Markers of human endometrial hypoxia can be detected in vivo and ex vivo during physiological menstruation.

STUDY QUESTION: Can markers of human endometrial hypoxia be detected at menstruation in vivo? SUMMARY ANSWER: Our in vivo data support the presence of hypoxia in menstrual endometrium of women during physiological menstruation. WHAT IS KNOWN ALREADY: Current evidence from animal models and human in vitro studies suggests endometrial hypoxia is present at menstruation and drives endometrial repair post menses. However, detection of human endometrial hypoxia in vivo remains elusive. STUDY DESIGN, SIZE, DURATION: We performed a prospective case study of 16 women with normal menstrual bleeding. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reproductively aged female participants with a regular menstrual cycle underwent objective measurement of their menstrual blood loss using the alkaline haematin method to confirm a loss of <80 ml per cycle. Exclusion criteria were exogenous hormone use, an intrauterine device, endometriosis or fibroids >3 cm. Participants attended for two MRI scans; during days 1-3 of menstruation and the early/mid-secretory phase of their cycle. The MRI protocol included dynamic contrast-enhanced MRI and T2* quantification. At each visit, an endometrial sample was also collected and hypoxia-regulated repair factor mRNA levels (ADM, VEGFA, CXCR4) were quantified by RT-qPCR. MAIN RESULTS AND THE ROLE OF CHANCE: Women had reduced T2* during menstrual scans versus non-menstrual scans (P = 0.005), consistent with menstrual hypoxia. Plasma flow (Fp) was increased at menstruation compared to the non-menstrual phase (P = 0.0005). Laboratory findings revealed increased ADM, VEGF-A and CXCR4 at menstruation on examination of paired endometrial biopsies from the menstrual and non-menstrual phase (P = 0.008; P = 0.03; P = 0.009). There was a significant correlation between T2* and these ex vivo hypoxic markers (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This study examined the in vivo detection of endometrial hypoxic markers at specific timepoints in the menstrual cycle in women with a menstrual blood loss <80 ml/cycle and without significant uterine structural abnormalities. Further research is required to determine the presence of endometrial hypoxia in those experiencing abnormal uterine bleeding with and without fibroids/adenomyosis. WIDER IMPLICATIONS OF THE FINDINGS: Heavy menstrual bleeding (HMB) is a common, debilitating condition. Understanding menstrual physiology may improve therapeutics. To our knowledge, this is the first in vivo data supporting the presence of menstrual hypoxia in the endometrium of women with normal menstrual bleeding. If aberrant in those with HMB, these non-invasive tests may aid diagnosis and facilitate personalized treatments for HMB. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by Wellbeing of Women grant RG1820, Wellcome Trust Fellowship 209589/Z/17/Z and undertaken in the MRC Centre for Reproductive Health, funded by grants G1002033 and MR/N022556/1. 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 articles on abnormal uterine bleeding. TRIAL REGISTRATION NUMBER: N/A.

The presence and regulation of connective tissue growth factor in the human endometrium.

BACKGROUND: The human endometrium efficiently repairs each month after menstruation. The mechanisms involved in this repair process remain undefined. Aberrations in endometrial repair may lead to the common disorder of heavy menstrual bleeding. We hypothesized that connective tissue growth factor (CTGF) is increased at the time of endometrial repair post-menses and that this increase is regulated by prostaglandins (PGs) and hypoxic conditions present during menstruation. METHODS AND RESULTS: Examination of 41 endometrial biopsies from 5 stages of the menstrual cycle revealed maximal CTGF mRNA expression (using quantitative RT-PCR) at menstruation and peak protein levels during the proliferative phase. CTGF was immunolocalized to epithelial and stromal cells, with intense staining of occasional stromal cells during the proliferative phase. Dual immunohistochemistry identified these cells as macrophages. Treatment of endometrial epithelial cells with 100 nM PGE(2), PGF(2α) or hypoxia (0.5% O(2)) revealed a significant increase in CTGF mRNA expression (P < 0.01 for all, versus vehicle control). Cells treated simultaneously with PGE(2) and hypoxia revealed a synergistic increase in CTGF expression (P < 0.05 versus PGE(2) or hypoxia alone) and maximal secreted CTGF protein levels (P < 0.05 versus control). CONCLUSIONS: CTGF is increased in the human endometrium at the time of endometrial repair post-menses. The increase in CTGF may be mediated by PG production and the transient hypoxic episode observed in the endometrium at menstruation.