Genetics

Pre-implantation Genetic Diagnosis

Homapage / Pre-implantation Genetic Diagnosis

From the same embryonic cell or tissue, it is possible to diagnose different mutations, HLA compatibility and to screen 24 chromosomes for aneuploidy at the same time

What is preimplantation genetic diagnosis (PGD)?

Preimplantation genetic diagnosis (PGD) is used to identify chromosomal abnormalities or genes responsible for genetic defects in embryos created through in vitro fertilization (IVF) before pregnancy.

When may PGD be advised?

PGD can be performed for 

  • Monogenic Disorders 
  • Preimplantation HLA typing for Haemopoietic Stem Cell Transplantation (HSCT)
  • Numerical and Structural Chromosomal Abnormalities 

What are the advantages of PGD over prenatal diagnosis (diagnosis during pregnancy)?

Prenatal diagnosis tests are available for couples who have high risk of transmitting a disease. Unfortunately, if the fetus is found to be affected by a genetic disorder, the couple may then be faced with the psychological and financial burden of the termination of the pregnancy. PGD offers couples the possibility of having disease-free embryos selected and transferred and thus of having healthy children without the complications of a termination.

How long has PGD been in use?

PGD was first performed by Alan Handyside and his colleagues in 1989, using PCR (polymerase chain reaction) technique for a sex-linked disorder in cleavage stage embryos. In the beginning of 90s, Kuliev and Verlinsky developed another technique in which polar bodies are analyzed. In Turkey, PGD for aneuploidy screening, PGD for single gene disorders and preimplantation HLA typing were first performed by Dr. Kahraman.

How can PGD be performed?

PGD can be performed via biopsy of polar bodies from oocytes or blastomere and trophectoderm tissues from embryos.

At which developmental stages can a biopsy be performed?

Biopsy of embryos can be performed at 3 different stages of embryonic development: 

  • Polar body analysis prior to or after fertilization (figure 1A).
  • Blastomere analysis at cleavage stage (figure 1B)
  • Trophectoderm tissue analysis at blastocyst stage (figure 1C)

These three methods can be used individually or in combination to increase the reliability of PGD. The experience of the laboratory staff is of key importance in successful biopsy and PGD procedures.

Figure 1: Polar body biopsy (A), blastomere biopsy (B), trophectoderm biopsy (C). (Images from Istanbul Memorial Hospital, IVF Laboratory)

What is the advantage of polar body analysis? How is it performed? 

During ovulation and after fertilization, oocytes undergo cell divisions. After these divisions, two byproducts, named polar bodies (PB), are formed. The two polar bodies can be removed sequentially or simultaneously.

The majority of chromosomal abnormalities occur during these two cell division processes. It is possible to eliminate chromosomally abnormal oocytes by analyzing PBs.

In what conditions polar body analysis is applicable? 

For couples with advanced maternal age, maternal translocation carriers, or maternally transmitted genetic conditions polar body analysis could be performed. 

What is the impact of chromosomal analysis in polar body samples in woman with advanced maternal age?

There is a strong correlation with maternal age and aneuploidies in embryos. Especially half of the oocytes of women aged 35 and over carry chromosomal abnormalities. In addition to other trisomies, the incidence of babies born with trisomy 21 (Down syndrome) increases dramatically by advancing maternal age (Table I). For that reason, analysis of oocytes through PB is especially valuable for women with diminished ovarian reserve and of advanced maternal age. 

Table I: The risk becomes more pronounced after the age of 35. For example, a. woman aged 44 has a 40 times the risk of conceiving a baby with Down Syndrome compared to young women

What is the advantage of blastomere analysis? How is it performed?

Cleavage-stage biopsy is generally performed on embryos with 6-8 cells on the 3rd day of embryonic development.  A hole is made in the zona pellucida and one or two blastomeres containing a nucleus are gently aspirated or extruded through the opening (Figure 1B). The main advantage of cleavage-stage biopsy over PB analysis is that the genetic contribution of both parents can be studied.  

When is blastomere biopsy advisable?

