Identification of trophoblast differentiation pathways regulated by Glial Cells Missing 1 using comparative studies of horse and human pregnancy

Project grant funded by Wellcome Trust

In this project we investigated the effect of the ligand Bone Morphogenetic Protein 4 (BMP4) on trophoblast differentiation either alone or in combination with activation of cAMP. We showed a key role for BMP4 signaling in the regulation of terminal differentiation of primary equine trophoblast cells and subsequent secretion of eCG via activation of the SMAD1/5 pathway (Cabrera-Sharp et al, 2014, Endocrinology). The observation of BMP4 signaling in primary trophoblast provides a previously unreported mechanism of TGFβ signaling in the placenta. We also confirmed the presence of BMP receptors in early human placenta, suggesting the pathway may be conserved. A number of additional signaling pathways that may regulate trophoblast differentiation and that have not been described before have been identified using microarray. We have validated the data using multiplex RT-PCR and are currently assessing the function of selected cell cycle molecules in trophoblast differentiation.

We found GCM1 acts to regulate eCGB mRNA expression. Luciferase assays demonstrated GCM1 transactivates a 335 bp segment of the eCGB promoter. We are currently performing binding assays to determine whether GCM1 is acting directly or indirectly on the promoter. We have additionally used our microarray data described above to identify a number of other potential regulators of eCGB transcription. We identified 7/225 transcription factors that had a >2-fold change in expression in the chorionic girdle between day 31 and 27 of gestation, were tissue specific, correlated with eCGB expression (R²>0.5) and had predicted binding sites in the eCGB promoter. We have confirmed that 6/7 transcription factors are expressed in the chorionic girdle at the time of initiation of transcription and we are currently testing their functionality.

Our long-term goal is to find a method to produce eCG in the laboratory, replacing the need to farm pregnant mares as serum (eCG) donors. Our Wellcome work is the first to identify a key mechanistic pathway that regulates differentiation of eCG-producing trophoblast cells and eCG secretion. Thus it represents the first major step towards achieving this goal. In additional experiments, we have identified a number of candidate pathways likely to act in concert with BMP4 to achieve maximal in vitro eCG production. The functionality of these pathways are being tested and we are hopeful that with this additional information, we will have a viable in vitro method to produce eCG.

Summary of our work showing mechanisms regulating trophoblast differentiation in the mare (see Cabrera-Sharp et al, 2014 for more details)

The effects of intrauterine infusion of peanut oil on endometrial health, salivary cortisol and interovulatory period in mares

Funded by European Breeders Fund supported by HBLB

We have recently completed a study investigating the effect of peanut oil on oestrus behaviour and endometrial health. The project was initiated and led by collaborator Dr Madeleine Campbell with whom we secured funding from the European Breeders Fund and HBLB to perform the work. The work stemmed out of a very interesting publication by Wilsher and Allen (2011) that showed that intrauterine infusion of peanut oil at Day 10 post-ovulation prolongs dioestrus in mares. In agreement with the authors, we hypothesised that this would be a very useful method of suppressing unwanted behaviours associated with oestrus in mares. The objectives of this study were, (i) to determine the effect of intrauterine infusion of peanut oil on endometrial health, (ii) to determine whether use of intrauterine peanut oil is painful and (iii) to confirm that peanut oil causes prolonged dioestrus. Six mares aged 3-12 years old were used in a cross-over design with each mare administered both 1 ml of intrauterine peanut oil and a sham treatment on different oestrous cycles. The effect of intrauterine infusion of 1 ml peanut oil or sham treatment were measured. Our results found the individual mare response to intrauterine infusion of peanut oil was variable. Peanut oil infusion did not statistically prolong the luteal phase, nor elevate salivary cortisol levels but did cause superficial erosion of the endometrial surface epithelium in all mares and significantly increased eosinophil numbers in the endometrium. The Kenney grade for biopsies from 2/6 mares worsened transiently following infusion. In conclusion, intra-uterine peanut oil does not statistically increase the duration of the luteal phase but results in an inflammatory response and increase in endometrial eosinophil numbers suggesting treatment is associated with inflammation. Interestingly, the results are consistent with an independent follow up study by Diel de Amorim (2016) that showed intrauterine coconut oil was similarly unable to prolong the dioestrus period in mares. Taken together, this would suggest that until further data is produced on the efficacy of this treatment, clinicians should avoid its use and continue to use more traditional methods of modulating oestrus behaviour such as progestin supplementation. Further details can be found in our publication in Theriogenology.

Risk factors and pathologies associated with early pregnancy loss

PhD studentship funded by the Thoroughbred Breeders Association supported by the HBLB

Whilst maternal age has long been recognised as an important risk factor in relation to the incidence of EPL, few other risk factors have been conclusively identified, specifically, multivariable data analysis of risk factors for EPL is sparse. By taking into account correlated factors, for example, mare age and status, multivariable analysis allows various related factors to be investigated together to allow a true interpretation of the individual affect of each. Work was conducted on data from 2246 thoroughbred pregnancies in the Newmarket area of the UK over the 2013 and 2014 breeding seasons and a total of 28 variables investigated. Increasing mare age, previous live foals and the presence of uterine cysts were associated with an increased risk of EPL whilst the use of ovulatory induction agents, flunixin used at the time of reduction of a multiple pregnancy and larger vesicle sizes decreased the risk of EPL. Further investigations found the use of ovulatory induction agents, lavaging the uterus at the time of cover with sterile saline and having a previous pregnancy resulted in a larger sized embryo up to day 34 of pregnancy. Results from this study can be used by stud farm personal when assessing their broodmare population and by clinicians when deciding upon therapeutic strategies. Additional work can be focused around these risk factors to further our understanding of the pathophysiology of EPL. Results from this project can be found in Rose et al, 2016 and Rose et al, 2017.