Preimplantation Genetic Diagnosis (PGD) is a set of techniques specifically developed to test the genetic background of preimplantation human embryos. In order for PGD to be performed, human embryos must be available for analysis prior to the implantation. Therefore, only parents undergoing IVF procedure can benefit from this technology.

PGD or PGS, which one to choose?In recent years there have been many arguments regarding the proper name for the techniques for testing genetic background of a preimplantation embryo. The arguments are entirely about semantics, not the underlying science. If you are interested in the indepth discussion on PGD vs. PGS, you may read an article on the subject in the “FAQ” section of our website. However, for the purposes of this publica-tion, please consider PGD and PGS to be synonyms, because this is exactly what they are.

Four major reasons for PGD• One or both parents are the carriers of a single-

gene disorder (Cystic Fibrosis, beta-Thalassemia, Tay-Sachs, etc).

• One of the parents is a carrier of a chromosomal aberration (translocation, inversion, deletion).

• A baby of a specific sex is desired by parents for family balancing, or because of a sex-linked genetic disorder, or due to some personal reasons.

• Aneuploidy screening (PGD-AS, or PGS) for the patients going through IVF infertility treatment, who wish to improve their chances for normal pregnancy.

For the first three groups, PGD provides the only alternative to recurrent miscarriages and abortions. It is only for the last group of patients, that the reasons for going through PGD are not so obvious. These are the patients of advanced maternal age (AMA, women over the age of 36), patients with recurrent pregnancy loss (RPL), and patients with failed IVF cycles. These patients have no known genetic disorders, but they wish to improve their chance for a successful IVF cycle. Currently the majority of PGD cases are performed for this group of patients.

Why do genetically normal parents select PGD?Genetically normal parents produce genetically abnormal embryos in great numbers, and the evidence for this is disturbingly abundant:

Thousands of babies with Down Syndrome are born in the US every year. About the same number of babies with lesser-known chromosomal disorders (Kleinfelter’s, Turner’s, Patau’s, Edward’s) are also born to genetically normal parents.

At least 15-20% of all human fetuses end up as detectable spontaneous abortions. About half of

Why PGD?

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these embryos abort because they are chromosom-ally abnormal. A chance in the 7-10% range for miscarriage due to genetic abnormalities is a serious risk, but this number still does not quite reveal the urgency for PGD.

Failed pregnancies. This happens to every couple trying to conceive. According to some estimates, over 75% of all human conceptions abort before the first missed menstrual cycle. Thanks to PGD we know why this happens. By being able to test human embryos at the very first stages of development PGD reveals the magnitude of the pool of genetic abnormalities generated by humankind. On average, more than half of the embryos of an IVF patient in her mid- to late-thirties are genetically abnormal, or aneuploid. Nature, it seems, found an extremely wasteful, but probably the easiest, way of dealing with genetic abnormalities. Athough generated in large numbers, most genetically abnormal conceptions perish prior to or shortly after implantation.

During an IVF cycle, when so much hope, efforts, and financial investments go into a single attempt, selection should be at the level of individual embryos, not failed pregnancies! By identifying aneuploid embryos, PGD insures that only those embryos which have a chance to establish a normal pregnancy are transferred.

How is it possible that genetically abnormal embryos are generated by genetically normal parents?Mitotic errors (mistakes in chromosome segregation during routine cell division) occur constantly in our bodies, but the resulting abnormal cells are quickly recognized and eliminated by the immune system. During the first five days of development, the human embryo cleaves and transforms from a single-cell zygote into a blastocyst with hundreds of cells. At these stages, however, the embryo does not have an immune system yet, and genetically abnormal cells are free to compete with normal cells for the privilege of building a baby. A single mitotic error during early cleavage may result in an embryo with a large proportion of genetically abnormal cells.

Surprisingly, the majority of chromosomal abnor-malities are generated prior to fertilization, even before an embryo has a chance to establish itself as a new genetic entity. These are the errors of oocyte maturation, which can be addressed by PGD.

How do these abnormal oocytes originate? Why do older women have so few pregnancies and so many abnormal babies?In the 19th century it was noticed that babies with mongolism (presently recognized as Down’s Syndrome, or trisomy for chromosome 21) are

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mostly last-born children in the family. In 1930, the maternal age effect was shown to be the critical factor: women over 35 years of age gave birth to over half of all babies with Down’s Syndrome, although that group accounted for only 15% of all births. The odds of giving birth to a baby with Down’s Syndrome increases exponentially with the woman’s age. For a 25-30 year old woman the chance is 1 in 1000, but for a 45-year-old woman it skyrockets to 1 in 32.

As it usually happens in biology, the reality behind this phenomenon is rather complicated. Human oocytes (eggs) are well protected from the insecurities of the outside world deep inside the mother’s body. Oocytes are first surrounded by the eggshell (called the zona pellucida) then by layers of cells whose only function is oocyte nurturing. In its turn, the major function of the ovaries is oocyte storage, growth and matu-ration. There are good reasons for all these safety measures. Oocytes are perhaps the most important cells in the female body, and they have to be kept safe and viable for a very long time. Like nerve cells, oocytes do not regenerate, and their population is never replenished. The last new oocyte is formed in the ovary at the 7th month of prenatal development, before a baby girl is even born. Then, for decades, oocytes are stored under the most optimal condi-tions, but they cannot be fully protected from the passage of time. A 40-year old oocyte, although well protected from the outside world, has been affected by a lifetime of body fevers, distresses, drugs, etc.

