Genetics
Preimplantation Genetic Testing (PGT)
Preimplantation Genetic Testing (PGT) is the earliest form of prenatal diagnosis, done on embryos. During the IVF cycle, a single cell or a few cells are removed (biopsied) from the embryo and are checked for specific genetic abnormalities. The embryos that are reported normal are transferred into the mother’s uterus. PGT offers a new alternative to chorionic villus sampling or amniocentesis. For any PGT testing, one needs to have multiple embryos developed by ICSI.
The steps involved are:
1. Medical evaluation of the couple to check the suitability for an ICSI cycle.
2. Genetic evaluation of the couple
3. The ICSI cycle.
4. Embryo biopsy which is followed by embryo cryopreservation.
5. Genetic testing of embryos (PGT)
6. Transfer of unaffected embryos
7. A Pregnancy test 14 days later.
Utility of PGT
PGT improves pregnancy rates and decreases spontaneous miscarriage rates in:
1. Older age women
2. Previous unsuccessful IUI/IVF attempts
3. History of recurrent spontaneous miscarriages
4. Male factor infertility
5. Past history of repeated terminations because of an abnormality diagnosed during pregnancy
6. Known carriers of a genetic defect (e.g. Beta-Thalassemia, Sickle cell anemia, Cystic Fibrosis, Spinal Muscular Atrophy, Duchenne Muscular Dystrophy, Fragile X Syndrome, Huntington Chorea or a chromosomal translocation.
Objectives
• Prevents chromosomally or genetically abnormal births in couples having a history of failed fertilization after IVF-ICSI, missed abortions or carriers of balanced translocations.
• Increases the chances of implantation.
• Reduces the incidence of spontaneous miscarriages.
• Reduces the incidence of multiple offspring.
• Eliminates the risk of a child being affected with a familial gene disorder.
• A previously affected child can be cured by a Saviour Sibling (HLA matched sibling) who can then donate the stem cells to the affected child. eg. Thalassemia major.
Different techniques offered at our Centre
- PGT-SR by Fluorescence In Situ Hybridization (FISH) for telomeric/cryptic translocations and inversions
PGT by the FISH technique is now mainly carried out in cases where the husband or wife is a carrier of a balanced translocation or inversion. This is required to select embryos free of related unbalanced rearrangements. Pre-PGT work up of the couple using specific probes for the rearrangements is necessary to check for an additional tiny or cryptic rearrangement which may not have been noticed earlier. The embryo biopsy is done on Day-5. About 5-8 trophectoderm cells are biopsied from each embryo and are fixed on slides and subjected to the FISH procedure using probes specific for each couple, as per the chromosomes involved. The fluorescent signals are observed using fluorescent microscopy technique. A normal cell will show two signals of each colour for each chromosome. If there are 3 signals or 1 signal present, it indicates abnormal embryos which should not be transferred to the mother.
Our team has more than 20 years of experience in this field and has reported the first live births for Robertsonian /Reciprocal / Cryptic Translocations and Inversions in India.
- PGT for all 24 chromosome aneuploidies / structural rearrangements (PGS or PGT-A / PGT-SR) using the array Comparative Genomic Hybridization (aCGH) technique
For 24 chromosome PGT, the biopsied cells are “tubed”, the DNA is amplified and tested for chromosomal aneuploidies using the microarray technique. Detection for all 24 chromosomes is possible on Day 5 of embryonic development. The results are available within 24 hours. The technique can also be used for detection of unbalanced translocations except those involving the telomeres (tips) of the chromosomes.
- PGT for all 24 chromosomal aneuploidies (PGS or PGT-A) using Next Generation Sequencing (NGS)
For PGT, the biopsied cells are “tubed”. The embryonic DNA is further subjected to a series of procedures and is finally sequenced using NGS technology to check for chromosomal aneuploidies. Detection of all 24 chromosomes is possible on Day 5 of embryonic development. The results are available within 24 hours.
- PGT for single gene disorders (PGT-M)
For PGT, the biopsied cells are “tubed”. Whole genome amplification is carried out to increase the quantity of DNA externally in a PCR machine. Then the amplified product is subjected to further testing for the single gene disorder. The parental genetic markers are matched with the embryonic markers in order to make a diagnosis.
Prenatal Diagnosis (PND)
This involves genetic analysis of cells of fetal origin in various disorders by chorionic villus sampling, amniocentesis, or cord blood sampling, by karyotyping and FISH or molecular analysis such as chromosome microarray or DNA sequencing.
Indications
- Women with advanced maternal age
- Previous child with a chromosome abnormality.
- Women who have had previous still births or early neonatal deaths
- Parents with balanced translocations
- Cases in which the Biochemical Marker tests or NIPT (Non Invasive Prenatal Test) shows a high risk for Trisomy
- Abnormalities seen on ultrasonography
- Both husband and wife are carriers of a single gene autosomal recessive disorder such as Beta- Thalassemia
The FISH technique is very useful for rapidly detecting aneuploidies in high-risk pregnancies. A ‘high risk’ for Trisomy 21 by the Double marker / Quadruple Marker test or Ultrasonography causes a lot of anxiety to the expectant couple. This can be relieved in a day by FISH on uncultured CVS or amniotic fluid cells. Karyotyping is simultaneously done to rule out many structural and numerical chromosome abnormalities. If the quantity of chorionic villi obtained in a biopsy is insufficient for karyotyping, FISH is still possible from interphase nuclei.
