20 years on – Reflections on Hyaluronan

It has been 20 years since my team first published on the effects of hyaluronan (also known as hyaluronic acid) on preimplantation embryo culture and transfer¹, and so this is a poignant time to reflect on the role of this remarkable macromolecule in assisted reproduction.

Does hyaluronan have a role in biology?

Hyaluronan is a fascinating molecule, whose biological role was initially perceived as a ubiquitous “tissue filler and joint lubricant” found throughout the body, but whose biological activity is only relatively recently being appreciated. Most everyone in the world has heard of this molecule, as it is advertised as a dermal filler (goodbye old wrinkles!) and is frequently on TV as an active component in anti-ageing creams. So, whether they realise it or not, millions of people never leave home without it, literally. But what is it and what does it actually do?

Classified as a glycosaminoglycan, it is technically a polysaccharide (as it lacks any protein component), and is built up of two repeating sub-units, N-acetyl -glucosamine and β-glucuronic acid. As a macromolecule, hyaluronan can exist in relatively small polymers of 5,000 Da, up to a whopping 4 million Da! It transpires that key functions of hyaluronan are related to its polymer length, more on that later.

The levels of hyaluronan increase during implantation

Our initial interest in hyaluronan was piqued by the fact that its levels within the uterus increase around the time of implantation, indicative of a biological function around this time². So, we embarked on a study to evaluate the effect of hyaluronan and other macromolecules on the development of the preimplantation mouse embryo¹. We found that not only was the inclusion of hyaluronan associated with increased embryo development and viability, but that there existed distinct benefit of having hyaluronan in the transfer medium. The latter observation paving the way for the creation of EmbryoGlue.

However, it would be remiss to limit the discussion on hyaluronan to embryo transfer alone, as there followed a stream of papers about hyaluronan and its impact on the culture of preimplantation embryos from several mammalian species, which arrived at two other fascinating conclusions: The first was the presence of hyaluronan in the culture medium maintained the cellular ultrastructure of the embryos. The second conclusion, consistent with these observations of improved physiology, was that the embryos of the human, and both domestic and laboratory animals, when cultured in the presence of hyaluronan were better able to cope with the stresses associated with cryopreservation (reviewed in (3)). From every perspective, the inclusion of hyaluronan in both culture and transfer media, made physiological sense, and the data clearly support multiple benefits to the embryo.

Proven effect on clinical embryo transfer

So, 20 years on, is hyaluronan (EmbryoGlue) of proven benefit to clinical embryo transfer? A review of PubMed over this time revealed that of 19 clinical studies on EmbryoGlue, 13 reported significant benefits particularly in women over 35 years of age, for frozen embryo transfers and for patients with recurrent implantation failure. Of those studies not reporting a benefit, importantly none reported any adverse outcomes. Indeed, studies not reporting a benefit tended to be small retrospective studies, and none were powered for transfer outcome. The only prospective randomised trial to date on EmbryoGlue, based on 1282 cases and powered for transfer outcome, reported significant increases in implantation and clinical pregnancy rates for both day 3 and day 5 transfers, with the benefit most marked on patients over 35 and for those patients with previous implantation failures⁴, findings confirmed by the other 12 studies reporting a benefit. Consistently, the latest Cochrane report in 2014 concluded “Evidence suggests improved clinical pregnancy and live birth rates with the use of functional concentrations of HA as an adherence compound in ART cycles.”⁵.

Proven effect on transfer outcome

Given that the majority of clinical data is in support of the use of EmbryoGlue for embryo transfer, how does hyaluronan confer a significant benefit to transfer outcome? We and others have provided several plausible mechanisms, ranging from the physical properties it confers at transfer to ensure integration of the transfer medium with the uterine environment, thereby ensuring the embryos stays where it is placed, to complex cellular mechanisms which promote both cell adhesion, provide immune tolerance and increase angiogenesis at the site of implantation. What is evident from a growing literature on this polysaccharide, is that it is a key regulator of tissue remodelling⁶, and that as it breaks down to smaller chain lengths, its bioactivity changes⁷. Our current research is focussing on the varied, and unique, abilities of this macromolecule to facilitate the implantation process.

In summary, hyaluronan has been shown to significantly improve embryo development in culture, increase cryotolerance and improve transfer outcome in several mammalian species, including the human. As a result, I for one, would never want to use a system devoid of this macromolecule, and all my clinical transfers are performed in EmbryoGlue, as failure to do so could seriously compromise a patient’s chance of conceiving. It transpires that hyaluronan can not only make you look younger when applied cosmetically, it can significantly increase your chance of having a baby if you require IVF!

Webinar with Prof. David K Gardner

Watch the recorded webinar ‘Optimising transfer outcome with the use of EmbryoGlue’ where Prof. David Gardner guides you through the role of hyaluronan in biology and the benefits of using EmbryoGlue in preimplantation embryo culture and transfer.

References

<sup>1. Gardner DK, Rodriegez-Martinez H, Lane M. Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer. Hum Reprod 1999;14:2575-80.
2. Carson DD, Dutt A, Tang JP. Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function. Dev Biol 1987;120:228-35.
3. Gardner DK. Dissection of culture media for embryos: the most important and less important components and characteristics. Reprod Fertil Dev 2008;20:9-18.
4. Urman B, Yakin K, Ata B, Isiklar A, Balaban B. Effect of hyaluronan-enriched transfer medium on implantation and pregnancy rates after day 3 and day 5 embryo transfers: a prospective randomized study. Fertil Steril 2007.
5. Bontekoe S, Heineman MJ, Johnson N, Blake D. Adherence compounds in embryo transfer media for assisted reproductive technologies. Cochrane Database Syst Rev 2014:CD007421.
6. Dicker KT, Gurski LA, Pradhan-Bhatt S, Witt RL, Farach-Carson MC, Jia X. Hyaluronan: a simple polysaccharide with diverse biological functions. Acta Biomater 2014;10:1558-70.
7. Cyphert JM, Trempus CS, Garantziotis S. Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology. Int J Cell Biol 2015;2015:563818.</sup>