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Study Question: Are there any phenotypic and structural/architectural changes in the vessels of endometrium and superficial myometrium during the normal menstrual cycle in healthy women and those with heavy menstrual bleeding (HMB)?, Summary Answer: Spatial and temporal differences in protein levels of endothelial cell (EC) markers and components of the extracellular matrix (ECM) were detected across the menstrual cycle in healthy women and these are altered in HMB., What Is Known Already: HMB affects 30% of women of reproductive age with ~50% of cases being idiopathic. We have previously shown that the differentiation status of endometrial vascular smooth muscle cells (VSMCs) is altered in women with HMB, suggesting altered vessel maturation compared to controls. Endometrial arteriogenesis requires the co-ordinated maturation not only of the VSMCs but also the underlying ECs and surrounding ECM. We hypothesized that there are spatial and temporal patterns of protein expression of EC markers and vascular ECM components in the endometrium across the menstrual cycle, which are altered in women with HMB., Study Design, Size, Duration: Biopsies containing endometrium and superficial myometrium were taken from hysterectomy specimens from both healthy control women without endometrial pathology and women with subjective HMB in the proliferative (PP), early secretory (ESP), mid secretory (MSP) and late secretory (LSP) phases (N = 5 for each cycle phase and subject group). Samples were fixed in formalin and embedded in paraffin wax., Participants/materials, Setting, Methods: Serial sections (3μm thick) were immunostained for EC markers (factor VIII related antigen (F8RA), CD34, CD31 and ulex europaeus-agglutinin I (UEA-1) lectin), structural ECM markers (osteopontin, laminin, fibronectin and collagen IV) and for Ki67 to assess proliferation. Immunoreactivity of vessels in superficial myometrium, endometrial stratum basalis, stratum functionalis and luminal region was scored using either a modified Quickscore or by counting the number of positive vessels., Main Results and the Role of Chance: In control samples, all four EC markers showed a dynamic expression pattern according to the menstrual cycle phase, in both endometrial and myometrial vessels. EC protein marker expression was altered in women with HMB compared with controls, especially in the secretory phase in the endometrial luminal region and stratum functionalis. For example, in the LSP expression of UEA-1 and CD31 in the luminal region decreased in HMB (mean quickscore: 1 and 5, respectively) compared with controls (3.2 and 7.4, respectively) (both P = 0.008), while expression of F8RA and CD34 increased in HMB (1.4 and 8, respectively) compared with controls (0 and 5.8, respectively) (both P = 0.008). There was also a distinct pattern of expression of the vascular structural ECM protein components osteopontin, laminin, fibronectin and collagen IV in the superficial myometrium, stratum functionalis and stratum basalis during the menstrual cycle, which was altered in HMB. In particular, compared with controls, osteopontin expression in HMB was higher in stratum functionalis in the LSP (7.2 and 11.2, respectively P = 0.008), while collagen IV expression was reduced in stratum basalis in the MSP (4.6 and 2.8, respectively P = 0.002) and in stratum functionalis in the ESP (7 and 3.2, respectively P = 0.008)., Limitations, Reasons for Caution: The protein expression of vascular EC markers and ECM components was assessed using a semi-quantitative approach in both straight and spiral arterioles. In our hospital, HMB is determined by subjective criteria and levels of blood loss were not assessed., Wider Implications of the Findings: Variation in the protein expression pattern between the four EC markers highlights the importance of choice of EC marker for investigation of endometrial vessels. Differences in expression of the different EC markers may reflect developmental stage dependent expression of EC markers in endometrial vessels, and their altered expression in HMB may reflect dysregulated vascular development. This hypothesis is supported by altered expression of ECM proteins within endometrial vessel walls, as well as our previous data showing a dysregulation in VSMC contractile protein expression in the endometrium of women with HMB. Taken together, these data support the suggestion that HMB symptoms are associated with weaker vascular structures, particularly in the LSP of the menstrual cycle, which may lead to increased and extended blood flow during menstruation., Study Funding/competing Interest(s): This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare., Trial Registration Number: Not applicable.

