reepithelialisation and new blood vessel formation in the endometrium (Fan et al. 2008), consistent with an essential role for VEGF in endometrial angiogenesis and repair.
Hypoxia has been detected in the mouse model of simulated menses (Chen et al. 2015, Cousins et al. 2016b, Maybin et al. 2018) and coincides with increased VEGF mRNA (Cousins et al. 2016b). Hypoxia and VEGF have also been detected in human perimenstrual endometrial biopsies (Punyadeera et al. 2006) highlighting their possible interrelation. In vitro studies have also shown that subjecting endometrial and epithelial stromal cells to hypoxia increases VEGF mRNA and protein (Popovici et al. 1999, Sharkey et al. 2000, Graubert et al. 2001) and that silencing of HIF1A abrogates this hypoxia-induced VEGF expression (Maybin et al. 2011b, Chen et al. 2015). Through a chromatin immunoprecipitation (ChIP) assay, Chen et al. detected the direct binding of HIF1A to the VEGF promoter, which was maximal during the endometrial breakdown of the mouse model of menses (Chen et al. 2015). Inhibition of HIF1A using 2-methoxyestradiol (2-ME) significantly suppressed VEGF levels during menses. Therefore, hypoxia, and more specifically HIF1A, seems to promote endometrial VEGF during menses.
In addition, VEGF expression is induced by different cytokines and chemokines (Li et al. 1995, Stavri et al. 1995, Zagzag et al. 2006), some of which contain hypoxic response elements. Optimal blood vessel formation requires the trafficking of endothelial progenitors cells through the interaction of the chemokine CXCL12 with its receptor CXCR4 (Ruthenborg et al. 2014). Both ligand and receptor have been found to be upregulated by HIF1A, contributing to angiogenesis and blood vessel repair partly through VEGF (Ceradini et al. 2004, Zagzag et al. 2006). CXCL12 and CXCR4 have been described in the human endometrium (Ruiz et al. 2010) and endometrial CXCR4 was found to be decreased in patients with heavy menstrual bleeding (Maybin et al. 2018). Hence, the interactions between hypoxia pathways and inflammatory processes may significantly influence endometrial vascular function.
During decidualisation there is in vitro evidence that endometrial stromal cells increase VEGF mRNA and protein (Popovici et al. 1999, Matsui et al. 2004) and that hypoxia induces further increases in VEGF (Popovici et al. 1999). This VEGF production may be responsible for macrophage recruitment and polarisation towards a pro-angiogenic M2 phenotype (Wheeler et al. 2018). Thus, the responsiveness of the decidualised stroma to hypoxia suggests a possible role in the preparation of the endometrial vasculature for implantation. Uterine HIF2A deficiency has been shown to impair decidualisation in mice, revealing a downregulation of prolactin-related factors which can compromise the maintenance of the corpus luteum and therefore endometrial receptivity (Matsumoto et al. 2018).
When studying implantation in mice, HIF factors were found to be differentially expressed at the time of peri-implantation: HIF1A was detected in the luminal epithelium, whereas HIF2A expression was limited to the stromal compartment and neither correlated with VEGF expression (Daikoku et al. 2003). Therefore, HIF effects on implantation seem to be more versatile than simply contributing to vessel formation, playing a substantial role in decidualisation, endometrial receptivity and embryo survival (Matsumoto et al. 2018). After implantation, HIF1A was found in the luminal epithelium and the decidual layer. However, the strongest signal came from HIF2A, with expression localised to stromal cells surrounding the blastocyst. This post-implantation HIF2A expression was correlated with VEGF induction, switching to a proangiogenic stimulus once implantation had taken place (Daikoku et al. 2003).
The role of hypoxia in endometrial pathology
As outlined above, the literature regarding the influence of hypoxia on inflammation, proliferation and vascular function is increasing (Fig. 3). The influence of oxygen levels on implantation, placentation and disorders such as pre-eclampsia has been comprehensively reviewed within this series by Burton (2009). The impact of hypoxia on embryo function has been covered in detail by Dunwoodie (2009). Therefore, this section is focused on the role of endometrial hypoxia during menstruation and its potential in the identification of novel diagnostic and therapeutic strategies.
