Endometrial Vasculature Research Articles

An investigation of endometrial vascularity in normally menstruating women and those in other physiological states using 3D ultrasound imaging.

This study aimed to determine the normal vasculature indices of the endometrium and to correlate them with those in various physiological states. Women undergoing ultrasound at the Feto-Maternal Center, Qatar in 2020-2021 as part of their gynecologic evaluation were enrolled into the study. They were divided into those with normal menses and no additional pathology, those following spontaneous miscarriage, postpartum and menopausal. Three-dimensional (3D) evaluation of the endometrial vasculature was done and the parameters quantified included vascularization index (VI), flow index (FI), vascularization flow index (VFI), endometrial thickness, endometrial volume and uterine volume. JASP, an open-source statistical analysis software, was used for analysis and an independent t-test to compare the vascularity indices. A multivariate regression analysis was also done to look at the factors affecting the endometrial vascular indices within the luteal phase. A total of 461 women were studied: 122 in the follicular phase, 199 in the luteal phase, 90 after a spontaneous miscarriage, 29 postpartum, and 16 menopausal. The vascularity indices were highest after miscarriage and lowest postnatally. There were no significant effects of age, gravida, para, or abortions on VI and VFI. However, there was a significant positive effect of age on FI (P = 0.019) There was a significant increase in endometrial volume and thickness in the luteal phase as compared to follicular phase (P < 0.01), but there was no difference in the vascularity indices. The uterine and endometrial volume in the postnatal group were nearly double that of the luteal group (P value <0.01 and 0.014, respectively). There was a significant decrease in flow index in the postnatal group compared to the luteal group (P < 0.01), suggesting low flow intensity in the postnatal group. Endometrial vascular indices measured using 3D Doppler can be used to determine normal vascular indices and vary with physiological states such as after miscarriages, postnatally and in the menopausal states.

P-361 Is HIF-2α involved in pre-menstrual conditioning of the endometrium to ensure optimal repair and prevent heavy menstrual bleeding?

Abstract Study question Does HIF-2α play a key role in pre-menstrual conditioning of the non-pregnant endometrium to optimise menstrual breakdown/repair and prevent heavy menstrual bleeding (HMB)? Summary answer HIF-2 was higher in secretory endometrium of women with HMB versus controls, but manipulation of HIF-2α in the mouse model did not impact endometrial breakdown/repair. What is known already Hypoxia inducible factor (HIF) is the master regulator of the cellular response to hypoxia and has two common alpha isoforms (HIF-1α/HIF-2α) with overlapping but distinct target genes. We revealed that menstrual endometrial HIF-1α is necessary for normal menstrual endometrial repair. We detected HIF-2α in human endometrium exclusively during the secretory phase. The role of HIF-2α in the non-pregnant endometrium remains undetermined but was important for successful uterine implantation and lung vascular remodelling in mice. We hypothesised that endometrial HIF-2 is required for optimising the endometrial vasculature pre-menstrually to ensure appropriate vasoconstriction and endometrial repair during menstruation. Study design, size, duration HIF-2 was examined in human endometrial tissue and a mouse model of simulated menstruation. Hif2α +/- mice provided genetic reduction of HIF-2 during simulated menses versus wild-type littermates. HIF-2α was also pharmacologically inhibited during the secretory phase (PT2385 vs vehicle controls). To increase HIF-2, Hif1α +/- mice were treated with a HIF stabiliser (DMOG) during the secretory phase to increase HIF-2α followed by decreased HIF-1α at menses –mimicking human endometrial findings in women with HMB. Participants/materials, setting, methods Human secretory endometrium was collected (ethically approved and consented) from women with objectively measured HMB (n = 5) and controls (n = 4) and HIF-2 detected by Western blot. Mouse uterus was collected at the time of progesterone withdrawal (T0, decidualisation) and 8h (T8, endometrial breakdown) and 24h (T24, endometrial repair) after progesterone withdrawal. Histological endometrial repair and menstrual blood loss (MBL) were quantified. Decidualisation markers were examined by RT-qPCR. Endometrial vessel maturity and hypoxia were assessed using immunohistochemistry/immunofluorescence. Main results and the role of chance Human secretory endometrial HIF-2α protein levels were higher in those with HMB compared to controls (P &amp;lt; 0.05). Genetic reduction of HIF-2α in our mouse model of menses (Hif2α +/-) revealed no significant differences in endometrial breakdown (T8) or repair grade (T24) and no difference in MBL at T8 versus controls. There were no significant differences in expression of decidualisation markers (Prlr/Prl3c1) and vessel maturity at T0 (CD31/α-SMA immunofluorescence staining), or the presence of hypoxia at T8 (pimonidazole immunohistochemistry staining). Pharmacological inhibition of HIF-2 at the time of decidualisation also showed no significant difference in endometrial repair at T24 versus controls. Increasing pre-menstrual HIF-2 via pharmacological stabilisation of HIF-2α in Hif1α+/- mice similarly did not significantly affect endometrial breakdown (T8) or repair (T24) or MBL at T8 versus vehicle treated controls, in contrast to our findings in women with HMB. Limitations, reasons for caution Histological scoring of endometrial breakdown/repair in the menstruation mouse model may not be sensitive enough to detect subtle endometrial changes with increased HIF-2α. As HIF-1α delays endometrial repair, an additional timepoint 36h after progesterone withdrawal may better capture any delay in endometrial repair as a result of increased secretory HIF-2. Wider implications of the findings HIF-2α may play a greater role in implantation than pre-menstrual conditioning of the endometrium to optimise menstrual breakdown/repair and limit menstrual blood loss. Although endometrial breakdown was not significantly affected by HIF-2, the impact on repair warrants further investigation to fully delineate the role of HIF-2 in the non-pregnant endometrium. Trial registration number Not applicable