Study Question: Does the use of a vascular contrast agent facilitate earlier detection of maternal flow to the placental intervillous space (IVS) in the first trimester of pregnancy?, Summary Answer: Microvascular filling of the IVS was demonstrated by contrast-enhanced ultrasound from 6 weeks of gestation onwards, earlier than previously believed., What Is Known Already: During placental establishment and remodeling of maternal spiral arteries, endovascular trophoblast cells invade and accumulate in the lumen of these vessels to form 'trophoblast plugs'. Prior evidence from morphological and Doppler ultrasound studies has been conflicting as to whether the spiral arteries are completely plugged, preventing maternal blood flow to the IVS until late in the first trimester., Study Design, Size, Duration: Uteroplacental flow was examined across the first trimester in human subjects given an intravenous infusion of lipid-shelled octofluoropropane microbubbles with ultrasound measurement of destruction and replenishment kinetics. We also performed a comprehensive histopathological correlation using two separately archived uteroplacental tissue collections to evaluate the degree of spiral artery plugging and evaluate remodeling of the upstream myometrial radial and arcurate arteries., Participants/materials, Setting, Methods: Pregnant women (n = 34) were recruited in the first trimester (range: 6+3 to 13+6 weeks gestation) for contrast-enhanced ultrasound studies with destruction-replenishment analysis of signal intensity for assessment of microvascular flux rate. Histological samples from archived in situ (Boyd Collection, n = 11) and fresh first, second, and third trimester decidual and post-hysterectomy uterine specimens (n = 16) were evaluated by immunohistochemistry (using markers of epithelial, endothelial and T-cells, as well as cell adhesion and proliferation) and ultrastructural analysis., Main Results and the Role of Chance: Contrast agent entry into the IVS was visualized as early as 6+3 weeks of gestation with some variability in microvascular flux rate noted in the 6-7+6 week samples. Spiral artery plug canalization was observed from 7 weeks with progressive disintegration thereafter. Of note, microvascular flux rate did not progressively increase until 13 weeks, which suggests that resistance to maternal flow in the early placenta may be mediated more proximally by myometrial radial arteries that begin remodeling at the end of the first trimester., Limitations Reasons for Caution: Gestational age was determined by crown-rump length measurements obtained by transvaginal ultrasound on the day of contrast-enhanced imaging studies, which may explain the variability in the earliest gestational age samples due to the margin of error in this type of measurement., Wider Implications of the Findings: Our comprehensive in situ histological analysis, in combination with the use of an in vivo imaging modality that has the sensitivity to permit visualization of microvascular filling, has allowed us to reveal new evidence in support of increasing blood flow to the IVS from 6 weeks of gestation. Histologic review suggested the mechanism may be blood flow through capillary-sized channels that form through the loosely cohesive 'plugs' by 7 weeks gestation. However, spiral artery remodeling on its own did not appear to explain why there is significantly more blood flow at 13 weeks gestation. Histologic studies suggest it may be related to radial artery remodeling, which begins at the end of the first trimester., Study Funding/competing Interest(s): This project was supported by the Oregon Health and Science University Knight Cardiovascular Institute, Center for Developmental Health and the Struble Foundation. There are no competing interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Study Question: Do platelets play any role in the development of adenomyosis?, Summary Answer: As in endometriosis, adenomyotic lesions show significantly increased platelet aggregation, increased expression of transforming growth factor (TGF)-β1, phosphorylated Smad3, markers of epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT), and smooth muscle metaplasia (SMM), in conjunction with increased fibrosis as compared with normal endometrium., What Is Known Already: Both EMT and FMT are known to play vital roles in fibrogenesis in general and in endometriosis in particular. EMT has been implicated in the development of adenomyosis. SMM is universally seen in endometriosis and also in adenomyosis, and is correlated positively with the extent of fibrosis. However, there has been no published study on the role of platelets in fibrogenesis in adenomyosis, even though adenomyotic lesions undergo repeated cycles of tissue injury and repair, which suggests the involvement of platelets and their possible roles in fibrogenesis., Study Design, Size, Duration: Cross-sectional studies of ectopic endometrial and control endometrial tissue samples from three sets of women with and without adenomyosis (n= 34 and 20, 12 and 10, and 8 and 8, respectively) were carried out from 2014 to 2015., Participants/materials, Setting, Methods: Immunohistochemistry analysis of ectopic endometrial tissues from women with (n= 34) and without (n= 20) adenomyosis with respect to biomarkers of EMT, FMT and highly differentiated smooth muscle cells as well as TGF-β1, phosphorylated Smad3, markers of proliferation, angiogenesis and extracellular matrix (ECM) deposits. Masson trichrome staining, Van Gieson staining and Pico-Sirius staining were performed to evaluate and quantify the extent of fibrosis in lesions. Progesterone receptor isoform B (PR-B) staining also was performed. In addition, CD42b-positive platelets in ectopic (n= 12) and control (n= 10) endometrium