Mechanisms underlying genetic maturation of a human oocyte, or meiosis, are very complex but the result of this process is the reduction by half the number of chromosomes the oocyte initially had in its “immature” state. Out of 46 human chromosomes each woman has 22 pairs of so-called autosomes and two sex determination chromosomes: X+X. Men also have 44 autosomes, one X chromosome and one tiny Y chromosome. Autosomes do not have any interesting names, like X or Y, and are known by their number. Thus, each human cell has two chromosomes No.1, two chromosomes No.2, ..., two chromosomes No.22, two chromosomes X (girls) or one X plus one Y (boys).

During meiosis, the oocyte discards its “second set” of 23 chromosomes and after that it is ready to be fertilized by a sperm, which will introduce its own, paternal set of 23 chromosomes. The resulting embryo will have a restored set of 46 chromosomes, 23 maternal and 23 paternal.

Any error made during oocyte maturation will be lethal for the resulting embryo. Let us take chro-mosome 21 as an example. If, by mistake, the oocyte gets rid of not just one, but of both of its chromosomes 21, the resulting embryo will end up with only one chromosome 21, introduced by a sper-

matozoon. This condition is called monosomy 21. Monosomy of any other autosome is generally lethal in humans, but a few babies with monosomy 21 have survived beyond birth, with severe abnormalities. No monosomies or nullisomies (embryos with both chromosomes missing) of any other chromosome have ever been found in newborns or in the aborted material. However, these abnormalities do exist in preimplantation embryos, but such embryos perish at the earliest stages of embryo development. These abnormalities “just” add to the statistics of failed pregnancies and failed IVF cycles.

If, on the other hand, an oocyte makes the opposite mistake and fails to extrude one chromosome 21 (while extruding the second set of every other chromosome), the resulting embryo will end up with one extra chromosome 21 - this condition is called trisomy 21. Most of these conceptions (about 75%) will be spontaneously aborted, but those surviving to birth will be the babies with Down’s Syndrome, the most common genetic defect in newborns. Other autosomal anomalies with any significant frequency among newborns are trisomies 13 (Patau’s Syndrome) and 18 (Edward’s Syndrome). Trisomy of any other autosome is lethal at the early stages of prenatal embryo development.

Meiotic error may also cause an abnormal number of sex chromosomes. Per each 100,000 recognized human pregnancies, around 1,500 will be aborted due to an abnormal number of sex chromosomes. Out of the same 100,000 human pregnancies, about 100 boys will be born with Kleinfelter’s Syndrome (instead of XY will have XXY set of sex chromo-somes) and around 50 girls will be born with Turner’s Syndrome (instead of XX will have X_, or XXX).

Apart from chromosomes 13, 18, 21, X, and Y, the only other chromosomes noticeably affecting the outcome of established human pregnancies are chromosomes 16 and 22. Trisomy 16 is found in one in every 11 spontaneous abortions and trisomy 22 is found in one out of every 35 spontaneous abortions. Embryos with trisomy 16 or 22 never reach term, but they affect the outcome of established pregnancies and add to the list of failed IVF cycles.

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Genetics 101

Immortal CoilsThe building block of every living organism on Earth is the cell. Whether it is a single bacteria or a congre-gation of billions of cells in the shape of an elephant or a giant sequoia, a single cell is how we all start and what we, living beings, are made of. Cells are microscopically small; thousands of cells may dance on the head of a pin!

Like humans, each cell has a “spark of life” inside, it is a long polymer molecule called Deoxyribonucleic Acid (DNA). DNA contains the information about the cell itself, on how to divide and make more cells, on how to differentiate and build the organs these cells form, information about the organism these organs serve, and finally the instructions (called instincts) on how these living creatures go about living their lives. It is a lot of information, information of vital importance! It should be stressed here that the only function of DNA is to store information, nothing more. All the dirty work of actually doing something, including synthesis of the DNA itself, or DNA replica-

tion, is done by the proteins and RNA.

Human DNA, if stretched out in a line, is almost four feet long and contains enough information to be compared to the Library of Congress or the Encyclopædia Britannica (depending on which side of the Pond you are on), or to gigabits of data, for those young enough not to care about printed pages. This enormously long strand of DNA fits inside a tiny cell by coiling, supercoiling and folding itself into more manageable lengths. Eukaryotic DNA shortens itself by winding around the special proteins called histones, then by compacting into the so-called 30nm chromatin fiber, then by supercoiling into a left-handed super-helix.

However, this level of compaction is still not compact enough to be manageable during cell division, so the strand of human DNA is actually broken into 23 pieces. Each of these pieces is called a chromosome and is visible as a separate entity only during cell division (called mitosis) when DNA undergoes its

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highest levels of compaction. In its normal, non-dividing state (called interphase) each cell keeps and protects its DNA inside the cell nucleus, visible under the microscope as a transparent almost homogenous structure packed with DNA.

To be an enormously long but at the same time an extremely stable molecule is a remarkable feat, however, this is not the reason why DNA is consid-ered the most amazing molecule on Earth. After all, polyethylene and cellulose are also long polymers comprised of ethylene or sugars. It is the ability to store and faithfully multiply information that makes DNA so special. If we look at the building blocks of this huge molecule, we shall see that the backbone of DNA is a molecule of deoxyribose (sugar). Billions of these interconnected (polymerized) molecules form the backbone of DNA. Left to itself, this would be just another type of cellulose, but each molecule of deoxyribose, apart from being attached to other deoxyriboses, is also connected to one out of four purine/pyrimidine bases, best known by their initials: A, T, G, C. These so-called nucleotides are the letters of the genetic code through which DNA (hopefully by now its full name, deoxyribonucleic acid is more manageable) codes the almost-infinite amount of information necessary for the building of a living creature.

(DNA is not one, but actually two polynucleotide strands. In physical terms this means that each polynucleotide strand lies next to another poly-nucleotide strand, and these strands are intercon-nected. A of each strand (so-called “sense” strand) is always connected to T from the opposite strand

(called “antisense strand”), and G is interconnected with C into base pairs. This is why DNA is also called a “double helix”: it consists of two interconnected polynucleotide strands running parallel to each other and also shaped like a spiral.)