Abnormal uterine bleeding
Abnormal uterine bleeding (AUB) affects 20–30% of pre-menopausal women and over 800,000 women seek treatment in the UK each year (National Heavy Menstrual Bleeding Audit 2011). Available medical treatments are often discontinued due to side effects or lack of efficacy. Research in this area was previously hindered by lack of a consistent classification system for the diagnosis of causes of AUB. This was rectified by the development of the FIGO classification system of structural and non-structural causes (Munro et al. 2011, 2018) (Fig. 4).
Abnormal uterine bleeding (AUB) and the potential role of hypoxia. Abnormal uterine bleeding may be due to structural (polyps, adenomyosis, leiomyoma, malignancy) or non-structural (coagulopathy, ovulatory, endometrial, iatrogenic or not otherwise classified) disorders. The role of hypoxia in AUB is unknown but its potential role in four disorders is illustrated. (I) Leiomyoma (fibroids): the decreased levels of endothelin and PGF2α receptors may compromise endometrial vasoconstriction and increase menstrual blood flow. (II) Malignancy: tumour hypoxia leads to disorganised angiogenesis and increased metastasis. (III) Endometrial disorders: endothelial cell focal discontinuities and impairment of vascular smooth muscle cells may influence vasoconstriction. This may decrease HIF-1α and prevent optimal post-menstrual repair. (IV) Adenomyosis: VEGF and HIF-1α overexpression may contribute to increased vessel formation and AUB. G, glands; BV, blood vessels; Myo, myometrium; E, endometrium; VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factor.
Citation: Reproduction 161, 1; 10.1530/REP-20-0268
Structural causes of AUB
Structural causes of AUB can be detected on examination or investigation of the uterus, for example, polyps, adenomyosis, leiomyoma (fibroids) and malignancy (Munro et al. 2011, 2018). These conditions have previously been under-diagnosed, with clinicians often treating the symptom of AUB without identifying the underlying cause. This has limited our knowledge on why these conditions develop and why they result in AUB.
Adenomyosis is the presence of ectopic endometrial glands and stroma within the myometrial layer of the uterus. It occurs in 7–27% of reproductive-aged women and presents with painful, heavy menstrual bleeding (Naftalin et al. 2012, Mavrelos et al. 2017). The impact of the adenomyotic lesions on the eutopic endometrium and the mechanisms causing AUB is not well understood. AUB due to adenomyosis (AUB-A) is particularly challenging as it is often resistant to medical treatment and surgical options (ablation or hysterectomy) are unacceptable to those wishing to preserve their fertility.
There is some evidence that the hypoxic response is aberrant within adenomyotic lesions. A study of hysterectomy samples from 14 women with adenomyosis and 9 without revealed increased VEGF protein in the eutopic endometrium of women with adenomyosis and increased VEGF and HIF1A protein in ectopic vs eutopic endometrium (Goteri et al. 2009). This suggests that a hypoxic environment in the adenomyotic lesions could contribute to increased vessel formation. In endometriosis, where ectopic endometrium implants outside of the uterus, HIF1A was also found to be increased in ectopic vs eutopic endometrium (Wu et al. 2007, Young et al. 2014). Inhibition of HIF1 in a mouse model of endometriosis suppressed the growth of lesions (Becker et al. 2008), identifying the hypoxia pathway as a potential therapeutic target. The peritoneum is a common site for implantation of ectopic endometrial deposits in endometriosis. Women with endometriosis have been shown to have increased HIF1A in non-affected peritoneum compared to peritoneum from women without disease (Young et al. 2014), consistent with a role of the hypoxia pathway in the development of peritoneal disease. Studies examining the non-affected myometrium in women with adenomyosis are not yet available, but similar alterations in hypoxic response would highlight hypoxia pathways as a potential target for preventative and therapeutic interventions.