were counted by confocal microscopy and compared. The protein expression levels of TGF-β1 and phosphorylated Smad3 in both ectopic (n= 8) and control (n= 8) endometrium were measured by western blot analysis. Immunofluorescent staining of both platelets and hepatocyte growth factor (HGF) was also performed for adenomyotic tissue samples (n= 10)., Main Results and the Role of Chance: Adenomyotic lesions had a significantly higher extent of platelet aggregation and increased staining for TGF-β1 and phosphorylated Smad3 (both P-values <0.001 versus control). In addition, E-cadherin staining was decreased while vimentin staining in adenomyotic epithelial cells was increased, along with increased staining of proliferating cell nuclear antigen, vascular endothelial growth factor and CD31 (all P-values <0.001), markers of proliferation and angiogenesis. Staining for α-SMA, a marker for myofibroblast, desmin, smooth muscle myosin heavy chain and oxytocin receptor was significantly increased in adenomyotic lesions versus control, concomitant with increased staining of collagen I and lysyl oxidase (all P-values <0.001). Histochemistry analysis indicates that the extent of fibrosis is high in adenomyotic lesions (P < 0.001), and the extent appeared to correlate negatively with the microvessel density (P < 0.05). PR-B staining was significantly decreased in adenomyotic lesion as compared with control endometrium (P < 0.001). Platelets and HGF were co-localized mostly in the stromal component of adenomyotic lesions, near the glandular epithelium., Limitations, Reasons for Caution: The results are limited by the cross-sectional nature of the study and the use of histochemistry and immunohistochemistry analyses only, but nonetheless is a validation of our previous finding in mouse experiments., Wider Implications of the Findings: The data presented are consistent with the notion that platelet-induced activation of the TGF-β/Smad signaling pathway may be a driving force in EMT, FMT and SMM in the development of adenomyosis, leading to fibrosis. This study provides the first piece of evidence that adenomyotic lesions are wounds that undergo repeated injury and healing, and, as such, platelets play critical roles in the development of adenomyosis by promoting proliferation, angiogenesis, increasing ECM deposits, and SMM, resulting in fibrosis. Platelets may also be involved in uterine hyperactivity and myometrial hyperinnervation. Our results provide one explanation as to why adenomyosis is a challenge for medical treatment, and shed new light onto the pathophysiology of adenomyosis., Study Funding/competing Interests: Support for data collection and analysis was provided by grants from the National Science Foundation of China. None of the authors has anything to disclose., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Study Question: What are the in vitro effects of estrogen receptor β (ERβ) activation on the function of endothelial cells (ECs) from different vascular beds: human endometrial ECs (HEECs; endometrium), uterine myometrial microvascular ECs (UtMVECs; myometrium) and human umbilical vein ECs (HUVECs)?, Summary Answer: Studies conducted in vitro demonstrate that the ERβ agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) has EC type-specific effects on patterns of gene expression and network formation. Identification of a key role for the transcription factor Sp1 in ERβ-dependent signaling in uterine ECs offers new insights into cell-specific molecular mechanisms of estrogen action in the human uterus., What Is Known Already: Estrogens, acting via ERs (ERα and ERβ), have important, body-wide impacts on the vasculature. The human uterus is an estrogen target organ, the endometrial lining of which exhibits physiological, cyclical angiogenesis. In fixed tissue sections, human endometrial ECs are immunopositive for ERβ., Study Design, Size, Duration: Cells were treated with a vehicle control or the ERβ agonist, DPN, for 2 h or 24 h (n = 5) followed by gene expression analysis. Functional assays were analyzed after a 16 h incubation with ligand (n = 5)., Participant/materials, Setting, Methods: Analysis of DPN-treated ECs using Taqman gene array cards focused on genes involved in angiogenesis and inflammation identified cell type-specific ERβ-dependent changes in gene expression, with validation using qPCR and immunohistochemistry. Molecular mechanisms involved in ERβ signaling were investigated using bioinformatics, reporter assays, immunoprecipitation, siRNA and a specific inhibitor blocking Sp1-binding sites. The endometrium and myometrium from women with regular menses were used to validate the protein expression of candidate genes., Main Results and the Role of Chance: HEECs and UtMVECs were ERβ+/ERα-. Treatment of ECs with DPN had opposite effects on network formation: a decrease in network formation in HEECs (P ≤ 0.001) but an increase in UtMVECs (P ≤ 0.05). Genomic analysis identified opposite changes in ERβ target gene expression with only three common transcripts (HEY1, ICAM1, CASP1) in all three ECs; a unique profile was observed for each. An important role for Sp1 was identified, consistent with the regulation of ERβ target genes via association with the transcription factor ('tethered' mechanism)., Limitations, Reasons for Caution: The study was mainly carried out in vitro using ECs of which one type was immortalized. Although the analysis of the protein expression of candidate genes was carried out using intact tissue samples from patients, investigations into in vivo angiogenesis were not carried out., Wider Implications of the Findings: These results have implications for our understanding of the mechanisms responsible for ERβ-dependent changes in EC gene expression in hormone-dependent disorders.