This method can be used for; 

  • Maternally/paternally inherited single gene disorders
  • HLA typing
  • Chromosomal rearrangements (translocations and inversions)
  • Advanced maternal age
  • Repeated implantation failures
  • Recurrent pregnancy losses
  • Severe male factor infertility

What are the advantages of trophectoderm tissue analysis? How is it performed?

Blastocyst stage biopsy is performed on day 5 of embryonic development by the excision of approximately five cells from the trophectoderm tissue without harming the inner cell mass (Figure 1C). This has become the most favored method in IVF laboratories since it increases implantation rates. It has several advantages over the other two biopsy methods: the biopsy of multiple cells facilitates diagnostic procedures, eliminating some problems that can occur in single cell analysis, such as reduced amplification efficiency and allele drop out (ADO). It does not remove a critical volume of the embryo and does not harm the inner cell mass from which the fetus will develop. Another advantage is that culturing embryos to blastocyst stage automatically eliminates a significant portion of chromosomally abnormal embryos, and so decreases the cost of the test by decreasing the number of embryos analyzed.  Finally,  through the transfer of fewer embryos, but which have higher implantation potential,  multiple pregnancies are avoided.

PGD for monogenic disorders

What are monogenic disorders?

A monogenic disorder is the result of a single mutation in a gene responsible for the disease. There are estimated to be over 4,000 human diseases caused by single gene defects.

What is a mutation?

Mutations are changes in the DNA sequence of a gene. There are estimated to be 30,000 different genes in the human genome. Mutations in these genes are responsible for genetic diseases and they are transferable from generation to generation, with a broad range of inheritability.

How are monogenic disorders inherited?

There are different mechanisms of inheritance:

  • Autosomal recessive (Figure 2a)
  • Autosomal dominant (Figure 2b)
  • X-linked
  • Y-linked
  • Mitochondrial

What are the most common autosomal recessive disorders?

The most frequently diagnosed are beta-thalassemia, cystic fibrosis, sickle cell anemia and spinal muscular atrophy.

What are the most common autosomal dominant disorders?

The most common dominant diseases are myotonic dystrophy and Huntington's disease.

What are the most common X-linked disorders?

Haemophilia A, fragile X syndrome and Duchenne muscular dystrophy are some of the most frequently diagnosed X-linked diseases.

Figure 2A: Autosomal recessive inheritance
Figure 2B: Autosomal dominant inheritance

For what type of monogenic disorders is PGD applicable?

In Istanbul Memorial Hospital, preimplantation genetic diagnosis can be performed to identify any single gene disorders for which molecular testing is possible.  

For which diseases has Istanbul Memorial Hospital performed PGD and HLA Typing?

Istanbul Memorial Hospital has performed PGD and HLA typing for a variety of different types of disorders. A list of these, which continues to grow, can be found in Tables II and III.

Is it possible to diagnose two different mutations related with different disorders at the same time?

It is possible to identify the presence of two different mutations or even mutations related to two different disorders. Furthermore, from the same embryonic cell or tissue, it is possible to diagnose different mutations, HLA compatibility and to screen 24 chromosomes for aneuploidy at the same time. 

Table II: Some of the monogenic disorders for which PGD has been performed in Istanbul Memorial Hospital

Adrenoleukodystrophy

Arthrogryposis-Renal dysfunction-Cholestasis (ARC) syndrome

Bartter syndrome

Becker muscular dystrophy

Beta Thalassemia

Breast Cancer

Charcot Marie Tooth (CMT)

Citrullinemia

Co-enzyme Q deficiency

Congenital adrenal hyperplasia

Congenital deafness

Congenital factor VII deficiency

Cystic fibrosis

Delta beta talassemia

Donohue syndrome

Duschenne Muscular Dystrophy

Ehler-Danlos Syndrome (EDS) Type VIIC

Epidermolysis bullosa simplex

Facioscapulohumeral muscular dystrophy (FSHD)

Familial Hemophagocytic lymphohistiocytosis

Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis

Familial Mediterranean Fever

Fanconi Anemia

Fragile X syndrome

Fraser Syndrome

Galactosemia

Glucose-6-phosphate dehydrogenase (G6PD) deficiency

Hereditary multiple exostoses

Hunter Syndrome

Huntington's disease

Hyper IgM syndrome

Hypohidrotic ectodermal dysplasia

Hypomyelination and congenital cataract

Infantile neuroaxonal dystrophy

Jubert Syndrome

Keratitis–ichthyosis–deafness (KID) syndrome

     

