Research quadruples speed and efficiency to develop embryos
By Samantha Kummerer | Bond LSC
What started as a serendipitous discovery is now opening the door for decreasing the costs and risks involved with in vitro fertilization (IVF).
And it all started with cultured pig cells.
Dr. Michael Roberts’ and Dr. Randall Prather’s laboratories in the University of Missouri work with pigs to research stem cells. During an attempt to improve how they grew these cells, researchers stumbled across a method to improve the success of IVF in pigs.
“Sometimes you start an experiment and come up with up with a side project and it turns out to be really good,” Researcher Ye Yuan said.
Their discovery doubles the number of piglets born and speeds up the entire IVF process by 400 percent, which significantly increases both the efficiency of experiments and their potential application to other species. The journal Proceedings of the National Academy of Sciences published their work July 3 in its online early edition.
From the beginning
The Prather lab in the MU Animal Sciences Research Center uses genetically modified pig embryos to improve pig production for agriculture and also to mimic human disease states, such as cystic fibrosis. Roberts’ team in the Bond Life Sciences Center occasionally collaborates with Prather’s lab to produce genetically modified pigs for this valuable research. However, the efficiency of producing these pigs is very low because it depends on multiple steps.
First, scientists remove oocytes (“eggs”) and the “nurse” cells that surround them from immature female pig ovaries and place the eggs in a chemical environment designed to mature the eggs, allowing them to be fertilized in vitro with sperm from a boar. This process creates zygotes, which are single-celled embryos, that are allowed to develop further until they become hollow balls of cells called blastocysts about six-days later. These tiny embryos are then transferred back into a female pig with the hopes of achieving a successful pregnancy and healthy piglets.
However, Roberts said the chance of generating a successful piglet after all those steps is very low; only 1-2 percent of the original eggs make it that far.
The quality of the premature eggs and the process of maturing them significantly reduces the rate of success.
“In other words, all this depends on having oocytes that are competent, that is they can be fertilized, form blastocysts and initiate a successful pregnancy,” Roberts explained.
Normally, researchers overcome the low success rate by starting out with a very large number of eggs, but this takes lots of time and money.
So, lab researchers, Ye Yuan and Lee Spate, began tinkering with the way the eggs were cultured before they were fertilized, making use of special growth factors they used when culturing pig embryonic stem cells.
Yuan and Spate added two factors called fibroblast growth factor 2 (FGF2) and leukemia inhibitory factor (LIF).
This combination helped more than the use of just a single factor and so they decided to add a third factor, insulin-like growth factor 1 (IGF1).
Together the three compounds create the chemical medium termed “FLI”.
“It improved every aspect of the whole process,” Roberts said. “It almost doubled the efficiency of oocyte maturation in terms of going through meiosis. It appeared to improve fertilization and it improved the production of blastocysts.”
In all, the use of FLI medium doubles the number of piglets born and quadruples the efficiency of the entire process from egg to piglet.
While the researchers are still figuring out why the three factors work together so well, Roberts believes it has to do with the fluid that surrounds the immature eggs while they are still in the ovary.
Roberts explained that unusual metabolic changes happen in the eggs and their nurse cells when the three components are used in combination but not when they are used on their own. These components are also found in the follicular fluid surrounding the egg when it is in the ovary.
However, follicular fluid actually contains factors that hinder egg maturation until the time is right, so it would seem counterintuitive to add the fluid to a chemical environment aimed at maturing the eggs. However, when freed from the other components of follicular fluid, the three growth factors act efficiently to promote maturation.
“It just creates this whole nurse environment for that egg. Once you’ve done that you’ve sort of patterned them to do everything else after that properly — fertilization, development of that fertilized egg to form a blastocyst, and the capability of those blastocysts to give rise to a piglet,” Roberts said.
Researchers hope the FLI medium can be translated beyond genetically modified pigs.
“If we could translate this to other species it could be more meaningful,” Yuan explained.
For the cattle industry, FLI has the potential to decrease the time between generations in highly prized animals.
Currently, if an immature dairy cow has desirable traits, the industry has to wait a year or so for that cow to mature and for its eggs to be collected. Using FLI medium immature eggs could be retrieved when the prized female is still a calf. After fertilizing them with semen from a prized bull, production of more cows with desirable traits could be achieved in a shorter amount of time.
The potential implications of this discovery aren’t just for farm animals.
Yuan said if this treatment could be applied to humans it would be a big help for both the patient and the whole field of human IVF.
Currently, in vitro fertilization for humans comes with high costs and risks.
“You try to generate a lot of eggs from the patients by using super-high doses of expensive hormones, which is not necessarily good for the patient and can, in fact, be risky. ” Roberts explained.
These eggs are then collected, fertilized, and the best-looking embryo transferred back to the patient. As in pigs, this overall process isn’t all that efficient. The hope is that the treatment of the patient with hormones can be minimized if immature eggs are collected directly from the ovary by using an endoscope and matured in FLI medium, allowing them to be just as competent as those retrieved after high hormone treatment.
“The idea is it would be safer for the woman, it would be cheaper, and it might even achieve a better success rate,” Roberts said.
The team still has some time before knowing for sure if FLI medium is applicable in other mammals.
Yuan said the focus is now on understanding the mechanism behind how the three compounds work so well together.
For now, preliminary data are being collected with mice and a patent is awaiting approval. Still, the team has high hopes for this almost accidental finding.
“Whenever you’re doing science, you’d like to think you’re doing something that could be useful,” Roberts said. “I mean when we started this out it wasn’t to improve fertility IVF in women, it was to just get better oocytes in pigs. Now it’s possible that FLI medium could become important in bovine embryo work and possibly even help with human IVF.”
Michael Roberts is a Bond LSC scientist and a Curators’ Distinguished Professor of Animal Science, Biochemistry and Veterinary Pathobiology in the College of Agriculture, Food and Natural Resources (CAFNR) and the College of Veterinary Medicine. He is also a member of the National Academy of Sciences.
Randall Prather is a Curators’ Distinguished Professor of Animal Science in the College of Agriculture, Food and Natural Resources (CAFNR) and Director of the National Institutes of Health funded National Swine Resource and Research Center.