Leiomyomas (uterine fibroids) are common, benign tumours of the myometrium that form as a consequence of the proliferation of uterine smooth muscle cells and collagen matrix. They occur in approximately 70% of women (Stewart et al. 2017) and are extremely heterogeneous in size, location and pathophysiology. Leiomyomas are symptomatic in approximately 50% of women (Day Baird et al. 2003) and may cause symptoms of AUB, pressure, pelvic pain and be associated with subfertility.
Genome wide association studies have identified genetic subgroups that may predispose to leiomyoma formation (reviewed in Stewart et al. 2016) but local mechanisms regulating their development remain an area of active research. Uterine leiomyomas contain broad avascular areas and HIF1A protein was found to be increased in leiomyoma nuclear protein extracts when compared to adjacent myometrium (Ishikawa et al. 2019). However, it is not yet clear whether hypoxia is necessary for leiomyoma development and/or growth. In contrast, an in vivo study of women with leiomyoma using DCE-MRI has revealed increased Ktrans (a combination of perfusion and permeability) in fibroids compared with the normal uterus (Majd et al. 2017) which does not support the presence of hypoxia within fibroids. There is evidence that treatment of leiomyomas with gonadotrophin-releasing hormone (GnRH) analogues, often used pre-operatively to reduce fibroid size and decrease AUB, lead to a decrease in perfusion parameters (Munro et al. 2014). These contrasting in vitro and in vivo findings may reflect the heterogeneity of leiomyomas and it remains unclear if altered perfusion is associated with AUB.
The cause of AUB experienced by a proportion of women with leiomyomas is not understood. Vasoconstriction may be impaired at the time of menstruation in women with fibroids, with leiomyoma tissue expressing altered levels of endothelin receptors and prostaglandin F2α when compared to normal myometrium (Pekonen et al. 1994, Miura et al. 2006). A small decrease in spiral arteriole vasoconstriction can significantly increase menstrual blood flow, causing heavy menstrual bleeding. A greater understanding of the role of hypoxia in leiomyoma formation and growth may identify new, specific treatments to reduce their presence, size and symptoms.
Endometrial cancer
The importance of hypoxia in the tumour microenvironment is well established, including its influence on immune cell populations, angiogenesis, tumour progression and metastasis (De Bock et al. 2011, Casazza et al. 2014, Schito & Semenza 2016, Semenza 2016). The accuracy of translation of these principles to patients with endometrial cancer is less well determined. In a quest to identify a robust biomarker that would predict tumour behaviour, Chang et al. identified an eight-gene set of lymphocyte and tumour hypoxia markers and validated its performance in predicting overall survival in six cancers, including 370 women with endometrial cancer (Chang et al. 2019). They found a superior performance over current tumour staging parameters, highlighting the importance of hypoxia in determining risk and aiding clinical decision making.
Assessment of endometrial tissues from 386 patients with endometrial carcinoma using CA9 as a hypoxia marker and CD34 to determine vascular density, revealed that patients with the presence of both hypoxia and high vascular density (16.4%) had reduced disease-specific survival and distant disease-free survival (Reijnen et al. 2019). In vivo imaging with DCE-MRI revealed that a poor prognosis was associated with low microvascular blood flow to the endometrial tumour (Haldorsen et al. 2013, 2014, Berg et al. 2016). This was thought to reflect disorganised angiogenesis with coexisting vascular proliferation and hypoxia. These studies highlight normalisation of the vasculature to limit hypoxia as a potential therapeutic target in endometrial cancer.
Non-structural causes of AUB
These non-structural disorders are not usually identified by routine pelvic imaging. They include coagulopathies, ovulatory dysfunction, endometrial and iatrogenic causes (Munro et al. 2018). Evidence for a role of hypoxia in these disorders is limited but its contribution to AUB of endometrial origin (AUB-E) is discussed below.