Krabbe Disease

Lafora Disease

Leber's congenital amaurosis

Li–Fraumeni syndrome

Limb-girdle muscular dystrophy

Maple syrup urine disease

metachromatic leukodystrophy

MTHFR deficiency

Mucolipidosis Type I 

Mucopolysaccharidosis Type I (Hurler Syndrome)

Mucopolysaccharidosis Type IIIA

Mucopolysaccharidosis Type IIIB

Mucopolysaccharidosis Type VI

Myotonic Dystrophy

Nemaline myopathy

Neurofibromatosis

Niemann–Pick disease

Nonketotic hyperglycinemia

Osteogenesis imperfecta

Osteopetrosis

Phenylketonuria

Polycystic kidney disease

Pompe disease

Propionic acidemia

Retinoblastoma

Sickle Cell Disease

Spastic Paraplegia Type III

Spastic Paraplegia Type V

Spinal Muscular Atrophy

Spinocerebellar ataxia type 2 (SCA2)

Tay–Sachs disease

TNF receptor associated periodic syndrome (TRAPS)

Tuberous sclerosis

Usher Syndrome Type 1B

Zelweger syndrome

     
 

PGD analysis for beta-thalassemia, alfa-mannosidosis and cystic fibrosis have been performed for the first time in Turkey by Dr. Semra Kahraman and her team in Istanbul Memorial Hospital (IMH). Furthermore, PGD for Bartter Syndrome has been performed for the first time in the world in IMH. 

How many different conditions were diagnosed in embryos in Istanbul Memorial Hospital Reproductive Genetics Center? 

To date 277 PGD cycles were performed for 196 couples carrying 71 different genetic disorders.  The indications included both common diseases such as beta thalassemia and rare disorders such as Ehler Danlos Syndrome Type VIIC (Dermatoparaxis), Co-enzyme Q deficiency,  Bartter Syndrome, Fraser Syndrome, Krabbe Disease and Lafora Disease (table II). 

What are the chances of achieving pregnancy in treatment cycles?

Embryo transfer was performed in 252 cycles and pregnancy was achieved in 45% of these cycles.  109 healthy babies were born and several more have not yet reached to term.

Preimplantation HLA matching

What is preimplantation HLA typing?

PGD can be used in combination with HLA typing. In this method, embryos that are both mutation free and HLA identical are selected and transferred to the mother. Stem cells from the resulting baby’s umbilical cord blood or bone marrow can be used for a sibling affected by a genetic disorder and in need of hematopoietic stem cell transplantation (HSCT).

What is allogenic HSCT and are there any advantages of HSCT from a related donor over non-related donors?

aHSCT from a related HLA identical donor is currently the only proven cure for some of the inherited and acquired diseases. HSCT with alternative donors is associated with higher morbidity and mortality.

What is the probability of finding a HLA identical donor in relatives and non-relatives?

The probability of finding an HLA identical related sibling is low. Only one-third of patients can find HLA-identical sibling. If there is only one sibling in the family, the probability of being HLA identical is only 25% whereas in presence of two sibling, this rate is 43.7%, and overall possibility is 30-36%. As for unrelated donors, there is a limited availability of finding a donor from national or international registries. 

For which type of disorders HSCT following HLA typing could be used as a cure?

HLA typing is performed for two different indications. The first indication is PGD for HLA typing together with mutation analysis for a single gene disorders such as beta-thalassemia. PGD has also been performed for HLA typing-only for couples with a child who has an acquired disease such as leukemia. A wide range of malignant and non-malignant diseases can be treated by aHSCT.

