A critical role for hyaluronan in sheep embryo development and implantation

Lead Research Organisation: Royal Veterinary College
Department Name: Comparative Biomedical Sciences CBS


The establishment of pregnancy needs both the presence of a good embryo and a uterus at the correct stage to receive it. Very often, the embryo will not implant because either the embryo is abnormal or the uterus is not properly prepared to receive the embryo. Poor fertility leads to much human suffering and a significant economic loss for farmers. This project is about identifying the nature of the dialogue between the mother and the embryo (so-called blastocyst) during the critical period of development that leads up to the implantation of the embryo in the wall of the uterus. We believe that a complex sugar molecule called Hyaluronan (HA) that is present in most animal tissues plays a vital role within the mother-embryo communication system. Information from ourselves and others suggests that HA has a very important role in this process, and we have evidence that modified HA can assist embryo implantation in sheep. We shall investigate its role in implantation. Exactly how HA works in signalling to the mother that an embryo is implanting is a further question that we will try to answer. We believe that progesterone (dominant hormone during the period of embryo growth and implantation), is vital for HA production and function. In order to answer this second question we will investigate how HA is made, and how it is processed to become active in the mother-embryo communication system. The purpose of this application is to secure the funding required to further our investigation into the function of HA in the embryo implantation process. Our investigation requires that we use animals for the experiments. However, we will also use animal tissues from abattoirs for a significant part of the project thus keeping the number of animals required for the project to an absolute minimum. We have planned the following experiments for these investigations: 1. Two methods will be used to determine the effects of progesterone on HA production: A) Uterine cells will be treated with progesterone in culture dish. B) Sheep which have had their ovaries removed (ovaries produce progesterone) and therefore have been deprived of progesterone, will receive progesterone replacement. Uterine cells from the culture, as well as the uterus and oviducts of the progesterone /treated sheep, will be collected and analysed for HA as well as HA modifying enzymes. 2. Two methods will be used to generate embryos and to determine the effects of HA on development of embryos to blastocyst stage and blastocyst quality: A) Sheep treated with hormones to induce multiple ovulations will be allowed to mate and become pregnant naturally. B) Oocytes will be obtained from abattoir ovaries and mixed with sperm to generate embryos (IVF). Embryos produced through either method will be exposed to chemicals that are known to affect the levels of HA. Embryo development to blastocyst stage will be quantitatively monitored and the quality of the blastocysts will be assessed after embryo staining. 3. Two methods will be used to determine the role of HA on blastocyst implantation. A) Blastocysts, produced in the lab and will be cultured together with uterine cells. Agents that modify levels of HA will be added during days 14-17, when implantation would occur in the uterus. The embryos will be examined for development and the uterine cells for their production of HA and associated molecules. B) Uterus in naturally mated sheep will be infused with similar agents one day before the expected day of embryo attachment (day 15 after mating). Uterine tissue will be collected after slaughter of half the sheep (day 17) to assess blastocyst attachment and to study of expression of known genes and proteins related to embryo implantation. The remainder of the sheep will be slaughtered on day 35 to assess foetal survival and pregnancy rate. This project will potentially lead to improvements in fertility following either in vitro fertilization or natural conception.

Technical Summary

The overall aim of this project is to determine the effects of hyaluronan (HA) on embryo quality, elongation and attachment in a sheep model. Incorporating parallel in vivo and in vitro studies, we hypothesise that progesterone (P4) activates HAS-3 and Hyal-2 to generate small HA fragments which increase blastocyst survival and quality by reducing apoptosis, and improve embryo attachment through induction of COX-2 and synthesis of PGE/I. Experiment 1. Effects of P4 on HA biosynthesis and degradation. Ovariectomised sheep and in vitro cultured uterine cells will be treated with P4. Uterine and oviductal tissues as well as secretions will be collected to extract HA and to measure its size and concentration by HPLC. Expression of HAS-3 and Hyal-2 will be assessed by quantitative RT-PCR and Western blotting. Experiment 2. Effects of HA size on embryo development and blastocyst quality. Sheep embryos, produced in vitro or after superovulation and natural mating, will be exposed to small or large HA, 4-MU (an inhibitor of HA synthesis) or Hyal-2. Development to blastocyst stage will be recorded and quality of the blastocysts will be assessed by differential staining and by counting the number of apoptotic cells in the trophoblast and the ICM. Experiment 3. Role of small HA fragments on embryo elongation and attachment: Blastocysts, produced in vitro and co-cultured on uterine cells, or produced from natural mating will be exposed to small and large HA molecules, 4-MU, NS-398 (COX-2 inhibitor) and Hyal-2 by supplementation in vitro from day 6 or in vivo via uterine infusion from day 14. Blastocysts will be assessed morphologically and by TEM for elongation and attachment. Uterine tissue will be collected on day 17 to assess the rate of blastocyst attachment and to study the expression of CD44, COX-2, IL-1 by immunohistochemistry and in situ hybridisation. Some of the sheep will be slaughtered on day 35 to determine foetal survival and pregnancy rate.