Differential expression of pro- and anti-angiogenic factors in the endometrium between repeat breeder and normally fertile cows

In cattle, the establishment of appropriate endometrial vasculature during the estrous cycle is required for preparing a receptive endometrium. This study aimed to investigate 1) mRNA expression of potent pro- and anti-angiogenic factors, 2) protein localization of the anti-angiogenic factor thrombospondin (TSP), and 3) vascularity in the endometrium of repeat breeder (RB) and normally fertile (non-RB) cows. Caruncular and intercaruncular endometrium was collected from RB and non-RB cows during the luteal phase of the estrous cycle. RB cows had greater mRNA expression levels of TSP ligands (TSP1 and TSP2) and receptors (CD36 and CD47) than non-RB cows. Although the mRNA expression levels of most angiogenic factors did not change by repeat breeding, RB cows had greater mRNA expression of fibroblast growth factor receptor 1 (FGFR1), angiopoietin 1 (ANGPT1), and ANGPT2 and a less mRNA expression of vascular endothelial growth factor B (VEGFB) than non-RB cows. By immunohistochemistry, TSP1, TSP2, CD36, and CD47 were detected in the luminal epithelium, glandular epithelium, stromal cells, and blood vessels of the endometrium. Two indexes of vascularity, the number of blood vessels and the percentage of area stained positive for the von Willebrand factor, were lower in the endometrium of RB than in that of non-RB cows. These results demonstrate that RB cows have a greater expression of both ligands and receptors for the anti-angiogenic factor TSP and a reduced vascular distribution in the endometrium compared with non-RB cows, suggesting suppressed endometrial angiogenesis.

Development and Validation of a Nomogram Prediction Model for Endometrial Malignancy in Patients with Abnormal Uterine Bleeding.

This study aimed to identify the risk factors and sonographic variables that could be integrated into a predictive model for endometrial cancer (EC) and atypical endometrial hyperplasia (AEH) in women with abnormal uterine bleeding (AUB). This retrospective study included 1837 patients who presented with AUB and underwent endometrial sampling. Multivariable logistic regression was developed based on clinical and sonographic covariates [endometrial thickness (ET), resistance index (RI) of the endometrial vasculature] assessed for their association with EC/AEH in the development group (n=1369), and a predictive nomogram was proposed. The model was validated in 468 patients. Histological examination revealed 167 patients (12.2%) with EC or AEH in the development group. Using multivariable logistic regression, the following variables were incorporated in the prediction of endometrial malignancy: metabolic diseases [odds ratio (OR)=7.764, 95% confidence intervals (CI) 5.042-11.955], family history (OR=3.555, 95% CI 1.055-11.971), age ≥40 years (OR=3.195, 95% CI 1.878-5.435), RI ≤0.5 (OR=8.733, 95% CI 4.311-17.692), and ET ≥10 mm (OR=8.479, 95% CI 5.440-13.216). A nomogram was created using these five variables with an area under the curve of 0.837 (95% CI 0.800-0.874). The calibration curve showed good agreement between the observed and predicted occurrences. For the validation group, the model provided acceptable discrimination and calibration. The proposed nomogram model showed moderate prediction accuracy in the differentiation between benign and malignant endometrial lesions among women with AUB.

The Menstrual Endometrium: From Physiology to Future Treatments.