Beta-thalassemia Acute Myeloid Leukemia 
Alfa Mannosidosis  Acute Lymphoid Leukemia
Cd3 protein deficiency  Aplastic Anemia
Diamond Blackfan Anemia  Burkitts Lymphoma
Fanconi Anemia  Histiocytosis
Gaucher Disease  Chronic Myeloid Leukemia
Hurler Syndrome (mucopolysaccaridosis)  Myelodysplastic Syndrome
Hyper IgD Syndrome  Non-Hodgkin's Lymphoma
Sickle cell anemia  
Thrombocytopenia  
Wiskott Aldrich Syndrome  
X-Adrenoleukodystrophy   

When and where was it first performed?

The first PGD with HLA matching was reported for Fanconi anemia by Yuri Verlinsky and his colleques in Chicago, USA, in 2000, making possible the successful hematopoietic reconstitution in an affected sibling by transplantation of stem cells. In Turkey, for the first time, HLA matching in combination with PGD was performed by Dr. Semra Kahraman and her team in 2003. 

What are the chances of finding an HLA matched embryo?

The theoretical probability of finding an HLA identical embryo in cases of acquired diseases is 25% (1/4), whereas the probability of finding an embryo that is both HLA identical and mutation free in cases of single gene disorders is approximately 18% (3/16). Because of the low number of embryos suitable for transfer, the number of oocytes generated through IVF is of key importance. To achieve a sufficient number of embryos of high quality, well designed stimulation protocols must be followed for each patient and developing follicles must be closely monitored.

Table III: Some of the disorders for which HLA typing in combination with PGD has been performed in Istanbul Memorial Hospital

Beta-thalassemia  
Alfa Mannosidosis  
Cd3 protein deficiency  
Diamond Blackfan Anemia  
Fanconi Anemia  
Gaucher Disease  
Hurler Syndrome (mucopolysaccaridosis)  
Hyper IgD Syndrome  
Sickle cell anemia  
Thrombocytopenia  
Wiskott Aldrich Syndrome  
X-Adrenoleukodystrophy   

HLA typing-only for acquired diseases

Acute Myeloid Leukemia  
Acute Lymphoid Leukemia  
Aplastic Anemia  
Burkitts Lymphoma  
Histiocytosis  
Chronic Myeloid Leukemia  
Myelodysplastic Syndrome  
Non-Hodgkin's Lymphoma  

 

 

How many patients requiring HLA matched donor were treated in Istanbul Memorial Hospital?

Until March 2012, 203 patients were treated in 400 cycles, which resulted in more than 93 pregnancies and 82 healthy babies, a dozen of pregnancies are still ongoing and have not yet reached to term. To date 35 children have been cured via stem cell transplantation from HLA matched siblings and 17 more children are awaiting an appropriate time for transplantation. 

What are the chances of achieving pregnancy?

Although the chance of finding a transferable embryo is very low, clinical pregnancy and live birth rates are quite high when at least one embryo is transferred. The clinical pregnancy rate in the overall group is approximately 37%.

What are the chances of achieving pregnancy?

Although the chance of finding a transferable embryo is very low, clinical pregnancy and live birth rates are quite high when at least one embryo is transferred.

How are PGD and HLA typing carried out?

There are three stages:

What happens during the set up procedure?

Prior to preimplantation HLA typing, a set up study is performed for every family. Set up procedure includes consultation with a clinical geneticist, confirmations of familial mutations, identifying the haplotypes of the mother, father and affected child and designing specific primers for PGD study (Figure 3). 

What tests are performed during the set up process?

Peripheral blood samples of the mother, father, affected child, and when available of other family members, such as an unaffected child or grandparents, are collected. The genomic DNA is isolated from these samples and subjected to haplotype analysis using STR markers to ensure the presence of enough informative markers which will be used in the PGD study.  For each family at least 12 heterozygous markers spanning the HLA-A, HLA-B, HLA-C, HLA-DR,HLA-DQ regions (HLA Classes I, II, and III) are selected.. This set-up procedure may take 3-8 weeks depending on the mutation being tested for.

Figure 3: Summary of set up process

Should couples undergo IVF treatment even if they are not infertile?

Yes. Couples at risk of transmitting a genetic disease should take part in and IVF program to produce embryos which will then be selected according to their genetic status. After controlled ovarian hyperstimulation, oocytes are fertilized in the IVF laboratory with sperms collected from the father. After fertilization, polar bodies, blastomeres or trophectoderm cells are tested for mutations in the genetic laboratory using single cell analysis methods. 

