September 2015

A Preimplantation Genetic Diagnosis Pioneer Takes
a Look BackDr. Leeanda Wilton of Melbourne IVF codeveloped genomic methods that were key predecessorsof technologies that are now contributing to successful IVF pregnancies.
Introduction
Breakthrough technologies are often the result of hard work, failure,serendipity, collaboration, and, finally, success. Leeanda Wilton, PhD
experienced all of the above in codeveloping 2 technologies that would
transform the field of preimplantation genetics. In the mid-1980s, achance encounter led to a crosstown alliance and the development of
single-cell fluorescent in situ hybridization (FISH), the first technology thatenabled researchers to identify multiple numerical chromosome errors inhuman embryos. A decade later, cooperation and hard work led to thedevelopment of metaphase comparative genomic hybridization (CGH),
the predecessor of array CGH technologies such as the 24sure® Array.
Dr. Wilton is now Scientific Director of Preimplantation Genetics
at Melbourne IVF, where she established the first PGD program inAustralia. Under her leadership, Melbourne IVF’s PGD program hasgrown significantly by using the latest technologies to help womenconceive babies.
iCommunity spoke with Dr. Wilton about her pioneering work in thisfield and some of the technologies that have enabled Melbourne IVF
to become leaders in preimplantation genetics.
Q: How did you become interested in molecular biology?
Leeanda Wilton (LW): I started out as a cell biologist and moved intomolecular biology by default. After I finished my PhD, my first project
was to learn how to remove a cell from a mouse embryo without
damaging the embryo. I was ultimately successful in developing
embryo biopsy and karyotyping of single mouse blastomeres. Thatresearch became the foundation of PGD in human embryos. With
the advent of PCR and other technologies, my focus naturally shiftedfrom cell biology to molecular biology to perform disease detectionin embryos.
Q: What was the spark that led to the development of single-cell
FISH technology for PGD?
LW: I was at the Institute of Zoology at the London Zoo in themid-1980s, developing reproductive technologies for preservationof endangered species. I was still very interested in PGD and had
the opportunity to collaborate with Drs. Robert Winston and AlanHandyside at the Hammersmith Hospital IVF unit. I was struggling torepeat my mouse karyotyping work on single human cells. Every time
I tried to get metaphase spreads from human embryonic cells, all I got
was a clump of DNA and I could barely distinguish any chromosomes.
I was tossing microscope slide after microscope slide into the trash
one day when Alan walked by and asked how things were going. Hecould see my frustration and suggested that I talk with Darren Griffin,then a PhD student at University College London. Darren was tryingto get the new FISH technique to work with human embryonic cells,

For Research Use Only. Not for use in diagnostic procedures.

but was struggling to prepare clean DNA spreads. I took some slides
over to his lab and overnight we’d solved each other’s problems. My
cell processing approach was ideal for leveraging the FISH method
he was using to look at the sex chromosomes in human embryoniccells. We analyzed more samples to determine the efficiency andeffectiveness of the method. Soon we were performing the first clinicalcases of preimplantation gender selection by FISH for patients with
X-linked diseases.
Q: What value did FISH provide in analyzing embryo genetics andwhat were its limitations?
LW: FISH enabled us to get our first indepth look at embryo
chromosomes and provided insight into embryo genetics. Initially, wecould only look at 2 chromosomes; then that expanded to 5, and then
8 chromosomes. By the late 1990s, it was clear that there was a lot ofimportant information that we weren’t seeing with FISH. We realized weneeded a technology that could analyze all chromosomes in a single cell.
Q: How was metaphase CGH developed?
LW: After we realized how inefficient FISH was, our team at Melbourne
IVF began collaborating with Drs. Lucille Voullaire and Bob Williamson
at the Murdoch Institute at the Royal Children’s Hospital in Melbourne
to develop the metaphase CGH technology. It enabled us to obtain
information on all chromosomes simultaneously. Developing metaphase
CGH was a tough project. We’re very proud that our team was the firstin the world to have a baby born from an embryo selected by analyzing
all the chromosomes in a single cell with metaphase CGH.