Abnormal uterine bleeding (AUB) is experienced by up to a third of women of reproductive age. It can cause anaemia and often results in decreased quality of life. A range of medical and surgical treatments are available but are associated with side effects and variable effectiveness. To improve the lives of those suffering from menstrual disorders, delineation of endometrial physiology is required. This allows an increased understanding of how this physiology may be disturbed, leading to uterine pathologies. In this way, more specific preventative and therapeutic strategies may be developed to personalise management of this common symptom. In this review, the impact of AUB globally is outlined, alongside the urgent clinical need for improved medical treatments. Current knowledge of endometrial physiology at menstruation is discussed, focusing on endocrine regulation of menstruation and local endometrial inflammation, tissue breakdown, hypoxia and endometrial repair. The contribution of the specialised endometrial vasculature and coagulation system during menstruation is highlighted. What is known regarding aberrations in endometrial physiology that result in AUB is discussed, with a focus on endometrial disorders (AUB-E) and adenomyosis (AUB-A). Gaps in existing knowledge and areas for future research are signposted throughout, with a focus on potential translational benefits for those experiencing abnormal uterine bleeding. Personalisation of treatment strategies for menstrual disorders is then examined, considering genetic, environmental and demographic characteristics of individuals to optimise their clinical management. Finally, an ideal model of future management of AUB is proposed. This would involve targeted diagnosis of specific endometrial aberrations in individuals, in the context of holistic medicine and with due consideration of personal circumstances and preferences.

The Plasminogen Activator System, Glucocorticoid, and Mineralocorticoid Receptors in the Primate Endometrium During Artificial Menstrual Cycles.

As a key mechanism in fibrinolysis and tissue remodeling, the plasminogen activator system has been suggested in the process of endometrial shedding and tissue remodeling. Previous studies have explored the role of estrogen, progesterone, and androgen receptors as well as elements of the renin-angiotensin-aldosterone system in shaping the morphology of the endometrium. This study investigates the distribution and concentrations of the mineralocorticoid receptor, glucocorticoid receptor, tissue plasminogen activator, urokinase plasminogen activator, and plasminogen activator inhibitor-1 within the endometrial stroma, glandular, and endothelial cells of the primate endometrium during artificial menstrual cycles. Our immunohistochemistry quantification shows mineralocorticoid and glucocorticoid receptors are ubiquitously distributed within the macaque endometrium with their patterns of expression following similar fluctuations to urokinase and tissue plasminogen activators particularly within the endometrial vasculature. These proteins are present in endometrial vasculature in high levels during the proliferative phase, decreasing levels during the secretory phase followed by rising levels in the menstrual phase. These similarities could suggest overlapping pathways and interactions between the plasminogen activator system and the steroid receptors within the endometrium. Given the anti-inflammatory properties of glucocorticoids and the role of plasminogen activators in endometrial breakdown, the glucocorticoid receptor may be contributing to stabilizing the endometrium by regulating plasminogen activators during the proliferative phase and menstruation. Furthermore, given the anti-mineralocorticoid properties of certain anti-androgenic progestins and their reduced unscheduled uterine bleeding patterns, the mineralocorticoid receptor may be involved in unscheduled endometrial bleeding.

P–387 Serum progesterone levels on the day of the endometrial receptivity analysis (ERA) biopsy do not correlate with the biopsy results

Abstract Study question Do the serum progesterone levels on the day of the endometrial receptivity analysis (ERA) biopsy correlate with the results of the ERA? Summary answer Serum progesterone levels on the day of the endometrial receptivity analysis biopsy do not correlate with the biopsy results. What is known already Endometrial receptivity is a time sensitive window characterised by maturation of the endometrium, during which the trophectodermal cells attach to the endometrial cells and invade the endometrial stromal vasculature. Progesterone is an essential element for receptivity and pregnancy. There is no consensus regarding the optimal progesterone levels in the luteal phase, for a successful pregnancy. Endometrial receptivity analysis is a diagnostic tool developed by profiling the transcriptome of over 238 genes that are expressed at different stages of the endometrial cycle. The results are reported as receptive, pre-receptive, early receptive, etc and are used to direct a personalised embryo transfer. Study design, size, duration We report a prospective study of 30 patients with a history of recurrent implantation failure (RIF). They underwent ERA testing in a medicated cycle, between early 2018 and late 2020. Participants/materials, setting, methods A large proportion of the patients we treat in our clinic (ARGC) have recurrent implantation failure. Thirty patients with RIF underwent ERA testing in a medicated cycle.They all followed the same protocol with down regulation, followed by estrogenic preparation for about 12–14 days, followed by progesterone for about 120 hours. An endometrial biopsy was taken at about 120 hours after progesterone exposure. Main results and the role of chance An ERA result was available on 28/30 patients. Eighteen were reported to be pre-receptive, seven receptive, 3 early receptive and 2 could not be analysed. The progesterone levels within 24 hours of the biopsy for the pre-receptive group ranged from 21.2–472 nmol/l, for the receptive group ranged from 27.8–152 nmol/l and for the early receptive group ranged from 54.9–162 nmol/l. Though the number of cases is small, we found no co-relation between the serum progesterone levels with the ERA results. Eighteen women underwent an embryo transfer based on the ERA results (pET-personalised embryo transfer). Eleven were positive with four live births, one early ongoing pregnancy, three miscarriages, one ectopic pregnancy, two biochemical pregnancies and seven negative results. Seven women had euploid embryo transfers-three had live births, one is viable at 11 weeks, one had a missed miscarriage and two were negative. There are no studies correlating the serum progesterone levels and the ERA results. In practice, we plan embryo transfers for women in frozen cycles by monitoring the serum progesterone levels alongside the day of the cycle. Hence, we wanted to review if the combination of the progesterone levels along with biopsy results would allow us the improve the results further. Limitations, reasons for caution This is a small study. Larger datasets are required to draw meaningful conclusions. Wider implications of the findings: If the above findings are confirmed by larger studies, we may not need to monitor serum progesterone levels during ERA biopsy cycles. Trial registration number NA

