Biology︱ CheMyong Jay Ko

Non-surgical sterilisation iSpay
Will pet-spaying operations soon be a thing of the past?
Dr CheMyong Jay Ko is the founder of Epivara and a professor in theCollege of Veterinary Medicine at the University of Illinois UrbanaChampaign, USA, working on reproductive endocrinology. His team hasrecently achieved a breakthrough in the search for a non-surgical methodof permanently sterilising female companion animals such as dogs and cats.
Here, we present their proof-of-concept study showing that a single doseof estrogen delivered at the prepubertal stage of life via a slow-releaseformulation prevents the development of reproductive organs. This noveltechnology, named iSpay, opens up a possibility for a practical alternative
to many millions of spaying operations currently used to prevent unwanted
pet pregnancies.

T

he life of a well-cared-for pet is
idyllic: a warm home, food served
up daily, and perhaps a cosy lap
to sit on. In stark contrast, dogs and catsthat can’t find a nurturing home will often
end up leading a painful and tough life.
Domesticated animals that are left tofend for themselves on the streets arevulnerable to abuse, starvation, disease,and early death. Some animals do get
rescued by charities that run animal
shelters and rehousing schemes, but theirresources are limited and not all stories
have a happy ending.
Many people love and enjoy their pets
but don’t necessarily want to share
their home with a constant streamof extra kittens or puppies. To avoid
unwanted litters, many pet owners andanimal charities decide to have female
animals spayed.

WHAT IS SPAYING?
Spaying is an operation where both theovaries and the uterus are commonlyremoved. Such surgeries are mostlysuccessful; as well as eliminating
unwanted sexual behaviour andpregnancies, they can lead to a reductionin the risk of some cancers. Unfortunately,
they also have downsides. Health risks
to the spayed animal include obesity,urinary incontinence, and unwanted
behavioural changes.
Furthermore, surgical procedures needhighly skilled vets, anaesthetics, andproperly equipped operating theatres
and recovery spaces. There are plenty ofcountries that do not have many of thesefacilities. Some countries may have theresources but have made the culturaldecision that performing surgery on
pets for the purpose of birth control is

unnecessarily invasive. Many Scandinavian
countries rely on very strict anti-roaming
laws to prevent unwanted pregnancies
instead of spaying.
A simpler, non-surgical method ofsterilising pets could greatly reduce
the population of unwanted animals,
eliminate the dangers of surgery, andsave a lot of time and money. The
Alliance for Contraception in Cats andDogs (ACC&D) is an organisation thathas joined forces with animal advocatesand researchers to foster the efforts ofdeveloping easier and less invasive ways
to control feline and canine reproduction
than surgery.

The process of becoming sexually mature
is an interesting, coordinated effort fromthe brain, the pituitary gland, and the ovary.
KISS1 stimulates GnRH secretion within thehypothalamus. iSpay inhibits the developmentof Kisspeptin neurons, the master regulator ofreproduction in female animals.

 HOW DO MAMMALS BECOMESEXUALLY MATURE?
Mammals are not able to have babies
immediately after birth. The processof becoming sexually mature is aninteresting, coordinated effort from thebrain, the pituitary gland, and the gonads.
We are just beginning to understand how
exactly this intriguing and complicatedsystem works, and it seems all threeof these areas of the body are needed
for success.
The brain’s role happens in thehypothalamus, an area known tocontrol the maturation and function ofreproductive organs. When allowed todevelop normally, specialised nerve cells
in this area called Kisspeptin neurons
produce a hormone called KISS1,which then locks onto a second typeof neurons called GnRH (gonadotropinreleasing hormone) neurons. GnRH
neurons are then triggered to startrhythmically producing GnRH. As thename suggests, this hormone thengoes on to tell the pituitary to makeand release gonadotropins – namely
luteinising hormone (LH) and follicle
stimulating hormone (FSH) – which inturn stimulate the ovary to synthesisesex steroid hormones and release eggs.
If the Kisspeptin neurons never developendocrinology. In 2016, he founded
Epivara Inc. to develop a practical nonsurgical sterilant.



