P–416 Radiotherapy inflicts long-term damage upon the uterus, causing uterine artery endothelial dysfunction and pregnancy loss in mice
Authors: Griffiths M , Marshall S , Cousins F , Care A , Winship A , Hutt K 
Published: August 2021

Abstract

Study question
Does the uterus sustain direct and long-term damage following radiotherapy, independent of ovarian damage?

Summary answer
Radiotherapy causes direct and long-term uterine damage. Ovariectomised and hormonally supplemented mice experience uterine artery endothelial dysfunction and pregnancy loss following transfer of healthy blastocysts.

What is known already
The detrimental off-target impacts of cancer therapies on the ovary are well established, with some fertility preservation techniques available to ensure patients maintain their fertility following gonadotoxic treatment. Low doses of radiotherapy kill the majority of primordial follicle oocytes in the ovary, reducing the ovarian reserve and fertile lifespan. Patients who have received radiotherapy experience higher rates of pregnancy complications including preterm birth, low birth weight, and stillbirth. However, no studies have investigated if radiation inflicts direct, fertility compromising damage to the uterus.

Study design, size, duration
Adolescent female mice were untreated or exposed to whole body y-irradiation (7Gy), then ovariectomised. Immediate damage was assessed up to 24 hours post-irradiation (n = 4/group). Four weeks following treatment, mice were hormone primed to induce endometrial receptivity (n = 7/group), artificial decidualisation (n = 7–8/group), or receive embryo transfers from healthy, unexposed donor mice to assess embryo implantation (n = 11–13/group), and mid-gestation development (n = 8–10/group).

Participants/materials, setting, methods
Four week old C57BL6/CBA (F1) female mice were used for this study. Immunofluorescence and in situ hybridisation were utilised to localise markers of immediate DNA damage and cell death following irradiation, and markers of receptivity in hormone primed uteri. Measurements of uterine artery blood flow were recorded using Doppler ultrasonography, and indices of pulsatility and resistance calculated. Uterine artery wire myography was performed to assess competency of endothelial and smooth muscle compartments following irradiation.

Main results and the role of chance
Within 24 hours of irradiation, DNA damage (yH2AX) and apoptosis (Puma/TUNEL) were elevated in uteri, compared to control. Irradiated mice that received embryo transfers from untreated donors had similar numbers of implantation sites 3-days post-transfer versus controls, however uteri were pale and atrophic suggesting impaired vascularisation. By 10-days post-transfer, implantation sites in irradiated mice were resorbing (p &lt; 0.001), although uterine artery Doppler ultrasound measurements demonstrated no change in pulsatility or resistance indices. When the brain was shielded from irradiation to protect the hypothalamic-pituitary-gonadal axis, resorption still occurred (p &lt; 0.001), suggesting direct uterine damage is the likely cause of pregnancy loss. To investigate uterine damage in the absence of an embryo, endometrial receptivity was induced artificially. Uteri from irradiated animals were lighter compared to control (p &lt; 0.05), however localisation of receptivity markers (E-cadherin, Mucin1, Ki67) was normal. When decidualisation was artificially induced irradiated uteri were also lighter (p &lt; 0.01) indicating impaired decidualisation and reduced capacity to adapt to pregnancy. Wire myography performed on uterine arteries demonstrated endothelial dysfunction in irradiated mice (p &lt; 0.0001).

Limitations, reasons for caution
Here, only a single age and dose of radiotherapy exposure are modelled. Patients of all ages can receive many doses of radiotherapy in combination with various chemotherapies. Our highly manipulable model enables any treatment variation to be modelled and the effect on the uterus and pregnancy examined.
Wider implications of the findings: Reproductive aged cancer patients need to be appropriately counselled regarding the risks to their long-term fertility following treatment. Until now, potential permanent impacts to the uterus following cancer treatment have not been considered. It is clear radiotherapy can impose long-term damage to the uterus and influence pregnancy success and fertility.

Trial registration number
NA