Endometrial Perivascular Progenitor Cells and Uterus Regeneration

Ovarian steroid-regulated cyclical regeneration of the endometrium is crucial for endometrial receptivity and embryo implantation, and it is dependent on the dynamic remodeling of the endometrial vasculature. Perivascular cells, including pericytes surrounding capillaries and microvessels and adventitial cells located in the outermost layer of large vessels, show properties of mesenchymal stem cells, and they are thus promising candidates for uterine regeneration. In this review, we discuss the structure and functions of the endometrial blood vasculature and their roles in endometrial regeneration, the main biomarkers and characteristics of perivascular cells in the endometrium, and stem cell-based angiogenetic therapy for Asherman’s syndrome.

Prostaglandin and antigestagen in pyometra bitches: vascular and stereological effect.

SummaryEffects of conservative treatment on uterine blood flow and morphometric findings are still unknown in bitches. Thus, this study aimed to compare uterine changes of pyometra bitches subjected to distinct modes of treatment. Pyometra bitches were assigned to: OHE (ovariohysterectomy immediately after diagnosis), Aglepristone (days 1, 2 and 8) and Associative (aglepristone treatment coupled with cloprostenol for 7 days) groups. After 9 days, bitches were ovariohysterectomized. Before surgery, uterine area was measured ultrasonographically and the uterine artery Doppler velocimetry analyzed blood flow velocity and indexes. Uterine horns were classified according to resistance index (RI) as more compromised and less compromised. Endometrial vasculature was quantitatively evaluated by color flow Doppler. Blood samples were collected to determine nitric oxide (NO) concentrations. Histological uterine structures were quantified by stereology and VEGF-A (vascular endothelial growth factor) and eNOS were (endothelial nitric oxide synthase) immunohistochemically analyzed. Aglepristone and Associative groups had lower uterine area and vascularization, and higher blood flow velocity and indexes compared to OHE group. Less compromised horn of Associative group had higher blood flow velocity compared to OHE group. Aglepristone group presented lower inflammatory infiltrate and larger uterine stroma. Associative group had lower volume density and absolute surface of endometrial cysts and lower VEGF-A expression for glandular epithelium and stromal cells. Blood NO and e-NOS immunostaining were not different among groups. In conclusion, association between aglepristone and prostaglandin is more effective in decreasing uterine vascularization and modulating uterine blood flow. Moreover, associative therapy promotes marked morphological changes.Lay summaryThis research compared two medical protocols of treatment for uterine infection (pyometra) in bitches, using a hormone blocker (anti-progesterone aglepristone) solely or in association with a uterine contraction inducer (prostaglandin; associative therapy). After treatment, bitches were gonadectomized and a microscopic analysis of uterine blood vessel formation and uterine tissue elements were performed as well as uterine blood flow evaluation through Doppler ultrasonography. According to vascular resistance, uterine horns were additionally classified as more compromised and less compromised. Both treatment protocols led to reduction of uterine dimensions and vascularization, and higher blood flow compared to untreated bitches. Less compromised uterine horn of the associative treatment had higher blood flow compared to untreated bitches. The hormone blocker treatment had lower inflammatory cells and larger uterine histological structure, while associative treatment had less uterine pathological cysts and lower blood vessel formation. The associative therapy is effective in decreasing uterine vascularization and modulating uterine blood flow as well as reestablishing endometrium structure in bitches with uterine infection.

Hemodynamic forces enhance decidualization via endothelial-derived prostaglandin E2 and prostacyclin in a microfluidic model of the human endometrium.