Dr. Leeanda Wilton is Scientific Director of Preimplantation
Genetics at Melbourne IVF.

September 2015
Q: How did the 24sure array change your practice?
LW: Although it was successful, the metaphase CGH method waslaborious and we had to wait a week or more to obtain results. Thatmeant we had to freeze all the embryos we were testing and freezing
technology then wasn’t as good as it is now. Metaphase CGH wasalso technically very challenging and results were difficult to interpret.
The 24sure Array is an automated CGH array technology and is much
faster than the old metaphase CGH method. It enables us to run 40–50
samples simultaneously. The faster turnaround means we can select andtransfer embryos via a fresh cycle. That makes a significant difference instreamlining the IVF process for our patients.
Within 3-4 months of shifting to the 24sure Array, we had a 5-foldincrease in the number of patients who we were treating. Althoughthat was challenging for our laboratory, it speaks to a huge increase
in interest for PGD for aneuploidy (PGD-A), or PGS, and our ability tosupport it.

“Within 3-4 months of shifting tothe 24sure Array, we had a 5-foldincrease in the number of patients
who we were treating.”
Q: What is the advantage the 24Sure Array for PGD-A?
LW: Certain types of patients are predisposed towards
chromosome errors, or aneuploidies, in their embryos. Most of
those errors are lethal, causing the embryo to die soon after it isplaced into the patient. In the past, we relied on an embryologist
to choose an embryo that was suitable for transfer by relying
solely on its morphology. We found in a retrospective analysis that
this approach meant that 75% of the time the chosen embryo hadan aneuploidy and effectively no chance of resulting in a healthy
pregnancy. By using the 24Sure array, we can identify embryosthat are free of aneuploidy and have a much better chance of
implanting and leading to a successful pregnancy.
Q: What impact has the 24sure Array had on your IVF decisions?
LW: The 24sure Array has made a difference in how we manage
embryo selection in older women who have an increased frequencyof egg chromosome errors that are then passed on to their embryos.
It’s faster and much more comprehensive, enabling us to improve
the pregnancy potential for patients who might be predisposed toaneuploidy, including older women and those who have sufferedrepeated miscarriage and failed IVF cycles. The 24sure Array enables
us to perform IVF with embryos that we know have a higher viability
potential, shortening the time it takes for these women to become
pregnant. That’s particularly significant for older women because
they really don’t have time to wait.
Q: Are you using VeriSeq® PGS in your clinic?
LW: We are using VeriSeq PGS as part of the Illumina STAR (SingleEmbryo TrAnsfeR Euploid Embryo) randomized control trial. We are alsousing VeriSeq PGS on a limited basis for analyzing embryos from older

For Research Use Only. Not for use in diagnostic procedures.

women who are interested in trying out new technologies. Although
we’re early adopters, we’re cautious about using new technologies.

Karyomapping is a phenomenal
technology and the biggest gamechanger in PGD for detection ofsingle gene diseases.
Q: What is karyomapping and what benefits does it provide
for detecting single-gene disorders?
LW: Karyomapping is a phenomenal technology and the biggest gamechanger in PGD for detection of single gene diseases, like cystic fibrosis,
Huntington disease, and Fragile X syndrome. This technology enables us
to analyze the single nucleotide polymorphism (SNP) pattern in embryos
to determine whether the embryos have inherited the single-gene defectthat affects the family. We’ve tripled our throughput of these types ofcases because karyomapping is so much simpler, faster, and cheaper,
than multiplex-PCR, which has been the standard technology in single
gene PGD for many years.
In the past, we had to develop individual tests for patients who had
family histories of single-gene disorders, which took many weeks andwas very expensive. The Infinium® HumanKaryomap-12 DNA Analysis
Kit enables us to assess the SNP pattern across the genome, so wecan examine the DNA region where the genetic error is located anddetermine if the embryo has inherited normal or mutated DNA fromits parents.
Q: Are you using BlueFuse® Multi Software to perform data analysisof VeriSeq PGS, 24sure Array, and HumanKaryomap-12 kits?
LW: One of the advantages of the VeriSeq PGS, 24sure, andHumanKaryomap-12 Kit technologies are that they all use the same
BlueFuse Multi Analysis Software. That’s a huge advantage for us
in laboratory and staff training. We love the fact that we can use thesame BlueFuse Multi Software platform to analyze Illumina genetic
data even though each technology is really very different.
Q: What’s most striking about the progress that’s been made in IVF?
LW: It’s hard to believe that the mouse embryo cell removal techniquesthat I was developing at my first job 30 years ago were adopted soquickly for human IVF procedures and have benefited so many people.
It’s rare in science to develop a new technology, see it through to clinicalapplication, and later witness its adoption throughout the world.
Q: Are there any technologies that have been practice-changing?
LW: The technologies that have had the most impact on IVF havebeen array CGH and karyomapping. The 24Sure Array made it
possible to look at every chromosome for genetic errors easily andinexpensively. It’s made an enormous difference in how quickly we cantest embryos and enabled us to take a huge step forward from themethods we used for many years.