While there is a growing realization that the origins of major obstetrical complications associated with defective deep placentation, such as pre-term labour, fetal growth restriction and pre-eclampsia, may lie in the very early pregnancy events, the underlying mechanisms are not understood. Impaired deep placentation is foremost a vascular pathology, characterized by a lack of endovascular trophoblast invasion and remodelling of a segment of the spiral arteries embedded within the inner myometrium of the uterus. Outside pregnancy, the inner myometrium represents a highly specialized, hormone-dependent structure, termed the junctional zone (JZ), which plays an integral part in the implantation process. The JZ changes with age and is disrupted in several reproductive disorders, such as endometriosis and adenomyosis, which in turn may account for the increased risk of adverse pregnancy outcome. Unlike the endometrium, the myometrial JZ is not readily accessible to biochemical or molecular studies, yet its structure and function can be assessed using imaging techniques, such as high-resolution ultrasound and magnetic resonance imaging. Thus, non-invasive assessment of the JZ prior to conception may turn out to be useful in identifying those women at risk of major obstetrical complications.

Background: The human uterine vasculature is highly structured, exhibiting circumferential and radial branching. Previously published angiograms of the arterial network describe a system of regular coils. Uterine fibroids lack this structured vasculature. In this study, we make a comparison between the vasculature in normal myometrium and in fibroids using robust stereological methods thus far lacking in the literature., Methods: Stereological and morphometric analysis of the vascular system was carried out on 15 normal and 27 small fibroid (5-40 mm) uteri taken from women suffering menorrhagia. Projected images of published angiograms were also re-examined, measuring tortuosity., Results: A decreasing gradient of vascular smooth muscle from outer to inner myometrium was found in normal uteri, with no corresponding gradient in capillary tissue fraction. An association between vascular luminal size, amplitude and frequency of vessel bending was also established. Conversely, fibroids were found to lack structured or muscularized vasculature., Conclusions: A quantitative gradient within the myometrial vascular system, which is absent in fibroids, has been demonstrated. These structural differences between diseased and healthy tissues are probably because of differing expression of angiogenic growth factors and may explain the distribution of particles seen after uterine artery embolization.

The role of calcium (Ca(2+)) released from intracellular stores and the entry of extracellular Ca(2+) for vasopressin (AVP)-induced responses in large and small, human, intramyometrial arteries was investigated. There was no statistical difference as revealed by pD(2) values (-log EC(50)), in the sensitivity of large and small vessels to AVP. Nimodipine caused an inhibition of contractions induced by low concentrations (10(-10) mol/l) of AVP in both types of vessels but, at higher concentration (>10(-10) mol/l), whereas responses in small arteries were diminished, in large arteries they remained unchanged. In Ca(2+)-free solution, responses of large and small arteries to potassium and to 10(-10) mol/l AVP were abolished. With 10(-6) mol/l AVP, response in small arteries was completely inhibited, whereas in large arteries it was reduced by approximately 50%. Additional experiments were done on large arteries. Thapsigargin (TSG), which causes depletion of internal Ca(2+) stores, caused a significant reduction in responses. Following treatment with TSG, responses to AVP in Ca(2+)-free solution were almost completely inhibited but arteries responded again when incubated in normal physiological salt solution. The results indicate that in contrast to large arteries, small arteries are highly dependent on extracellular Ca(2+). Response of large arteries showed considerable dependence on Ca(2+) stored internally particularly, for maximum activation.