Now that the DNA’s physical structure and the letters of the genome’s alphabet are revealed, it is time to introduce the concept of gene. We shall use the terminology of molecular biologists by saying that gene is a stretch of DNA coding information about a specific protein.

The nucleotide sequence ATGGATGCACACAAG ... TAA is as clear to any cell as the following message to us: START protein synthesis, grab amino acid Aspara-gine, link to amino acid Alanine, link to amino acid Histidine, link to amino acid Lysine ... STOP protein synthesis. Another molecule of human albumin is synthesized!

Humans have approximately 25,000 genes in their DNA, which takes up less than 10% of the genome. The other 90+% consist of broken/discarded genes (pseudogenes), viral genomes successfully inserted into the DNA of our ancestors before being neutral-ized, satellite DNA at chromosome centromeric regions, telomeric DNA, transposons and selfish DNA, and huge stretches of DNA for which we have no names and no plausible explanation.

Genetic Defects: Single-Gene MutationsThere is an infinite number of ways how the informa-tion stored in DNA may become corrupted. It may come at the level of an individual gene if, say, the first nucleotide A in the sequence above becomes substituted for G and albumin synthesis fails to start, or the sixth nucleotide T becomes substituted for G and instead of Asparagine, Glutamine becomes the first amino acid in the chain. Sometimes small variations in protein composition are acceptable, sometimes (in case of antibodies) desirable, and in other instances (histone H4) instantly lethal.

Over 10,000 human diseases are caused by defects in single genes. Single gene disorders, which are also described as monogenic diseases, and sometimes as mutations, are individually very rare but geneticists estimate that each of us carries about a dozen lethal genes in our DNA. It is only due to the fact that each of us has TWO full copies of human genome in each and every cell, that lethal genes cannot reveal them-selves. Having at least one Normal copy of a gene (called wild-type allele) is usually enough to prevent the expression of a mutant gene.

Genetic Defects: Chromosomal AbnormalitiesThere is no smaller defect in human genome than the substitution of one nucleotide out of a few billion. On the other end of the scale are genetic disruptions

DNA

Base Pair

Cytosine Guanine

Thymine Adenine

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of global proportions:

•Polyploid is a term used to describe cells or embryos where the whole genome was dupli-cated, or multiplied several times.

(As mentioned before, the whole human genome is broken into 23 pieces, called chromosomes. One set of 23 chromosomes is called a haploid set, and although it is a full set, containing the whole human genome, only mature oocytes and spermatozoa have haploid set of 23 chromosomes. Each somatic cell and each cell of the human embryo has a diploid set of chromosomes (as always, di- stands for double) thus it is correct to say that humans have 46 chro-mosomes, or 23 pairs of chromosomes.

To complicate things further, there are not 23, but 24 different human chromosomes. Chromosomes 1 through 22 are known by their number and called autosomes, and chromosomes X and Y are known as the sex chromosomes. During fertilization, 23 chromosomes are inherited from the mother and the other 23 chromosomes arrive tightly packed inside the father’s spermatozoon. As a result, a normal male human embryo will have 46 chromosomes: 44 autosomes (or 22 pairs of homologous chromo-somes), one X plus one Y chromosome. A normal female embryo will have 44 autosomes and two X chromosomes. In other words, although it is tradi-

tional and quite correct to say something like “my genome”, the fact remains that “your genome” is actually two full human genomes!)

The lower level of pangenomic disasters, right after whole genome amplification, would be the addition of a whole chromosome:

•Aneuploidy is the term for the cases when an embryo (or a cancer cell) has too many or too few chromosomes, compared to the normal set of 46 chromosomes. Down’s Syndrome, or trisomy of chromosome 21, is the best known, and the most feared case of aneuploidy in humans.

At even lower levels of chromosomal aberrations we shall see the changes in chromosome structure:

•Translocations occur when a piece of one chro-mosome becomes attached to another chromo-some. Reciprocal translocation means that two chromosomes swapped their segments. Robert-sonian translocation means that two acrocentric chromosomes fused to form one chromosome.

•Deletion means that a segment of a chromo-some is missing.

•Inversion means that a segment of chromosome is inverted without changing its original location.

Male Karyotype: Total 46 chromosomes or 22 pairs of homologous chromosomes plus two sex-chromosomes, X and Y.

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TranslocationsWhat are Translocations?One in every 500 newborns is a carrier of chromo-somal translocation. A translocation occurs when two nonhomologous chromosomes exchange their segments. As explained in “Genetics 101” section, out of our 46 chromosomes, we inherited 23 from our mother and 23 from the father. Chromosome 7 from a mother is homologous to the chromosome 7 from a father and nonhomologous to any of the other chromosome.

There are two kinds of translocations: reciprocal and Robertsonian. A reciprocal translocation means that two chromosomes swapped their segments, while in a Robertsonian translocation two acrocentric chromosomes fused to form one big chromosome.

Unless, during DNA segment exchange, the break-point slices through some important gene, there is no loss of genetic information and no harm is done, but the newborn becomes a translocation carrier. Carriers of translocations may never know that they have this genetic condition until the time comes for them to have a baby.

Fertility Problems due to TranslocationsFor illustration purposes we are going to use a carrier of a reciprocal translocation. Starting from the zygote stage, and throughout development, the homologous chromosomes never actually meet. They do reside in one nucleus and they do join a single metaphase plate during cell division. However, at the molecular level they are as far apart as two people in a crowd. The one and only time homologous chromosomes come to close and intimate contact is during Meiosis.