Molecular structure ofthe protein Kisspeptin(top) and GnRH
(bottom).

A simpler, non-surgical method ofsterilising pets could greatly reduce thepopulation of unwanted animals.
in the first place, then the cascadeof neuronal and pituitary hormone
releases can’t be activated, and theanimal therefore can’t release eggs andbecome pregnant.

THE SEARCH FOR AN ‘OFF SWITCH’
Dr CheMyong Jay Ko is a professor
in the College of Veterinary Medicine
at the University of Illinois at UrbanaChampaign, USA working on reproductive

We might predict there to be manypotential molecules or compounds that
could disrupt this complicated chainreaction. Indeed, some are well-known
and used as contraceptives both inhumans and occasionally for pets.
Contraceptives are useful in situations
where the aim is only to temporarily
prevent pregnancy. For permanentsterilisation, they are relatively impracticalsince they require repeated dosing, which
is time-consuming and prone to mistakes.
In the search for new medications,existing drugs are often foundto have previously unknown
properties. Estradiol benzoate(EB) is such a drug. EB was
already a well-known form of the
reproductive hormone estrogen. It’s used
for many medical applications, including
hormone replacement therapy to reduce
menopausal symptoms, as part oftreatment against prostate cancer, and asa growth stimulant in the cattle industry.
Previous studies had found that
repeated daily dosing of newborn
female rats with EB for 11 days disrupted
the development of their reproductive
organs by targeting Kisspeptin neurons

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Behind the Research

to be equivalent to the 11 daily doses or
the silicone implants.

Estradiol benzoate is used for many
medical applications, including hormone
replacement therapy.

(Minabe et al, 2017), so Ko’s team (DrsChanjin Park, Rex Hess, Po-Ching Lin,
Sherry Zhou, Sandra Soto, Lindsey
Reinacher, and Katie Chai) decided totake these findings further and developa practical method to create a sterilant.
GETTING THE RIGHTMETHOD OF DOSINGThe team first tested if a silicone implantthat released a significantly smaller
amount of EB over 2-3 weeks than
the total amount of EB injected daily
for the 11 days in the previous study
would sterilise the female rats. However,they found that making EB-containing
silicone implants and inserting them into
small animals was inconvenient. Instead,

it would be preferable to deliver thedrug using an injection method.
Ko and his collaborators developed a
new method to deliver the EB using
biodegradable microspheres. The
microspheres used are less than 10µm in
diameter and contain many tiny dropletsof EB. The spheres are way too small forthe human eye to see individually andappear as a powder that is then diluted
before being injected with a routinely
used syringe and needle. The researchers
hoped that the extra time needed tobreak down the microspheres in thisnew formulation would deliver the EB
droplets over an extended time, therefore
smoothing out the EB dose enough for it

This non-surgical method of sterilisation
for companion animals could replacehundreds of thousands of spaying
operations each year.

The team found that a single dose of EB
in a microsphere formulation was indeed
enough to limit the development of bothovaries and the womb and permanently
sterilise young female rats. Encouragingly,
no significant side effects have been
detected for the animal’s overall healthand other hormone functioning. Recently,
they also developed a pellet form ofEB formulation which showed the same
effectiveness and safety in a trial as themicrosphere form.
THE FUTURE POSSIBILITIESKo hopes this new technique of single
injection using a biodegradable
formulation of EB will now provide anaffordable, effective, practical, andhumane method to prevent unwanted
pregnancy in domestic pets. The team
tested this non-surgical method – called
iSpay – on female Beagle dogs andfound that they also became sterilewith no obvious side effects. The dogs
that participated in the experiment are
currently known to be healthy and over 3years old. It is also likely that a sustained
dose of EB will sterilise young cats andother mammals that are currently spayedby surgery.
This non-surgical method of sterilisation
for companion animals could replacehundreds of thousands of spaying
operations each year and greatly
decrease the suffering and damage
caused by unwanted free-roaming dogs
and cats.
Unlike previous methods of reproductive
control, iSpay will prevent thedevelopment of the reproductive systembefore it fully matures. Therefore, it willlead to permanent infertility, a feat thathas not been accomplished before.