STUDY QUESTIONDoes the uterine vasculature play a localized role in promoting stromal cell decidualization in the human endometrium?SUMMARY ANSWEROur study demonstrated that hemodynamic forces induced secretion of specific endothelial cell-derived prostanoids that enhanced endometrial perivascular decidualization via a paracrine mechanism.WHAT IS KNOWN ALREADYDifferentiation of stromal cell fibroblasts into the specialized decidua of the placenta is a progesterone-dependent process; however, histologically, it has long been noted that the first morphological signs of decidualization appear in the perivascular stroma. These observations suggest that the human endometrial vasculature plays an active role in promoting stromal differentiation.STUDY DESIGN, SIZE, DURATIONPrimary human endometrial stromal cells were co-cultured for 14 days with primary uterine microvascular endothelial cells within a microfluidic Organ-on-Chip model of the endometrium.PARTICIPANTS/MATERIALS, SETTING, METHODSCultures were maintained with estradiol and a progestin, with or without continuous laminar perfusion to mimic hemodynamic forces derived from the blood flow. Some cultures additionally received exogenous agonist-mediated challenges. Decidualization in the microfluidic model was assessed morphologically and biochemically. ELISA was used to examine the culture effluent for expression of decidualization markers and prostaglandins. Immunofluorescence was used to monitor cyclooxygenase-2 expression in association with decidualization.MAIN RESULTS AND THE ROLE OF CHANCEA significantly enhanced stromal decidualization response was observed in the co-cultures when the endothelial cells were stimulated with hemodynamic forces (e.g. laminar shear stress) derived from controlled microfluidic perfusion (<0.001). Furthermore, the enhanced progestin-driven stromal differentiation was mediated via cyclooxygenase-2 and the paracrine action of prostaglandin E2 and prostacyclin. Altogether, these translational findings indicate that the vascular endothelium plays a key physiologic role during the early events of perivascular decidualization in the human endometrium.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis report is largely an in vitro study. Although we were able to experimentally mimic hemodynamic forces in our microfluidic model, we have not yet determined the contribution of additional cell types to the decidualization process or determined the precise physiological rates of shear stress that the microvasculature of the endometrium undergoes in vivo.WIDER IMPLICATIONS OF THE FINDINGSIdentification of specific endothelial-derived prostaglandins and their role during endometrial reproductive processes may have clinical utility as therapeutic targets for reproductive disorders such as infertility, endometriosis, adenomyosis, pre-eclampsia and poor pregnancy outcomes.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by the Veterans Affairs (I01 BX002853), the Bill and Melinda Gates Foundation Grand Challenges Exploration (OPP1159411), the Environmental Toxicology Training Grant (NIH T32 ES007028) and the Environmental Protection Agency STAR Center Grant (83573601).CONFLICT OF INTERESTThe authors report no conflicts of interest.TRIAL REGISTRATION NUMBERN/A.

Bone-marrow-derived endothelial progenitor cells contribute to vasculogenesis of pregnant mouse uterus†.

Angiogenesis is essential for cyclic endometrial growth, implantation, and pregnancy maintenance. Vasculogenesis, the formation of new blood vessels by bone marrow (BM)-derived endothelial progenitor cells (EPCs), has been shown to contribute to endometrial vasculature. However, it is unknown whether vasculogenesis occurs in neovascularization of the decidua during pregnancy. To investigate the contribution of BM-derived EPCs to vascularization of the pregnant uterus, we induced non-gonadotoxic submyeloablation by 5-fluorouracil administration to wild-type FVB/N female mice recipients followed by BM transplantation from transgenic mice expressing green fluorescent protein (GFP) under regulation of Tie2 endothelial-specific promoter. Following 1 month, Tie2-GFP BM-transplanted mice were bred and sacrificed at various gestational days (ED6.5, ED10.5, ED13.5, ED18.5, and postpartum). Bone-marrow-transplanted non-pregnant and saline-injected pregnant mice served as controls (n=5-6/group). Implantation sites were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence. While no GFP-positive EPCs were found in non-pregnant or early pregnant uteri of BM-transplanted mice, GFP-positive EPCs were first detected in pregnant uterus on ED10.5 (0.12%) and increased as the pregnancy progressed (1.14% on ED13.5), peaking on ED18.5 (1.42%) followed by decrease in the postpartum (0.9%). The percentage of endothelial cells that were BM-derived out of the total endothelial cell population in the implantation sites (GFP+CD31+/CD31+) were 9.3%, 15.8%, and 6.1% on ED13.5, ED18.5, and postpartum, respectively. Immunohistochemistry demonstrated that EPCs incorporated into decidual vasculature, and immunofluorescence showed that GFP-positive EPCs colocalized with CD31 in vascular endothelium of uterine implantation sites, confirming their endothelial lineage. Our findings indicate that BM-derived EPCs contribute to vasculogenesis of the pregnant mouse decidua.

Equine endometrial vascular pattern changes during the estrous cycle examined by Narrow Band Imaging hysteroscopy

The aim of this study was to evaluate the uterine blood supply and endometrial vessel architecture, during the equine estrous cycle. Narrow Band Imaging (NBI) hysteroscopy was used for evaluating changes in the endometrial vasculature during the estrous cycle [six mares, d 0 (representing the day of ovulation), d 6 and 11 in four locations]. In addition, endometrial biopsy samples were used for immunodetection of markers for angiogenesis (Vascular Endothelial Growth Factor A, its receptor 2, as well as angiopoietin-2 and its receptor-tyrosine-kinase Tie2) during the estrous cycle (three mares, d 0, 5 and 10; one biopsy per mare). Detailed analysis of hysteroscopic images revealed an increase in the vascular density from estrus towards diestrus. In contrast, microscopic specimens prepared from biopsies revealed no evidence for changes in the endometrial vessel number during the estrous cycle. Studies on expression of angiogenesis markers indicated that cyclic changes in the endometrial vascular density observed by NBI-hysteroscopy were not due to formation of new vessels. It is concluded that vessels are involved in blood supply of a smaller area during diestrus, facilitating better distribution of nutrients during this phase.