During Meiosis an oocyte has to get rid of half of its genetic material, because otherwise fertilized by sperm it will produce a triploid baby in the first generation, a tetraploid baby in the second, and so on. In a process called synapsis, homologous chro-mosomes are paired together and held by the special synaptonemal complex. Once all 46 chromosomes are paird into 23 pairs or bivalents, the oocyte gets

rid of one full chromosome from each bivalent by extruding it into the 1st Polar Body. This is followed by the 2nd meiotic division with the extrusion of the 2nd Polar Body, but the mechanism of this division is similar to any regular mitosis.

As illustrated in the diagram, this process of meiotic chromosome segregation becomes compli-cated for the translocated chromosomes. Instead of forming a bivalent, chromosomes with a reciprocal translocation, and their normal homologs, form a quadrivalent. When the time comes to extrude half the chromosomes into the 1st Polar Body, all 4 chro-mosomes from the quadrivalent may get extruded, or none, or anything in between. 32 different oocytes may result, as illustrated below.

Of the 32 types of gametes only the first two, high-lighted in green, are capable of producing a healthy baby. The rest are too genetically abnormal to support normal embryo development.

In general, for the carriers of the balanced transloca-tions the chances to have a normal child on their own is 10% to 60%. As a result, PGD is the only alternative to repeated miscarriages and abortions for a parent with a translocation.

Mitosis

Meiosis 1

Normal With Translocation

Mitosis

Meiosis 1

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Embryo Development

Day 0: Oocyte Day 1: Zygote Day 2: Two-Cell Embryo Day 3: 10-Cell Embryo

Day 4: Morula Day 5: Blastocyst Day 6: Hatching Blastocyst Day 7: Hatched Blastocyst

Below are the specifics of preimplantation embryo development in human IVF:

• Day 0 - Oocytes are placed in the same droplet of culture media with spermatozoa. Fertilization takes place at night. Assisted fertilization (ICSI) may be performed for the patients with oligozoo-spermia.

• Day 1 - Fertilized oocytes, now called Zygotes are at the two pronuclei (2PN) stage meaning that oocyte nucleus and sperm nucleus are visible as two separate nuclei inside one cell. By the end of Day 1 the zygote divides (cleaves) into two cells.

• Day 2 - Two- to four-cell stage embryos. This is the so-called “cleavage” stage when the embryo divides (cleaves) to produce more cells, called blastomeres, from the original one-cell zygote.

• Day 3 - The second day of the cleavage stage. The embryo is at the 6- to 16-cell stage. The embryo does not grow in size, so each blasto-mere of an 8-cell embryo is exactly 1/8th the size of the original zygote. Visually unremarkable but an extremely important event takes place at the 8-cell stage, the embryo genome wakes up! The process is called genome activation and marks the point from which embryo development will be controlled by the genes, not an ooplasm.

• Day 4 - The first so-called “morphogenetic event” in embryo development occurs. The 16 to 20+ cell embryo starts compaction to form the Morula.

• Day 5 - The morula starts the process called cavitation and forms a Blastocyst. This is the stage when cell differentiation (triggered already at the morula stage) becomes evident. The outside, Throphectoderm (TE), cells will form the embryonic side of the placenta, and the Inner Cell Mass (ICM) cells will form the embryo proper.

• Day 6 - The Blastocyst expands and hatches from its zona pellucida. The zona pellucida is equivalent to the eggshell in a chicken’s egg; it is formed during oocyte growth and protects the oocyte and then preimplantation embryo from its surrounding.

• Day 7 - The hatched Blastocyst implants into the uterine wall, this is the end of an independent, preimplantation stage of embryo development.

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The PGD Procedure

The Embryo is held by the Holding Pipette.

The Zona Pellucida is punctured by the PZD Tool.

The PZD Tool is pressed and rubbed against the Holding Pipette.

The PZD Tool is rubbed against the Holding Pipette until the Zona is breached.

The embryo is picked-up again by the Holding Pipette.

The Biopsy Pipette is inserted through the slit in the Zona.

The blastomere is aspirated into the Biopsy Pipette.

Preimplantation Genetic Diagnosis (PGD) can only be offered to patients going through an in vitro fertiliza-tion (IVF) cycle.

Embryo BiopsyThe first step in the PGD protocol is embryo biopsy. Embryo biopsy is the process of removing a single cell from a preimplantation human embryo. By analyzing the biopsied cell we can draw conclusions as to the genetic status of the whole embryo. If the cell removed is a male cell, we can be 100% sure that the embryo will eventually develop into a boy, if the same cell also has an extra chromosome 21, we conclude that such embryo will develop into a boy with Down’s Syndrome.

Embryo biopsy can be performed on Day 3, Day 4, or Day 5 after fertilization.

Day 3 BiopsyFor the embryo biopsy an embryologist uses one of three methods to break through the zona pellucida (a glycoprotein layer surrounding the embryo, a.k.a. “zona”):

• Partial zona dissection (PZD) is done mechani-cally using a glass needle. This is the quickest and safest method of performing biopsy. However, this is also the most technically challenging method.

• Acidified solution is used to dissolve a hole in the zona pellucida.

• A Laser is used to burn a slit in the zona.

Once the zona is “opened” an individual blastomere is taken from the embryo using a biopsy pipette which is blunt, flame-polished, and has an inner diameter of about 30-50μm for the safe removal of one blas-tomere. Some embryologists do not feel comfortable

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using a biopsy pipette, they either “flush out” or “squeeze out” one or a few blastomeres through the opening in the zona.

Day 4 BiopsyPerformed in exactly the same way as Day 3 biopsy but the cell loss to the embryo is reduced by the fact that only one out of 16-30 cells is biopsied instead of one out of 6-12.