The team tested their non-surgical
iSpay method on female Beagle
dogs who became sterile with noobvious side effects.

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As we gain a better understanding of
the interesting biology of reproduction,
it is likely that more and more options
for birth control will be discovered.
Perhaps the greater challenge will thenbe how to navigate the ethics of thesenew powers. With this in mind, ACC&D
has brought together experts in this field
to focus on improving ethical decisionmaking tools for researchers working inthis area.

Dr CheMyong Jay Ko

E: jayko@illinois.edu E: jay@epivara.com T: +1 859-494-0082 W: vetmed.illinois.edu/kolaboratory
W: www.epivara.com
www.linkedin.com/company/epivara

Research Objectives

References

Dr CheMyong Jay Ko and his team developed iSpay, a nonsurgical method for the permanent sterilisation of female
mammals.

• Park, CJ, et al, (2023) Single neonatal estrogen implantsterilizes female animals by decreasing hypothalamic KISS1
expression. Scientific Reports, 13, 9627. doi.org/10.1038/
s41598-023-36727-8• ACC&D (2023) Ethical decision-making in innovation foranimal welfare. [online] ACC&D. www.acc-d.org/ethicaldecision-making [Accessed 29/11/2023]• Minabe, S, et al, (2017) Long-term neonatal estrogen
exposure causes irreversible inhibition of LH pulses by
suppressing arcuate kisspeptin expression via estrogen
receptors α and β in female rodents. Endocrinology, 158(9),2918–2929. doi.org/10.1210/en.2016-1144

Detail
BioDr CheMyong Jay Ko is a professor in the College ofVeterinary Medicine at the University of Illinois UrbanaChampaign. His primary research focuses on reproductive
endocrinology. He received a PhD degree from Seoul
National University and postdoctoral training from theUniversity of Kentucky. Before moving to Illinois in 2011,
he worked as a professor in the Medical Center of theUniversity of Kentucky for 10 years. In 2016, Dr Ko founded
Epivara at the University of Illinois’ Research Park.
Funding
Epivara Inc, National Science Foundation (NSF)
Collaborators
The researchers are grateful to Dr Ozawa (Nippon MedicalSchool, Tokyo, Japan) for providing the anti-kisspeptin
polyclonal antibody (RRID: AB_2910199). They also thank Dr
C L Wright, Dr C J Fields, Dr J Drnevich, and Dr M Tseng fortheir assistance in performing single-cell RNA sequencing
and analysing data.

‘iSpayed’ adult Beagles remain infertile atthe age of 3 years and are very playful.

Personal Response
What inspired you to conduct this study?
My work at the university includes not only teachingveterinary students but also counselling them on their visionand challenges. I have found that many students dream ofbecoming veterinarians to help and treat sick or suffering
animals, but in reality, many veterinarians spend theircareers performing spay and neuter surgeries. My vision
primarily focuses on enabling the students I teach to leadmore meaningful lives as veterinarians, and I take great joy
and a sense of purpose in the belief that such work can also
make people and animals happier.
Do you think there may be applications for this newtechnique in nature conservation, perhaps in the control
of invasive mammals?
The current developed technology does not work inanimals beyond achieving sexual maturity. Therefore, there
are many limitations to its use in controlling the populationof wild animals. However, this technology can be used foranimal species where easy and efficient access to newlyborn individuals is possible.
When will your first product become available on
the market?
Our iSpay technology is protected by patents in the
USA and EU countries. The team is seeking FDA approvalof their first product, which may take approximately 3–5years. Immediately after receiving the FDA approval, theproduct will become available on the market.

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