Long-acting progestin-only contraceptives impair endometrial vasculature by inhibiting uterine vascular smooth muscle cell survival

Molecular mechanisms responsible for abnormal endometrial vasculature in women receiving long-acting progestin-only contraceptives (LAPCs) are unknown. We hypothesize that LAPCs impair vascular smooth muscle cell (VSMC) and pericyte proliferation and migration producing thin-walled hyperdilated fragile microvessels prone to bleeding. Proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (αSMA) double-immunostaining assessed VSMC differentiation and proliferation in endometria from women before and after DepoProvera (Depo) treatment and from oophorectomized guinea pigs (OVX-GPs) treated with vehicle, estradiol (E2), medroxyprogesterone acetate (MPA), or E2+MPA. Whole-genome profiling, proliferation, and migration assays were performed on cultured VSMCs treated with MPA or etonogestrel (ETO). Endometrial vessels of Depo-administered women displayed reduced αSMA immunoreactivity and fewer PCNA (+) nuclei among αSMA (+) cells (P < 0.008). Microarray analysis of VSMCs identified several MPA- and ETO-altered transcripts regulated by STAT1 signaling (P < 2.22 × 10(-6)), including chemokine (C-C motif) ligand 2 (CCL2). Both MPA and ETO reduce VSMC proliferation and migration (P < 0.001). Recombinant CCL2 reversed this progestin-mediated inhibition, whereas a STAT1 inhibitor abolished the CCL2 effect. Similarly, the endometria of MPA treated OVX-GPs displayed decreased αSMA staining and fewer PCNA (+) nuclei in VSMC (P < 0.005). In conclusion, LAPCs promote abnormal endometrial vessel formation by inhibiting VSMC proliferation and migration.

Detection of endometrial and subendometrial vasculature on the day of embryo transfer and prediction of pregnancy during fresh in vitro fertilization cycles

ObjectivesSuccessful implantation depends on interaction between a blastocyst and a receptive endometrium. Endometrial vasculature is important in the early endometrial response to blastocyst implantation, and vascular changes can affect uterine receptivity. This study aims to investigate whether vascular parameters measured using three-dimensional power Doppler ultrasound (3D PD-US) could predict pregnancy following fresh in vitro fertilization and embryo transfer (IVF–ET) using a gonadotropin releasing hormone (GnRH) agonist long protocol. Materials and methodsThis prospective observational study enrolled 236 nulliparous women who underwent a first IVF–ET using a GnRH long protocol with stimulation by recombinant FSH (rFSH) from May 2009 to April 2012. After excluding two cases of tubal pregnancy, 234 women were in either a pregnant group (n = 113) or a nonpregnant group (n = 121). Color Doppler ultrasound and 3D PD-US examinations were performed on the day of embryo transfer. Main outcomes were pulsatility index (PI), resistance index (RI), systolic/diastolic ratio (S/D) of the uterine artery, vascularization index (VI), flow index (FI), and vascularization flow index (VFI) of the endometrium and subendometrial region. Measurements were analyzed relative to IVF–ET outcome (pregnant vs. nonpregnant). ResultsNo significant differences were observed in patient age, infertility duration, body mass index (BMI), basal FSH levels, number of retrieved oocytes or good quality embryos, or endometrial thickness or volume between the two groups. The pregnant group had higher endometrial VI, FI, and VFI scores than the nonpregnant group (p = 0.001, p = 0.000, p = 0.021, respectively). By contrast, neither subendometrial region VI, FI, and VFI scores (p = 0.770, p = 0.252, p = 0.451), nor uterine artery PI, RI, or S/D scores (p = 0.256, p = 0.527, p = 0.365) differed between groups. Cut-off values of endometrial VI, FI, and VFI scores were 0.95, 12.94, and 0.15 for pregnancy achievement. ConclusionThree dimensional PD-US was a useful and effective method for assessing endometrial blood flow in IVF cycles. Good endometrial blood flow on the day of embryo transfer might be associated with high pregnancy success with a GnRH long protocol, because this is indicative of endometrial receptivity in fresh IVF cycles.

Altered vascular smooth muscle cell differentiation in the endometrial vasculature in menorrhagia.