This study aimed to compare vascular parameters between fibroid and myometrium. From 10 uteri, specimens were taken from small fibroids (<0.5 cm), from the inner and outer parts of large fibroids (>3 cm), and from myometrium. Antibodies to endothelial cell markers CD31, CD34, factor VIII-related antigen (FVIII), and Ulex europaeus lectin were used in routine immuno- and lectin chemistry protocols. Parameters calculated were vascular area (VA), microvascular density (MD) and vascular luminal diameter. VA measures showed that myometrium had a greater area stained than small fibroids (P = 0.03) using CD31 and both inner (P = 0.04) and outer (P = 0.01) regions of large fibroids using FVIII, and than all groups (small, P = 0.02; inner, P = 0.02; outer, P = 0.006) using the lectin U. europaeus. MD was higher in myometrium than all uterine fibroid groups (small, P = 0.009; inner, P = 0.01; outer, P = 0.01) using U. europaeus lectin, than both regions of large (inner, P = 0.04; outer, P = 0.02) fibroids using FVIII, and than outer regions of large fibroids using CD31 (P < 0.05). There were significantly larger diameter vessels in myometrium and large fibroids compared with small fibroids using CD34, FVIII and the lectin U. europaeus (P

Angiogenesis, defined as the growth of new vessels from pre-existing vessels, involves microvascular rather than large vessel endothelial cells. Accordingly, microvascular endothelial cell (MEC) proliferation assays are an appropriate in-vitro model of angiogenesis. We have developed a method for the isolation and long-term culture of large numbers of MEC from the human myometrium, tissue readily available from hysterectomy specimens. Human myometrial MEC were positively selected from tissue dissociated sequentially with collagenase and trypsin using Ulex europeaus antigen-1 (UEA)-coated dynabeads. Cultured myometrial MEC displayed characteristic endothelial phenotype and function for up to 14 passages: cobblestone morphology, formed capillary-like tubes on Matrigel, expressed CD31, Factor VIII-related antigen, bound UEA lectin, incorporated 1,1'-dioctadecyl-1,3,3,3', 3'-tetramethylindocarbocyanine perchlorate-labelled acetylated low density lipoprotein, migrated and proliferated in response to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), but not epidermal growth factor. Optimal growth of human myometrial MEC occurred in a simple medium comprising M199, 5 ng/ml bFGF, 15% human serum, 5% fetal calf serum (FCS) and heparin. Human serum was essential for growth, although there was a synergistic effect when FCS was included. Almost identical dose-response curves were obtained for bFGF- and VEGF-induced myometrial MEC proliferation in early and late passage cells. Therefore myometrial MEC are a good model for in-vitro studies of uterine angiogenesis, since they have a stable phenotype and proliferative responsiveness to VEGF and bFGF for up to 14 passages.

Menorrhagia is a significant problem in women of reproductive age. In half of the cases no specific aetiology is known. Vascular factors play a role but remain poorly understood. We chose to study whether any association exists between the flow impedance of uterine arteries and the amount of menstrual blood loss. The study population consisted of 60 spontaneously menstruating 35- to 49-year-old women without endometrial hyperplasia, polyps, or submucous fibroids. The pulsatility index (PI) from uterine arteries, arcuate arteries, and radial arteries was measured by transvaginal colour Doppler. Menstrual blood loss was measured by the alkaline haematin method. A significant inverse correlation was found between uterine artery PI and the amount of menstrual blood loss, suggesting that women with lower uterine flow impedance bleed more. A regression model confirmed that this association was specific and not explained by uterine size, fibroids or any other of the 11 potential confounders included in the model. The correlation between uterine artery PI and amount of menstrual blood loss suggests that vascular factors may be involved in the pathogenesis of menorrhagia.

Expression of vascular cell adhesion molecule (VCAM) and endothelial leukocyte adhesion molecule (ELAM)-1 on human uterine endothelium was examined by immunohistochemistry. Endomyometrial blocks were obtained from 26 hysterectomy specimens (n = 6, menstrual; n = 6, follicular; n = 14, luteal). VCAM and ELAM-1 expression was compared to that of the classic endothelial markers Factor VIIIRA, CD31, CD34 and CD36, which were found to be present in samples taken from all stages of the cycle. Factor VIIIRA, CD31 and CD34 were expressed on most endothelium whereas CD36 was expressed primarily on that of small vessels. CD36 was also found to be strongly expressed on endometrial epithelium and no changes were apparent during the menstrual cycle. ELAM-1 expression was not detected in any of the tissues examined. VCAM expression varied during the cycle, appearing on endometrial endothelium in the mid- and late luteal phases, stages at which large granular lymphocytes migrate into endometrium. VCAM or a VCAM-like molecule may be involved in migration of large granular lymphocytes into endometrium.