Investigating perfusion and hypoxia in the human uterus

Introduction: Heavy menstrual bleeding (HMB) is a common and debilitating condition. Current treatment options are limited by lack of effectiveness, side effects and surgical risks. Better understanding of the physiology of menstruation may help identify new therapeutic targets. Transient vasoconstriction of the endometrial spiral arterioles occurs prior to menstruation. There is in vitro evidence that endometrial hypoxia is present at menstruation and activates endometrial repair and that a decreased hypoxic response may result in HMB. Determination of hypoxia in vivo has not yet been possible in the human endometrium. Magnetic resonance imaging (MRI) techniques may be utilised to non-invasively detect markers of endometrial hypoxia in women. Obesity, defined as a body mass index (BMI) ≥30 kg/m2, has a profound impact on health and its prevalence is rising worldwide. However, there are limited data on the influence of obesity on menstrual blood loss (MBL). Therefore, it was hypothesised that: 1.Hypoxia is present in the human endometrium at menstruation and can be detected in vivo and ex vivo.2.Women with objectively measured HMB have a defective endometrial hypoxic response during menstruation.3.MRI assessments of endometrial hypoxia correlate with laboratory-based techniques in the detection of hypoxia markers. 4.Obesity in women is associated with increased MBL due to an impaired endometrial hypoxic response at menstruation. Methods: 24 participants were recruited and underwent measurement of MBL using the modified alkaline haematin method (HMB: ≥80 ml) and attended for MRI scans timed for the menstrual and early/mid-secretory phase of the menstrual cycle. To address hypotheses 1-3, the protocol included T2* quantification and dynamic contrast-enhanced MRI (DCE-MRI). An endometrial sample was also collected in the menstrual and early/mid-secretory cycle phase for laboratory analysis of hypoxia markers: CAIX (a hypoxia marker) analysed by immunohistochemistry (IHC) and hypoxia-regulated repair factors (ADM, VEGFA, CXCR4) and lactate dehydrogenase (LDHA) measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Endometrial vasculature was also analysed by immunofluorescence for CD-31 (endothelial cell marker) and α-SMA (marker of vascular smooth muscle cells).To assess hypothesis 4, women (n=122) completed a pictorial bleeding assessment chart (PBAC) and had their BMI measured. Endometrial samples were also collected from 28 women in the menstrual/late-secretory cycle phase to assess for hypoxic markers as above. Results: In vivo imaging revealed women with normal menstrual bleeding (NMB; < 80 ml) had reduced endometrial T2* at menstruation compared to the non-menstrual phase. Deoxyhaemoglobin increases local magnetic field inhomogeneity which decreases T2*, hence these data are consistent with hypoxia at menses. Endometrial plasma flow (Fp) was increased at menstruation compared to the non-menstrual phase which may reflect post hypoxic vasodilation of the spiral arterioles within the endometrium. Ex vivo findings in women with NMB showed increased levels of ADM, VEGF-A, CXCR4 and LDHA mRNA in paired menstrual compared to non-menstrual endometrium, supporting the presence of endometrial hypoxia at menstruation. Comparing women with NMB and HMB, in vivo imaging showed no difference in T2* at menstruation. However, endometrial Fp and plasma volume (vp) were reduced at menstruation in women with HMB compared to NMB. This may reflect delayed initiation of endometrial repair or a lack of post hypoxic vasodilation within the endometrium of women with HMB. There were no differences in laboratory analyses of endometrial hypoxic markers at menstruation between these two groups of women. A significant negative correlation between T2* and endometrial concentrations of ADM, VEGF-A, CXCR4 was observed providing validation of this non-invasive technique for the detection of endometrial hypoxia in vivo. There was a positive correlation between BMI and MBL but no evidence that obesity impairs endometrial hypoxia at menstruation. In summary, the novel in vivo and ex vivo data presented in this thesis support the presence of hypoxia in the endometrium of women with NMB. Women with HMB displayed altered endometrial perfusion at menstruation. Non-invasive imaging may improve diagnosis and provide opportunity for identification of personalised targets for the management of HMB.