Day 5 BiopsyBy Day 5 the embryo reaches the blastocyst stage. Biopsy on Day 5 is done with a laser to slice away a cluster of 2 to 10 or more cells. The cells biopsied on day 5 are taken from the trophectoderm (TE), which is the outer layer of the blastocyst. Following the biopsy the blastocyst is usually frozen, but sometimes it is cultured until Day 6 or even Day 7 waiting for the results of genetic analysis.

Embryo Biopsy on Day 3 vs. Day 4 vs. Day 5At ViaGene we strongly recommend embryo biopsy to be performed on Day 3 with Embryo Transfer (ET) on Day 4 (even Day 3, for our local IVF Centers). There is no need to unnecessarily prolong embryo stay in culture, unless a lot of embryos are available for transfer and an extra selection for the “toughest” embryos is possible. It should be noted, though, that being the “toughest” in vitro does not necessarily translate into the most viable. More importantly, being unable to reach the blastocyst stage in vitro does not mean the same for in vivo conditions. Embryos which arrest by Day 5 in the culture media may have survived, if transferred by Day 4. This is especially crucial for the patients of the advanced maternal age (AMA, women over the age of 36) who may only have 1 or 2 embryos available for transfer by Day 4, and potentially none by Day 5.

(Although it might not be relevant to humans, it should be noted that in the Research Laboratories and in animal husbandry preimplantation embryos are transferred back into the oviducts or uteri of their mothers as soon as technically possible. The best results in transgenic mice project was achieved when zygotes were transferred into the ampulla region of the oviducts immediately after transgenic DNA injection into the zygote’s pronucleus.)

It has been argued that Day 5 biopsy avoids the problem of embryo mosaicism by taking 10 or more cells for analysis. However, averaging all biopsied TE cells by WGA for Microarray analysis is not the best way to detect mosaicism. Furthermore, being mostly “sisters” and “granddaughter” blastomeres of a single blastomere, the biopsy of 5-10 TE cells provides us, even if analyzed individually, with genetics of the progeny of a single blastomere of the 8-cell embryo. Numerous attempts to show the effects of ooplasm polarization have shown us, if anything, that cells do

not migrate at the preimplantation stage.

At ViaGene, when we do 24-chromosome aneuploidy testing for IVF Centers concerned with embryo mosaicism or the myth of self-correction, we offer Day 4 biopsy of two cells from the opposite sides of the morula. This is less damaging than taking 5-10 cells one day later and also gives us a better chance of detecting mosaicism than any other PGD technique.

Does biopsy damage the embryo?Biopsy itself, if performed by a professional, poses no risk to the embryo. However, one cell has to be removed and we should never forget that Day 3 embryos have only 8 cells. One blastomere out of 8 may seem like a lot, but this does not mean that later in development an embryo would miss one-eighth of its size, or some vital organ. The preim-plantation human embryo is able to compensate for one missing cell. This is a benefit of being among the species, which use the so-called regulative type of development. In a human embryo, the fate of the blastomere is not preprogrammed by the egg. Instead, the function of the cell is determined and readjusted according to it’s the position in the devel-oping embryo.

It must be stressed that the benefit of selecting out genetically abnormal embryos must outweigh the negative effect of a single cell removal. It has been proven that unless each step of the PGD procedure is done by professionals, the results may be less than beneficial, and sometimes even damaging to the outcome of the IVF cycle. This is why it is still important to choose a reliable PGD laboratory and a qualified embryologist to perform the biopsy procedure.

Biopsied Samples in PCR Tubes

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PGD Technologies24 Chromosome Capable

FISH aCGH SNP OGS EGS

First Clinical Use 1991 2003 2008 2009 2011

24 Chromosome Aneuploidy Screening no yes yes yes no

Detects Haploid, Triploid, & Polyploid Embryos yes no yes/no yes yes

Detects Abnormal Karyokinesis yes no no yes yes

Detects Mitotic Trisomies & Mosaicism yes yes no yes yes

Detects Uniparental Disomies no no yes no no

Ease of Sample Preparation difficult easy easy dificult easiest

Sample Preparation Procedure fixation tubing tubing fixation sliding

Procedure Duration 3-12 hrs 12 hrs 12 hrs 6 hrs 1 hr

After biopsy, the removed cells must be analyzed using one of the available PGD methods. A list detailing the currently used PGD technologies clearly shows that no technology is perfect, each one has its specific drawbacks and limitations. For instance, while we believe that OGS technology is more reliable when it comes to aneuploidy testing, Microarray-based PGD methods have a potential for providing an unlimited amount of genetic information from a single cell.

FISHDuring Fluorescent In Situ Hybridization (FISH) the biopsied blastomere is fixed so that the cytoplasm is washed away leaving only the nucleus attached to the slide. Then the probes are hybridized to the nucleus. The probes are the strands of DNA identical to some specific regions of human chromosomes. For example, the probe for chromosome 18 has the same polynucleotide sequence as a centromeric DNA of chromosome 18. The probes are labeled with fluorophores and the results of FISH are detected by

a fluorescent microscope. Standard FISH is capable of detecting up to 12 chromosomes in a few hours.

DNA Microarray TechnologyAlso known as DNA Chips, this is basically FISH technology turned upside-down. Instead of hybrid-izing labeled DNA probes to the nucleus fixed on the slide, the probes are pre-attached to the slide (thus the name, array of DNA probes) and DNA from the biopsied cell nucleus is restricted into pieces, labeled by fluorophores and then hybridized to the microar-ray. The concept of Fluorescent In-Situ Hybridization (FISH) stays the same, but now we are producing fluorophore-labeled probes out of the cell nucleus to hybridize these “embryonic” probes to the microar-ray. After hybridization the microarray is scanned by a laser and analyzed by a computer. Microarrays are capable of analyzing all 24 human chromosomes in less than 24 hours.