What is special about preimplantation genetic diagnosis?

Preimplantation genetic diagnosis uses single cell PCR techniques. With this method as little as 6 ρg of DNA from only one cell can be amplified to millions of copies in several hours for analysis of the genetic status of the embryo.

Figure 4: Single Cell Analysis Steps

What are the main steps in PGD?

First, biopsied cells are put into tubes containing lysis buffer (Figure 4). 

Cells are lysed by incubation at 65Cº for 10 minutes in a sterile PCR tube containing 5 µl of lysis buffer. 

After lysis reaction, DNA testing is performed by two rounds of PCR reactions. After the second PCR reaction, amplified STR markers are subjected to capillary electrophoresis in the sequencer machine. Each embryo is analysed together with the results of the mother, father and affected child to identify embryos compatible with the affected child (Figure 5).  

Figure 5: The arrows show the inheritance pattern of the alleles in the affected child and the compatible embryo. 

Why choose Istanbul Memorial hospital for PGD and HLA typing?

Istanbul Memorial Hospital IVF and Reproductive Genetics Laboratory is one of the leading centers in the world for PGD and HLA typing. Through the work of Dr. Kahraman and her team in Istanbul, dozens of HLA compatible babies have saved the lives of  their siblings with their cord blood or bone marrow cells. To date, cord blood or bone marrow transplantation has been done for beta-thalassemia, sickle cell anemia, Wiskott-Aldrich Syndrome, Fanconi Anemia, Glanzmann's Thrombasthenia, X-adrenoleukodystrophy, acute myeloid leukemia, acute lymphoblastic leukemia and Diamond- Blackfan anemia. 

Preimplantation Genetic Diagnosis (PGD) for numerical and structural chromosomal abnormalities: FISH & Microarray techniques

Another important application of PGD is to increase assisted reproductive techniques (ART) success for infertile patients (aneuploidy screening) and couples carrying chromosomal abnormality such as translocations. 

 Figure 6: Karyotype of a normal male: 46,XY, 23 pairs of chromosomes. 

What is aneuploidy screening?

Numerical abnormality in chromosomes is called aneuploidy. Chromosomally normal human embryonic cells contain 46 chromosomes (22 pairs of autosomes and 1 pair of gonosomes) (Figure 6). Aneuploidy can originate from an excess number of chromosomes (e.g, trisomy) or from missing chromosomes (e.g. monosomy). 

What is the benefit of screening embryos for aneuploidy?

Approximately half of preimplantation embryos carry chromosomal abnormalities. Using PGD it is possible to eliminate chromosomally abnormal embryos. By transfering normal embryos, the success rate of IVF is increased in cases of
•    repeated implantation failures
•    recurrent pregnancy losses
•    advanced maternal age
•    severe male factor infertilty

What are structural chromosomal abnormalities?

There are three main types of chromosomal rearrangement

  • Reciprocal Translocation
  • Robertsonian Translocation
  • Inversions 

What is Robertsonian Translocation?

Robertsonian translocation is formed by the fusion of two acrocentric chromosomes (13,14,15,21,22). This rearrangement decreases the total chromosome number by one (45 chromosomes) without resulting in any phenotpic change in the carrier (figure 7A).

What is Reciprocal Translocation?

Reciprocal translocation is caused by the rearrangement of parts between nonhomologous chromosomes (Figure 7B).

Does translocation affect fertility?

Balanced translocation carriers may have fertility problems and may experience multiple pregnancy losses due to unbalanced segregation products in their sperms or oocytes. While the frequency of balanced translocations in the newborn population is 0,2%, this rate increases up to 2,5% for couples experiencing repeated implantation failures and 9,2% for couples experiencing recurrent abortions.

 
 

What are inversions?

Inversions are rearrangements within the same chromosome. They occur if two breakages occur in the same chromosome and the chromosomal part between these two points turns over 180 degrees and sticks to the ends again.