How does the smooth muscle content and differentiation stage of vascular smooth muscle cells (VSMCs) in endometrial blood vessels change according to the different phases of the menstrual cycle and is this altered in women with menorrhagia? The smooth muscle content (as a proportion of the vascular cross-sectional area) of endometrial blood vessels remained unchanged during the normal menstrual cycle and in menorrhagia; however, expression of the VSMC differentiation markers, smoothelin and calponin, was dysregulated in endometrial blood vessels in samples from women with menorrhagia compared with controls. Menorrhagia affects 30% of women of reproductive age and is the leading indication for hysterectomy. Previous studies have suggested important structural and functional roles for endometrial blood vessels, including impaired vascular contractility. Differentiation of VSMC from a synthetic to contractile state is associated with altered cellular phenotype that contributes to normal blood flow and pressure. This vascular maturation process has been little studied in endometrium both across the normal menstrual cycle and in menorrhagia. Endometrial biopsies were taken from hysterectomy specimens or by pipelle biopsy prior to hysterectomy in controls without endometrial pathology and in women with menorrhagia (n = 7 for each of proliferative, early-secretory, mid-secretory and late-secretory phases for both groups). Biopsies were formalin fixed and embedded in paraffin wax. Paraffin-embedded sections were immunostained for α smooth muscle actin (αSMA), myosin heavy chain (MyHC), H-caldesmon, desmin, smoothelin and calponin (h1 or basic). VSMC content was measured in 25 αSMA(+) vascular cross sections per sample and expressed as a ratio of the muscular area:gross vascular cross-sectional area. VSMC differentiation was analysed by the presence/absence of differentiation markers compared with αSMA expression. Smoothelin and calponin expression was also analysed in relation to total number of blood vessels by double immunostaining for endothelial cell markers. Study of VSMC differentiation markers revealed decreased expression of calponin both in αSMA(+) vessels (P = 0.008) and in relation to total number of vessels (P = 0.001) in late secretory phase endometrium in menorrhagia compared with controls. Smoothelin expression in αSMA(+) vessels was increased (P = 0.03) in menorrhagia, although this was not significant in relation to the total number of vessels. In normal endometrium, the proportion of blood vessels expressing αSMA increased from 63% in proliferative endometrium to 81% in the late secretory phase (P = 0.002). The overall arterial muscle content did not differ between control and menorrhagia at any phase of the menstrual cycle, occupying 78-81% of gross vascular cross-sectional area during the different menstrual cycle phases. This study included both straight and spiral arterioles and analysed only stratum functionalis. The VSMC differentiation with respect to αSMA expression is an observational study and the data are presented as presence or absence of the differentiation markers in each field of view, corresponding with the vascular cross sections included in the study of vascular muscle content. Smoothelin and calponin have been widely implicated as important regulators of vascular tone, vascular contractility and rate of blood flow. Our results have uncovered a disparate pattern of calponin expression, potentially indicating a dysfunctional contraction mechanism in the endometrial blood vessels in menorrhagia, thus implicating calponin as a potential therapeutic target. This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. Not applicable.

An Overview of Molecular and Cellular Mechanisms Associated with Porcine Pregnancy Success or Failure

Prenatal mortality remains one of the major constraints for the commercial pig industry in North America. Twenty to thirty per cent of the conceptuses are lost early in gestation and an additional 10-15% is lost by mid-to-late gestation. Research over the last two decades has provided critical insights into how uterine capacity, placental efficiency, genetics, environment, nutrition and immune mechanisms impact successful conceptus growth; however, the exact cause and effect relationship in the context of foetal loss has yet to be determined. Similar to other mammalian species such as the human, mouse, rat, and primates, immune cell enrichment occurs at the porcine maternal-foetal interface during the window of conceptus attachment. However, unlike other species, immune cells are solely recruited by conceptus-derived signals. As pigs have epitheliochorial placentae where maternal and foetal tissue layers are separate, it provides an ideal model to study immune cell interactions with foetal trophoblasts. Our research is focused on the immune-angiogenesis axis during porcine pregnancy. It is well established that immune cells are recruited to the maternal-foetal interface, but their pregnancy specific functions and how the local milieu affects angiogenesis and inflammation at the site of foetal arrest remain unknown. Through a better understanding of how immune cells modulate crosstalk between the conceptus and the mother, it might be possible to therapeutically target immune cells and/or their products to reduce foetal loss. In this review, we provide evidence from the literature and from our own work into the immunological factors associated with porcine foetal loss.

Vascular dysfunction as a cause of endometrial bleeding

Introduction. Heavy menstrual bleeding (HMB) and the spotting and bleeding (S/B) associated with the use of hormonal contraceptives are distinct entities affecting endometrial vasculature and hemostasis.Materials and methods. An overview of the major etiologies and potential treatments for each condition is provided.Results. HMB is potentially caused by several different hemostatic dysfunctions. Combination oral contraceptives, levonorgestrel-releasing intrauterine system, non-steroidal anti-inflammatory drugs, and anti-fibrinolytics all have been shown to have some degree of efficacy in treating HMB. The basic cause of HMB is unknown in the majority of cases. Endometrial S/B related to hormonal contraceptives is a common occurrence and may well have a common etiology in altered angiogenesis resulting in abnormal blood vessels with fragile vessel walls. There is no effective treatment for this problem.Conclusions. Medical therapy for HMB is limited and effective for reducing blood loss during menstruation. There is no effective treatment for the S/B associated with hormonal contraceptives.