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Description My research has discovered
1. The critical role of an important enzyme Hyaluronisade II in supporting preimplantation embryos development. It is produced by the uterine tube (oviduct) where the embryo spends early days after fertilisation. IVF culture media and practice removes this important factor through regular washing of the embryos. Supplementation of Hyal-2 to embryo culture media will compensate for its deficiency in IVF system and improves the rate of embryo development and embryo quality leading to improved pregnancy rate after embryo transfer.
2. In contrast to the beneficial effect of Hyals in embryo development, my research data has revealed an important fact about the obstructing role of HA in embryo implantation. Presence of HA at the embryo implantation site blocks embryo adhesion to uterine way and embryo implantation, whereas, blocking HA production at the time of embryo implantation significantly improves pregnancy rate.
3. Production of HA and Hyals are regulated by steroid hormones oestradiol and progesterone. High oestradiol during first stage (follicular phase) of reproductive cycle, stimulates production of large size HA which is important for oocyte maturation and fertilisation and dilation of cervix. This is followed by high progesterone after formation of the corpus luteum, which stimulates production of small size HA and Hyals to support embryo development and prepare uterus for reception of embryo. Small HA have angiogenic activity and prepare the uterus for embryo implantation.
Exploitation Route Manufacturers of embryo culture media both for human and animals will be able to produce Hyal-2 based culture media to improve blastocyst rate. I have been in discussion with Vitrolife one major producer of culture media for human assisted reproduction. Unfortunately, the lengthy discussios have not resulted in practical commitment by this company.

Further to this we have preliminary data which shows large and small size HA have different and contrasting roles in uterine receptivity and mocusal immunity . These information was submitted a grant proposal to BBSRC for funding last year. Despite achieving excellent scores by reviewers, the BBSRC failed in its mission to support this important work. It would have opened a new era in reproductive biology and fertility treatment in animals and in human.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology

URL http://www.ncbi.nlm.nih.gov/pubmed/23625939
Description The impact of HA and its mechanism of action on embryo development has helped wider use of HA and its' related product in human an animal embryology
First Year Of Impact 2011
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Description BBSRC Follow on Funfd
Amount £157,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2014 
End 11/2015
Title Improving success of IVF 
Description The data from my BBSRC Follow-on Fund grand has revealed that the elements from hyaluronan system can be used to improve embryo development rate resulting in improved pregnancy rate in sheep. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Human fertility treatment and animal IVF will benefit from this technology. Interests has been expressed from commercial companies involved in human and animals assisted reproduction technology. 
Description Universitat Autonoma de Barcelona 
Organisation Autonomous University of Barcelona (UAB)
Country Spain 
Sector Academic/University 
PI Contribution I had a research project with Prof. Maria Teresa Paramio. As a collaborator the project funded by Spanish Government, I contributed to the research on the effects of PUFAs on improving oocyte quality from prepubertal sheep and goat. I co-supervised a PhD student with Prof. Paramio.
Collaborator Contribution My PhD student spent the last two years of his PhD study in Prof. Paramio's laboratory. I also conducted an in vivo study in Barcelona on sheep. Samples collected from this study were transferred to my lab for analysis.
Impact One publications has been published from this work in 2014.
Start Year 2012
Description University of Warwick 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution I discussed the results of my research about involvement of hyaluronan system in embryo implantation with Professor Jan Brosens. He is one of the leading researchers in human fertility focusing on regulation of embryo implantation in the uterus and the role of leukocytes. This meeting was mediated through my research collaborator Professor Geraldine Hartshorne. Subsequently, I sent a number of reagents to test the effect on human uterine cells.
Collaborator Contribution Professors Jan Brosens and Geraldine Hartshorne recruited a MSc student to carry out the experiments. The results are highly promising and has led to new discoveries which is due to be submitted as a manuscript.
Impact I expect submission of the first manuscript with the next few months.
Start Year 2019
Title P60757GB 
Description 1. Use of very low molecular weight hyaluronan fragments as supplement for human and animals embryo culture 2. Use of hyals as supplement for human and animals embryo culture 3. Hyal-2 as a biomarker of embryo quality and predictor of pregnancy outcome after embryo transfer 4. Inhibition of hyaluronan synthesis in the uterus enhances embryo attachment and pregnancy rate 
IP Reference  
Protection Patent granted
Year Protection Granted 2016
Licensed No
Impact The Notable impact will be in human assisted reproductive technology, animal embryo transfer including endanger animal species