There are two types of inversions

Paracentric inversions

Pericentric inversions

 Figure 8: a)Paracentric Inversion, b)Pericentric Inversion

 

  • When the inverted chromosomal segment does not contain the centromere region, the inversion is called paracentric (figure 8a).
  • When the inverted chromosomal segment contains the centromere region, the inversion is called pericentric (figure 8b).

Do inversions affect fertility?

Though inversions are very rarely observed, inversion carriers may experience fertility problems.  During the meiosis of gametogenesis, a single crossover event that occurs between the breakpoints produces unbalanced gametes that carry deletions, insertions, and either zero or two centromeres. PGD can be helpful in the identification of unbalanced gametes.

How can chromosomal abnormalities in embryos be detected?

There are two main approaches for the detection of numerical and structural chromosomal abnormalities in embryos.

  • FISH (Flourescent in situ Hybridization)
  • Array-CGH (Array-Comperative Genomic Hybridization)

What is FISH?

FISH uses fluorescently labeled probes which are specific to each chromosome (figure 9). The steps of technique consist of fixation of the nucleus of the biopsied cell, probe application, hybridization, washing and analysis . Every step is of key importance and requires experience and skill.

 Normal blastomere
Monosomy 21, Trisomy 22
 Complex Aneuploid
Figure 9: FISH images of normal (A) and abnormal (B, C) blastomeres: chromosome 13 (red), chromosome 16 (light blue), chromosome 18 (blue), chromosome 21 (green), chromosome 22 (yellow), (Images from İstanbul Memorial Hospital, Reproductive Genetics Laboratory).

Which chromosomes are screened in FISH?

In Istanbul Memorial Hospital Reproductive Genetics Laboratory, embryos can be screened in 24 hours for the following chromosomes 8,13,14,15,16,17,19,18,20,21,22, X and Y. 

Why are these chromosomes chosen for screening?

The above chromosome panel can detect most of the chromosomal abnormalities that may be found in abortus materials. Those most frequently found in spontaneous abortus materials are “trisomy 16” and “monosomy X” (Turner syndrome).

What are the disadvantages of FISH?

Although it is a relatively low cost, less complex method, FISH cannot detect all abnormalities, since the number of chromosomes that can be analyzed is limited.

What is aCGH? What are its advantages?

Array-Comperative Genomic Hybridization (a-CGH),  is a recently developed technique that can detect changes in the amount of DNA in cells. This technique is able to detect all chromosomal abnormalities.

In aCGH, DNA from a test sample (green) and a normal reference sample (red) are labelled differentially, and hybridized to several thousand probes which are imprinted on a glass slide (Figure 10 and 11). The fluorescence intensity of the test and the reference DNA is measured to calculate the copy number changes for each chromosome.

 Figure 10: Array-Comperative Genomic Hybridization Technique (aCGH)
 
Figure 11: An image of a microchip glass used for aCGH at İstanbul Memorial Hospital, Reproductive Genetics Laboratory. 

How long does this analysis take?

All chromosomes can be analyzed within 12 to 24 hours at Istanbul Memorial Hospital Reproductive Genetics Unit 

What are the advantages of aCGH over FISH?

30-40% of all chromosomal abnormalities cannot be detected by FISH. However, aCGH can detect abnormalities of all chromosomes, both numerically and structurally, with high resolution. Deletions, duplications and unbalanced chromosomal regions can be easily detected using this method. Some examples of normal and abnormal embryos are shown below (Figures 12,13,14).

In IMH Reproductive Genetics Laboratory, aCGH has been used successfully since the beginning of 2011. Because results can be obtained within 12-24 hours, vitrification of the embryos is unnecessary.  CGH is the most reliable method for selecting embryos with the highest implantation potential.

Figure 12: Image of a “normal” trophectoderm sample. The average intensity ofr all chromosomes is within the optimal range.
 
 
Figure 13: “ Trisomy 16” detected in a blastomere using aCGH. The arrow indicates an increase in the amount of chromosome 16 compared to the reference DNA
 
 
Figure 14: “Monosomy 15” detected in a blastomere embryo using aCGH. The arrow indicates a decrease in the amount of chromosome 15 compared to the reference DNA.
SAYFA BAŞINA DÖN