As the name implies, microarray is small, but an individual cell nucleus is still smaller! Thus the First Challenge: the need to amplify DNA from a single nucleus using polymerase chain reaction (PCR) specifically tailored for the whole genome amplifica-tion (WGA). There are few ways for the WGA, but none of them is perfect: some parts of the whole genome are always overamplified and others are underrepresented. This leads to pseudo-duplications or pseudo-deletions following microarray analysis. The only reliable way to avoid this problem is to have more than one cell for PGD analysis. For this reason, microarray-based PGD is especially recommended for patients who choose blastocyst biopsy.

The Second Challenge is to make sense out of the hybridized microarray, because just hybridizing “embryonic” probes to an array is not very informa-Normal boy analyzed by 5-Chromosome FISH

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If the tested nucleus does not have a single chromo-some 21 then the respective spots on the microarray will fluoresce with Green only.

aCGH made its debut in the PGD field with BAC (bacterial artificial chromosomes) based arrays, designed specifically for PGD use. Quite recently the leaders in microarray technology took notice of PGD and entered the field by either buying the original PGD-oriented microarray technology or by offering highly competitive products of their own. One of these new developments is the so-called High Density Multiplex arrays.

At ViaGene we are using custom 8x60k SurePrint G3 Human CGH arrays from Agilent Technologies. The numbers mean that a single microscope slide (the base for all microarrays on the market) has 8 individual arrays printed on it, with each array consisting of 60,000 “spots”. One individual spot has millions of copies of long oligonucleotide printed on it, oligonucleotide specific for some unique region of human chromosome. The arrays are optimized for PGD aneuploidy screening, they were developed by ViaGene and produced by Agilent Technologies specifically for PGD aneuploidy testing.

SNP MicroarraySNP stands for Single Nucleotide Polymorphism. The concept of SNP is as different from aCGH as aCGH is different from FISH. SNP is a variation of single nucleotides in human population and is the most frequent type of variation in the genome. SNP Micro-arrays are made of very short DNA probes, called short oligonucleotides, these are the only probes capable of distinguishing the variants of SNPs and thus making a call regarding the genetics of a cell.

The technology is very new, very powerful and poten-tially the most promising. It is not yet known how SNP Microarray compares to the more established aCGH technologies used for PGD. A controversy is also raging regarding the need of “parental support technology” to make SNP microarrays applicable to PGD.

tive. If the analyzed cell is totally missing chromo-somes 21 (so-called Nullisomy 21) microarray will definitely reveal this by showing no signals at the spots specific for chromosome 21. However, probes made from a cell with trisomy or monosomy for chro-mosome 21 will hybridize to microarray as readily as probes made from a normal cell: all array spots will be covered and be fluorescing during scanning.

There are two conceptually different ways of getting aneuploidy revealed by microarrays: using tested and proven CGH (Comparative Genome Hybridiza-tion) or the new and promising SNP (Single Nucleo-tide Polymorphism) technologies. As a result, there are two types of microarrays, aCGH and SNP.

Array CGHaCGH stands for array Comparative Genome Hybrid-ization. Following WGA, sample DNA is labeled with Red fluorophore and applied to aCGH microarray along with normal male DNA labeled with Green fluorophore. Both tested DNA in Red and control DNA in Green compete with each other for DNA targets on the microarray. By analyzing the color of each spot, it is possible to determine if the analyzed nucleus has the same amount of DNA as the normal control (thus also being normal) or outperforms the control DNA (thus has an extra genetic material, like trisomy 21).

Agilent Technologies CGH Microarray

24 Chromosomes analyzed with aCGH

A

A

T

A

C

A

C

T

T

G

AT

G

T

G

CGC

G

GC

A

A

T

A

C

A

C

T

T

G

AT

G

T

G

TAC

G

GC

SNP

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OGSIn Oligonucleotide Genome Screening (OGS), as in FISH, the nucleus is used as a target for the fluorophore-labeled probes. However, the similar-ity to FISH ends here. The probes themselves are completely different: OGS uses oligonucleotide probes developed originally for microarray use. The protocol for target pretreatment, hybridization and wash was totally reinvented in order to completely eliminate the unavoidable drawbacks of FISH, namely “failed hybridizations” and “target degrada-tion”. Unlike FISH and Microarray, which require 3-12 and 6-16 hours of hybridization, hybridization time for OGS is just 5 minutes per cycle.

Because OGS does not depend on PCR/WGA, it is not affected by DNA contamination and thus provides more reliable results. Unlike Microarray-based PGD, OGS easily detects triploids and polyploids, genetic defects responsible for almost 10% of genetically abnormal first-trimester abortions. Although less time-consuming than Microarray, the procedure is much more labor-intensive and requires significantly more training. With OGS technology aneuploidy screening of all 24 chromosomes takes less than 6 hours.

EGSExpress Genome Screening (EGS) was specifically designed to be faster and less complicated than any other PGD technology on the market today. EGS relies on the techniques developed for OGS for the chromosomal analysis, but there are significant differences in the sample preparation procedure and the speed of analysis. Where OGS requires a difficult to master fixation procedure, which limits its availability throughout the country, EGS uses a new ‘sliding’ procedure which can be easily taught to any embryologist within minutes. This allows clinics who

were previously limited to offering only Microarray based PGD to now also offer EGS.

However, the speed of the EGS procedure is what really sets it apart. EGS can provide results 12 times faster than any of the currently available Microarray techniques. In most cases EGS results are available in about an hour, while Microarray based methods require 12 or more hours.

ViaGene offers EGS-XY, specifically designed for gender selection cases, and EGS-5, designed to screen for ALL types of congenital defects related to chromosomal euploidy, including Down’s Syndrome, Patau’s Syndrome, Edwards Syndrome, Trisomy X, Turner Syndrome, XYY Syndrome, and Klinefelter Syndrome.