September 2015
We really can’t believe the difference karyomapping is making. Morethan 90% of patients who need PGD for single gene diseases are able
have their testing done using karyomapping, which is faster and morecomprehensive.
Q: What is the risk to patients if IVF labs fail to keep up with thelatest technologies?
LW: Every patient who comes into an IVF program wants to havea baby as soon as possible. By not keeping up with the latest
technologies, labs can negatively impact the success of their IVF
programs. For example, some programs are still running FISH
even though it’s very clear that it overcalls errors. Because they’reunwittingly deselecting viable embryos, the labs are limiting thepatient’s embryo pool for IVF procedures, potentially delaying thepatient’s chances of a successful pregnancy.

Learn more about the Illumina products andsystems mentioned in this article:•24sure Array, www.illumina.com/products/24sure-microarrays.html.

•

iScan System, www.illumina.com/systems/iscan.html.

•VeriSeq PGS, www.illumina.com/products/veriseq-pgs.html.

•

MiSeq System, www.illumina.com/systems/miseq.html.

•

BlueFuse Multi Software, www.illumina.com/clinical/clinical_informatics/bluefuse.html.

•

HumanKaryomap-12 DNA Analysis Kit, www.illumina.com/products/human-karyomap.html.

•

NextSeq 550 System, www.illumina.com/systems/nextseqsequencer.html.

“One of the advantages ofthe VeriSeq PGS, 24sure,and HumanKaryomap-12 Kit
technologies are that they all
use the same BlueFuse Multi
Analysis Software.”
Q: What new technologies are on the horizon that might impact thefuture of PGD?
LW: I think NGS technologies, like VeriSeq PGS, will become more
widely used in the future. It’s possible that NGS will be used to detect
single-gene diseases in embryos. However, that will require significant
upfront development work for each disease that we are currently
identifying with karyomapping.
We’re very excited about the potential of the NextSeq® 550 Systemdual sequencing scanner to minimize the amount of equipment wehave. That’s why we were a part of the beta testing for the NextSeq
550 System. We have a lot of large pieces of equipment in different
labs, including the iScan® System and several MiSeq® Systems. The
possibility of doing sequencing and scanning with 1 machine would
be great.
Q: How important is collaboration in developing and implementing
future technological advances in IVF?
LW: Collaboration was critical for the successful development of
single-cell FISH and metaphase CGH. That’s a reflection of the fieldand everyone’s generosity with information. We want to benefit
patients around the world, not just in our geographic regions. We’re all
focused on improving outcomes and helping our patients have a baby
as soon as they can.

For Research Use Only. Not for use in diagnostic procedures.

September 2015

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
© 2015 Illumina, Inc. All rights reserved. Illumina, 24sure, BlueFuse, Infinium, iScan, MiSeq, NextSeq, VeriSeq, andthe pumpkin orange color are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries.
Pub. No. 1570-2015-006 Current as of 27 August 2015