Identification of Label-Retaining Perivascular Cells in a Mouse Model of Endometrial Decidualization, Breakdown, and Repair1

The human endometrium is incredibly dynamic, undergoing monthly cycles of growth and regression during a woman's reproductive life. Endometrial repair at the cessation of menstruation is critical for reestablishment of a functional endometrium receptive for embryo implantation; however, little is understood about the mechanisms behind this rapid and highly efficient process. This study utilized a functional mouse model of endometrial breakdown and repair to assess changes in endometrial vasculature that accompany these dynamic processes. Given that adult endometrial stem/progenitor cells identified in human and mouse endometrium are likely contributors to the remarkable regenerative capacity of endometrium, we also assessed label-retaining cells (LRC) as candidate stromal stem/progenitor cells and examined their relationship with endometrial vasculature. Newborn mouse pups were pulse-labeled with bromodeoxyuridine (BrdU) and chased for 5 wk before decidualization, endometrial breakdown, and repair were induced by hormonal manipulation. Mean vessel density did not change significantly throughout breakdown and repair; however, significantly elevated endothelial cell proliferation was observed in decidual tissue. Stromal LRC were identified throughout breakdown and repair, with significantly fewer observed during endometrial repair than before decidualization. A significantly higher percentage of LRC were associated with vasculature during repair than before decidualization, and a proportion were undergoing proliferation, indicative of their functional capacity. This study is the first to examine the endometrial vasculature and candidate stromal stem/progenitor cells in a functional mouse model of endometrial breakdown and repair and provides functional evidence suggesting that perivascular LRC may contribute to endometrial stromal expansion during the extensive remodeling associated with this process.

Tamoxifen Modulates Cell Migration and Expression of Angiogenesis-Related Genes in Human Endometrial Endothelial Cells

The selective estrogen receptor modulator tamoxifen is used for the prevention and treatment of breast cancer. The adverse effects of tamoxifen include vaginal endometrial bleeding, endometrial hyperplasia, and cancer, conditions associated with angiogenesis. The aim of this study was to examine the effects of tamoxifen on cell migration and angiogenesis-related gene expression in human endometrial endothelial cells (HEECs). The regulatory effects of tamoxifen on endometrial stromal cells and HEECs were also examined. HEECs and stromal cells were isolated and grown in monocultures or co-cultures, and incubated with 0.1 to 100 μmol/L tamoxifen for 48 hours. Quantitative PCR demonstrated that tamoxifen decreased the mRNA expression of vascular endothelial growth factor-A (VEGF-A) and increased the mRNA expression of VEGF receptor-1 and placental growth factor (PLGF) in HEECs. Tamoxifen's effects on VEGF-A were inhibited when HEECs were co-cultured with stromal cells. In addition, tamoxifen reduced VEGF-induced HEEC migration. The tamoxifen-metabolizing enzymes CYP1A1 and CYP1B1 were detected by immunohistochemistry in and around endometrial blood vessels and by quantitative PCR in HEECs. Our data suggest that tamoxifen changes the regulation of angiogenesis in the endometrium, likely by reducing angiogenic activity. The results also indicate that endometrial stromal cells regulate some of tamoxifen's effects in HEECs, and the presence of tamoxifen-metabolizing enzymes suggests tamoxifen bioactivation in the endometrial vasculature in vivo. These findings may help to elucidate the mechanism of the bleeding disturbances associated with tamoxifen treatment.

The diagnostic value of the Doppler ultrasonography in distinguishing the endometrial malignancies in women with postmenopausal bleeding

To investigate the diagnostic value of blood flow measurements in endometrial, myometrial and uterine vasculature by transvaginal Doppler ultrasonography in the differentiation of the neoplastic endometrial pathologies in women with postmenopausal bleeding. 106 women who presented with postmenopausal bleeding were enrolled in a prospective cohort study. Endometrial thickness, pulsatility and resistance indices of the uterine, myometrial and endometrial vasculature, endometrial histopathology were measured by transvaginal Doppler sonography. Dilatation and curettage were performed for all women. Sonographic and histopathological results were evaluated. Endometrial malignancy was diagnosed in 24 of the patients (22.7%). Endometrial thickness was found to be higher in the patients with malign histopathology compared with the patients of benign histopathology. Statistically, uterine artery PI, RI, radial artery PI, spiral artery PI, and RI were also significantly lower in patients with malign histopathology. According to ROC curve analysis the endometrial thickness of 5 mm, uterine artery PI of 1.450, uterine artery RI of 0.715, radial artery PI of 1.060, and radial artery RI of 0.645 were defined as the cut-off points. In multivariate regression model, only uterine artery PI was identified as independent determinant of malignant endometrium. Blood flow of uterine artery and also myometrial and endometrial vasculature displayed lower impedance in patients with malignant endometrium, but these lower indices are not already adequate for using as diagnostic tests.