Future of PGDMicroarray-based PGD is not “The Ultimate PGD,” it is just the latest idea in the PGD field, and the technol-ogy itself is still in its Research and Development (R&D) phase. Moreover, it is not one, but actually a few technologies with the “Microarray” name tag. At this time we are witnessing a fierce competition between different types of Microarrays, only time will tell whether it is aCGH, or SNP-based microarrays, BAC or high-density oligos, or parental support tech-nology or karyomapping that will become the next PGD standard. There are indications that Genome Sequencing may be the Next Best Thing. Also, lets not forget that the same companies making microar-rays are also developing new generations of better probes for the “golden standard” in PGD: the tried and true FISH. This means that our own OGS and EGS methods are constantly improving!

24 Chromosomes analyzed with OGS

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It is often stated that the most common causes for PGD misdiagnosis are outdated PGD technologies, embryo mosaicism, and embryo self-correction.

Outdated TechnologyBeing in the PGD field since its inception we would like to comment on the “outdated” part. Each new technology becomes “standard technology” after a few years of successful use by its inventors. At this point, it attracts attention of the people not necessarily qualified for its use, especially if training and certification programs have not been set up. If the number of the so-called “in-house PGD labs” outweigh the output from the “Reference Laborato-ries” the results become quite dramatic. A few years back rates of 40-50% misdiagnosis were reported by some PGD practitioners. This was followed by Prospective Randomized Studies reporting shockingly low pregnancy rates from the use of FISH technology for PGD. Luckily for the concept of PGD, those who actually developed PGD technologies, have come up with new PGD procedures and there is hope that the cycle will not repeat itself.

MosaicismThe presence of cells in an embryo which have different genotypes is called Mosaicism. Some newborns with Down’s Syndrome are actually mosaics, they have both normal and aneuploid cells. By analyzing a few hundred or a few thousand cells it is possible to determine the level of mosaicism in a specific tissue. Not so with PGD! Analysis of a single cell tells us nothing about embryo mosaicism, and if the embryo is actually a mosaic, then PGD results become misleading.

Self-CorrectionAnother rare genetic phenomenon is Self-Correction. The proof of concept comes from the unusual genetic condition called uniparental disomy when an embryo has two copies of a chromosome from one parent and no copies from the other parent. The most reasonable explanation for this odd condition is “self-correction.” Take Angelman Syndrome as an example. Suppose originally an embryo has a trisomy for chromosome 15, having 2 chromosomes 15 from the mother, and one from the father. Through self-correction the embryo extrudes one extra chromosome, the one inherited from the father. Although the embryo is left with the normal karyotype, both remaining chromo-somes 15 come from the mother. This results in a baby with Angelman Syndrome. Proponents of “self-correction” go one step further. They hypothesize that aneuploid preimplantation embryos actively correct their chromosomal abnormalities. We do not know how they explain restoration of euploidy by the embryos with chromosomal monosomy.

PGD ReliabilityEmbryo mosaicism is a well established fact of preimplantation human development. Luckily for the concept of PGD, it affects almost exclusively the embryos of suboptimal quality, which rarely progress to the blastocyst stage. At ViaGene we do follow-up analysis for blastocysts found aneuploid by PGD. This is why we called “self-correction” a myth. The results obtained in our laboratory contradict the hypothesis.

We believe that 99% of “mosaicism” and “self-correction” explanations/excuses stem from the fact that PGD laboratories mark all “no results” or “incon-clusive PGD results” as “abnormal.” This is one of the rules coming from the early nineties when PGD was done exclusively for single-gene disorder cases. The policy of “not proven normal means abnormal” makes perfect sense for, say, Cystic Fibrosis or chromosomal translocation carriers. However, when applied to aneuploidy testing, such approach leads to unacceptably high rates of false-positive results.

At present our OGS method for 24-chromosome aneuploidy screening is confidently below 2% of false-positives level. Needless to say, we have never had a false negative (abnormal embryo marked as normal) PGD result. Our goal is to have the level of false-positive PGD results at zero. The main reason for our concern with false-positive results is that the patients who need aneuploidy testing the most, those in the 38-44 age group, have only one or two genetically normal (euploid) embryos for transfer. To select these embryos out because of the high false-positive rate means taking the couple’s only chance of having a baby.

Other causes for misdiagnosis include unavoidable technical limitations of different PGD techniques. Improper blastomere fixation for FISH and OGS, allele drop-out (ADO) for single-gene disorders, preferential amplification during WGA and sample contamination with extragenous DNA (ranging from polar bodies to dead sperm heads to airborn DNA) for Microarray-based PGD are just a few examples. Paraphrasing the saying of the leading biologist of our generation: “however many ways there may be of being alive, it is certain that there are vastly more ways of being dead” we say: “there is an infinite number of ways of doing PGD wrong!”

In general, PGD carries with it an approximately 1% chance for misdiagnosis, though at ViaGene, we are proud to say that in 10 years with have had zero occurrences of misdiagnosis, out of thousands of embryos analyzed. When considering PGD as an integral part of IVF cycle, each couple should weigh potential benefits and risks of the procedure and decide for themselves.

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ConclusionWe have talked about some of the most common reasons why patients choose PGD, including aneuploidy testing, single gene disorders, and translocations. We covered the genetic processes which sometimes break down, resulting in the need for PGD, and the latest available techniques for detecting these genetic abnormalities, and assuring a healthy pregnancy. We hope that this booklet was informative and helpful. If you have any questions about PGD, please do not hesitate to ask your doctor or contact ViaGene Fertility directly by emailing to questions@viagenefertility.com

About the AuthorThis booklet was written by Dr. Sergei Evsikov and edited by ViaGene staff. Dr. Evsikov is one of the world’s foremost embryologists specializing in pre-implantation genetic diagnosis (PGD) and is the Director of ViaGene Fertility Center in Malibu, CA.

