More than a miss: Scientists identify a gene variant that causes early pregnancy loss

In recent years, the percentage of live foals to mares covered in Australia has remained stable at 66%, despite a shrinking pool of broodmares and stallions. Every lost or missed pregnancy is a large expense for the breeder, and it is the early stages of equine pregnancy that are most delicate and susceptible to loss.

Funded by the AgriFutures Thoroughbred Horses Program, researchers at the University of Newcastle and University of Sydney believe they have identified one potential genetic cause of early pregnancy loss, and are working towards developing a screening tool to help breeders have realistic expectations for reproductive success.

A lethal combination

Genes and gene variants that never appear in a healthy, living foal twice are referred to as lethal genes, meaning that having two copies always results in premature death of the horse or embryo. When the team at the University of Sydney, led by Dr Brandon Velie, investigated the genomes of several equines in search of potentially lethal genes, the gene variant LY49B became the subject of interest.

“Most lethal genes arise during embryonic development,” said Dr Zamira Gibb, an Associate Professor who leads the University of Newcastle half of the team. “In genetics, we have what is called the Hardy-Weinberg equilibrium; there are different types of genes that should exist in certain ratios depending on how they perform.

“For instance, blue eyes are recessive, so a person needs two copies of the gene to have blue eyes, which naturally happens in roughly 25% of the population. There will be 25% that have two brown eye genes and 50% that will have one brown and one blue gene, all will have blue eyes.

“When Brandon’s team screened the whole genome for alterations of that ratio, they didn’t find a single horse with two copies of LY49B.”

Recessive lethal genes that activate later in development include Hereditary Equine Regional Dermal Asthenia (HERDA), Warmblood Fragile Foal Syndrome, and the colour overo, but to see no horses with two copies of LY49B suggested it impacted the embryo.

As a result, this means that mating two horses together with one copy of the gene each has a 25% chance of the mating not creating a viable embryo at all.

“The work has been validated by a group led by Dr Mandi de Mestre,” Gibb said. Dr de Mestre is the Dorothy Havemeyer McConville Professor at Baker Institute for Animal Health at Cornell University.

“She looked at lots of reproductive tissues and she thought, following on from Brandon’s findings, that they should be able to find evidence of the gene in those tissues, but she didn’t. This suggests it is lethal very early in the embryo’s development, before the embryo becomes established.”

Confirming the findings

In Velie’s screening group, he found that 50% of Thoroughbreds appeared to carry the gene, and the research group’s next steps are to confirm this across a broader sample group.

“The pool he looked at was relatively small, so we would like to expand that and look at the different populations and geographical distribution around Australia,” Gibb said. “There is no reason to believe it will be isolated to one area, but something the industry has indicated an interest in is if the ratios are different in each area. So we will be sampling in Queensland, Victoria, New South Wales, and Western Australia.”

The team will be working with hair or blood samples, and are working towards developing a polymerase chain reaction (PCR) test to isolate the gene.

“This is the type of testing we use for coat colour genetics, for example,” Gibb said. “The way that they identified the gene originally was using whole genome sequencing, which is very expensive, while a PCR is much easier to do on a big scale. We currently have a biotechnology Honours student working on designing a primer that will recognise the unique sequence of the gene.”

Once broader surveying backs up Velie’s findings, Gibb takes over the next step of the process, which will look at how the gene practically impacts an embryo’s formation.

“There is very little literature about what this gene does during reproduction, but it does play a role in the immune system,” she said. “We do know that it is expressed by the outer cells of the embryo, which eventually turn into the placenta, so we believe it has something to do with modulating the mother’s immune system.

“We need to look at the mechanisms of what it is doing, but we can’t do that in a horse’s body. Instead, we will be making embryos in the IVF lab at the University of Newcastle and implanting them into what are called organoids.”

Organoids are “mini organs”; tissue grown from and with the same properties as the original organ, which allows the team to look at how the embryo, sperm, and seminal plasma interact with equine endometrium. This will allow the team to hopefully unlock what exactly LY49B is doing that results in embryo loss.

Knowledge and management

There is the potential that the team’s research could lead to a commercial screening test for the gene down the track, which could offer breeders a new tool to assist in making their matings each year.

“We have been doing a lot of communication with the industry, because sometimes the stallion farms can be quite skeptical,” said Gibb. “They can be concerned that finding the gene could reduce the popularity of their stallion, but the way we see it, it just means breeders are able to manage their expectations better when making informed breeding decisions."

At the national herd level, it has been calculated that the gene could be contributing to a 6.25% drop in pregnancy rates per cycle. Considering the final live foal to mares covered rate has sat at around 66% for some time, this is a chance to really shift the needle.

“AgriFutures is here to assist the industry in finding new ways to improve its productivity, and this is an important area to be looking into,” said Johnathan D’Arcy, Chair of the Thoroughbred Horses Advisory Panel. “By researching the prevalence of this gene, we could hopefully work towards improving the overall fertility rate by four or 5%.

“The important thing is that it gives mare owners more information about potential matings. If they know their mare is a carrier, they can be more selective, or they can take the chance that 25% of matings will result in no embryo, just from this gene. There is still a 75% chance of success.”

“It’s all about knowledge and management,” said Gibb. “It could be as simple as knowing both mare and stallion are carriers, but you believe together they’re going to make an amazing foal, so you recognise that there is a 25% chance each cycle that a pregnancy might not happen.

“I wouldn't be surprised if you have a very fertile stallion who is a carrier and he has great fertility rates, because maybe everything else is going well with his sperm.”

“This could be a really good tool for everyone,” said D’Arcy. “Stallion farms are already working very hard to ensure that their stallions aren’t overworked, so this could be another tool to ensure that he has the maximum chance of getting a viable embryo with every cover.

“And by knowing how you can improve your chances of a viable embryo, you can reduce the costs of production and improve the welfare of your animals by minimising the amount of trips to get covered, and reducing the amount of vet visits needed. It's a very expensive industry and anything we can do to alleviate some of those costs has to be a big positive for the industry.”