AUB-E includes disorders of local endometrial haemostasis, vascular function and/or inflammation (Fig. 4). Women with objectively defined heavy menstrual bleeding (HMB; >80 mL/cycle) had reduced levels of HIF1A protein and downstream target genes in menstrual phase endometrial biopsies when compared to those from women with normal blood loss (Maybin et al. 2018). Examination of endometrial repair in mice where hypoxia was prevented during simulated menses, or where HIF1A was pharmacologically or genetically reduced, revealed delayed repair (Maybin et al. 2018). This is consistent with hypoxia having a key role in the rapid endometrial repair necessary to limit menstrual blood loss. The delayed repair in a non-hypoxic mouse menstruation model could be rescued with a pharmacological compound that stabilises HIF1, identifying a potential non-hormonal therapeutic target for women with AUB-E.
The cause of the endometrial tissue hypoxia observed at menstruation is unknown. It is likely that spiral arteriole vasoconstriction limits blood supply to the functional layer of the endometrium following progesterone withdrawal (Markee 1940). Hence, factors that limit the ability of the specialised endometrial arterioles to constrict will have a significant impact on the presence of endometrial hypoxia. Women with the symptom of HMB have been shown to have significantly decreased smooth muscle myosin heavy chain in their spiral arterioles and also reduced vascular smooth muscle cell proliferation during the mid-late secretory phase compared to those with normal menstrual blood loss (Abberton et al. 1999). Another study showed that endometrial vessel wall circumference and endothelial cell focal discontinuities were both significantly larger in women with HMB compared to normal controls (Mints et al. 2007). Furthermore, calponin (a vascular smooth muscle cell contractile protein) was found to be significantly lower in endometrial blood vessels in women with HMB (Biswas Shivhare et al. 2014). This evidence is all consistent with an aberrant vasculature within the pre-menstrual endometrium of women with AUB-E, leading to a suboptimal hypoxic response during menstruation.
Conclusions
Herein, we have reviewed the mounting evidence for the presence of endometrial hypoxia and its potential impact on endometrial function. Furthering our understanding of hypoxia in endometrial physiology and pathology using the tools described in this review may provide novel preventative and therapeutic strategies for those suffering from endometrial disorders, including abnormal uterine bleeding (AUB). Furthermore, a complete understanding of optimal endometrial physiology may inform the management of other disorders where aberrant hypoxia is a prominent feature, such as tumour biology and chronic obstructive pulmonary disorder. Addressing the gaps in our knowledge of how hypoxia influences endometrial function represents an exciting area with huge translational potential.
Declaration of interest
R M-A, L K, J J R and J A M have nothing to disclose. H O D C has clinical research support for laboratory consumables and staff from Bayer AG and provides consultancy advice (but with no personal remuneration) for Bayer AG, PregLem SA, Gedeon Richter, Vifor Pharma UK Ltd, AbbVie Inc; Myovant Sciences GmbH. H O D C receives royalties from UpToDate for article on abnormal uterine bleeding. J A Maybin is a Guest Editor of this special issue of Reproduction. J A Maybin was not involved in the review or editorial process for this paper, on which she is listed as an author.
Funding
Some aspects of studies described herein were undertaken in the MRC Centre for Reproductive Health and have been supported by funding from the Medical Research Council: MRC Project Grant G0600048 and Centre Grants G1002033 and MR/N022556/1. This work was also supported by Wellcome Trust Grants 209589/Z/17/Z and 100646/Z/21/Z; Wellbeing of Women Grant RG1820; and Academy of Medical Sciences Grant AMS-SGCL13.
Author contribution statement
R M-A, L K and J A M wrote and edited the manuscript. J J R and H O D C modified the manuscript. All authors have read and approved the final version of this manuscript.
Acknowledgements
We are grateful to Alison Murray for manuscript review and Sheila Milne for assistance with manuscript formatting.
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