Previously, he has held positions as Director of Preim-plantation Genetics and Research at ART Reproductive Center in Beverly Hills, and Director of Experimental Embryology at the Reproductive Genetics Institute in Chicago, which, with Dr. Evsikov’s help, became the first facility in the United States to perform clinical PGD cases.

Before immigrating to the United States, Dr. Evsikov was the Senior Scientist and head of Experimental Embryology at the National Academy of Sciences of the Ukraine. Dr. Evsikov has been involved in most of the latest research in PGD, developing techniques and protocols that have become standard in the laboratory. He is the author or co-author of numerous articles and is a frequent speaker at conferences throughout the world.

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PGD TerminologyNormal Embryo: Humans have 46 chromosomes: 22 pairs of autosomes, and two sex chromosomes, X+X or X+Y. Any chromosome combination other than 46,XX (Normal Female) or 46,XY (Normal Male) is considered ABNORMAL.

Monosomy: A monosomy is an absence of one chromosome. A monosomy of any autosome is lethal in humans, only a few babies with autosomal monosomy have survived beyond birth, they all had monosomy of chromosome 21.

Trisomy: A trisomy is a presence of one extra chromosome. The most common among newborn is a trisomy of chromosome 21, leading to Down’s Syndrome. According to the latest published National Vital Statistics report, out of 2,748,302 births, 1,298 had Down’s Syndrome and 1,093 had other chro-mosomal anomalies. Among these “other chromo-somal anomalies” the most common is an abnormal number of sex chromosomes. Per each 100,000 recognized pregnancies, around 1,400 abort due to an abnormal number of sex chromosomes, 100-200 boys are born with Kleinfelter syndrome (instead of XY they have XXY, XXXY, XXYY, or even XXXXY sets of sex chromosomes) and about 50 girls are born with Turner syndrome (instead of XX they have X, XXX, or XXXX).

Other common trisomies include trisomy 18 (Edward’s Syndrome) and 13 (Patau’s Syndrome). Frequencies of either syndrome range from 1 in 2,000 to 1 in 15,000. The only other chromosomes noticeably affecting the outcome of established pregnancies are chromosomes 16 (trisomy 16 is found in 1,229 among 15,000 spontaneous abortions) and 22 (trisomy 22 is found in 424 out of 15,000 spontaneous abortions). Aneuploidy for any other chromosome is lethal at the very first stages of embryo development, before a pregnancy can even be established.

Haploid: Haploid embryos have only one set of 23 chromosomes. They originate from parthenogeneti-cally activated oocytes, sometimes from prematurely dividing zygotes.

Triploid, Tetraploid, or Polyploid embryos are those having full extra sets of all 23 chromosomes. These embryos originate from an oocyte fertilized by two spermatozoa, by diploid spermatozoon, or from an oocyte which failed to extrude the second polar body. Polyploidization also occurs during embryo cleavage, however, at the blastocyst stage it is a normal step in trophectoderm formation.

Chaotic Cleavage means that during mitosis, when the zygote and, subsequently, blastomeres divide

into two daughter cells, the chromosomes segregate between two sister blastomeres randomly or chaoti-cally, causing multiple monosomys and trisomys. Most of these embryos are also morphologically abnormal and very few of them progress beyond the cleavage stage. Such embryos are usually marked as having “Complex Abnormalities.”

Embryo Mosaicism: Mosaicism is a result of mitotic error and as such arises during embryo cleavage, when one of the blastomeres divides into two geneti-cally unequal daughter blastomeres. This may lead to an embryo having both normal and abnormal cells, i.e., mosaic embryo. Cells with autosomal monosomy may be selected out during further embryo development; however, if their population rises above some critical level, the embryo will die before or shortly after implantation. If an embryo is suspected of having genetically normal cells and cells with autosomal monosomy, such embryos should be avoided during embryo transfer. Since mosaic embryos with monosomy of chromosome 21 may result in a live birth, such mosaic embryos are marked as abnormal.

Embryo with genetically normal cells and cells with autosomal trisomy may develop into an abnormal mosaic baby. Some newborns with Down’s, Edward’s, and Patau’s Syndrome are actually mosaics. Mosaic embryos with trisomies should never be considered for transfer.

Multinucleate/Anucleate Blastomere: Some embryos may be revealed as abnormal even prior to genetic testing, during embryo biopsy or after blastomere fixation. If a single blastomere has more than one nucleus, it is called a multinucleate blas-tomere. Even if each nucleus (separated in the PGD Report by square brackets) is genetically normal, or if they add to a normal set of chromosomes, the corresponding embryo may be genetically abnormal. Multinucleation indicates some gross abnormalities in the timing between cell division (cytokinesis) and nuclear division (karyokinesis). If cleavage results in one blastomere retaining both nuclei, then its ‘sister blastomere’ will have none. Absence of a nucleus (anucleate blastomere) may be similar in its origins to multinucleation, but it may also be considered as an extreme example of embryo fragmentation. Although anucleate blastomeres cannot give any indications as to the embryo’s genetic background, it should be noted that their presence lowers embryo viability. If multiple morphologically normal blastomeres were analyzed, and none of them had a nucleus, such an embryo may be considered not viable due to gross errors in embryo cleavage.

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ViaGene Fertility, LLC29169 Heathercliff Rd., Suite 213Malibu, CA 90265

Tel: (888) 842-6961Fax: (888) 842-7297Email: pgdivf@viagenefertility.comWeb: www.viagenefertility.com