Sample
- Amniotic fluid (8 ml for FISH; 15 ml for karyotyping; in 2-4 plain – red top – vacutainers). In case of a sanguinous tap, it is better to collect in heparin vacutainers.
- Cord blood (1ml in sodium heparin – green top – vacutainer).
- CVS in sterile normal saline with 2 drops of Gentamycin or in media tubes supplied by the laboratory.
Products of Conception (POC)
Missed abortions and still-births can be studied by karyotyping and FISH to detect chromosomal abnormalities. Spontaneous abortion samples show a high frequency of aneuploidy. Aneuploidy detection by FISH can include combinations of chromosomes 13, 21 / 18, X, Y / 16 / 22.
Karyotyping from ‘products of conception’ (POC) of a spontaneous or missed abortion is done by fibroblast culture to rule out a chromosome abnormality, which may be a likely cause of the abortion.
Sample
To avoid contamination of tissue, the sample from early abortions should be taken directly with a sterile Ovum Forceps or Menstrual Regulation (MR) syringe and transported in a sterile container with normal saline to which 2-3 drops of Gentamycin are added. The sac surrounded by chorionic villi is the tissue of choice in early miscarriages while a 1” piece of placenta and fetal skin could be collected from 2nd and 3rd-trimester samples. Cord blood (0.5-1ml) could also be collected in a sodium heparin vaccutainer from stillborn fetuses for karyotyping.
The samples can be stored in the refrigerator overnight if required. Rapid transportation is required with a cool pack.
We have a high success rate of karyotyping from POC.
Postnatal Diagnosis
This involves analysis of samples such as blood, bone marrow, buccal cells, urine, formalin fixed paraffin embedded (FFPE) tumor tissue for various genetic disorders using different cytogenetic and molecular techniques like karyotyping, FISH, PCR and Sanger Sequencing.
Indications
- Primary or Secondary Infertility
- Primary amenorrhea
- Ambiguous genitalia
- Developmental delay
- Dysmorphic features
- Microdeletion Syndromes
- Chromosomal breakage disorders like Fanconi anemia, Bloom Syndrome, Ataxia Telangiectasia
- Hematological malignancies
- Breast / Oesophageal / Bladder Cancer and Lymphomas
- Hematological disorders like Beta thalassemia, Sickle cell anemia, MTHFR mutations, Prothrombin gene mutation
- Periodic Fever Syndromes
- Auto-inflammatory Disorders
Genetic Counselling
Genetic counselling helps parents to understand a genetic disorder which may be picked up on prenatal diagnosis, or during the investigation of an affected child and reach decisions about what to do next.
The family history is evaluated and the risks of recurrence in a subsequent pregnancy are explained, with information on available prenatal diagnostic tests. The possible cause of the condition is explained in order to reduce the feeling of guilt experienced by parents. Options of Assisted Reproductive techniques to bypass an inherited condition are discussed. Other family members needing prenatal diagnosis are identified and awareness is given in order to prevent the birth of another affected child in the extended family.
Genetic counselling is given to the couple and family members when needed. The couples are guided on tests to identify the disorder, and the availability of prenatal or preimplantation genetic testing (PGT) to prevent the birth of an affected child. They are also briefed about the consequences of genetic disorders and given information about support groups. A Pedigree Chart (Family Tree) is drawn based on the information given. This is to determine the pattern of inheritance and the risk of recurrence. eg. If there is a child affected with thalassemia, there is a 25% risk in every pregnancy of another child being affected. Therefore, prenatal diagnosis has to be carried out at 11-12 weeks from CVS, in every pregnancy in such cases, or they can opt for PGT to eliminate the risk of thalassemia int he subsequent pregnancy.
If there is a history of recurrent miscarriages, karyotyping of the couple may show a balanced translocation in one of the partners, who is only a carrier. PGT (Preimplantation Genetic Testing) is available at our Centre for Robertsonian or Reciprocal translocations and Inversions, using the couple’s own gametes. About 5-8 trophectoderm cells from each embryo are tested and the abnormal embryos (unbalanced) are not transferred.
In the case of a child suffering from a suspected genetic disorder, it is always advisable to test the DNA and identify the mutation, as it will help for prenatal diagnosis in the next pregnancy. This also applies to products of conception (POC) of recurrent miscarriages. The stored DNA of an affected child can come into use when couples want to prevent the recurrence of the same disorder in a subsequent pregnancy. For accurate prenatal diagnosis, it is necessary to know the exact DNA mutation or variant in the earlier affected child, and then test the couple to determine the risk of recurrence. Ideally, genetic testing such as next-generation sequencing should be carried out before planning a subsequent pregnancy. Targeted testing of other family members may also be necessary.
Carrier screening of the couple is also available. Some couples opt for this test prior to the 1st pregnancy itself even if there is no history of a genetic disorder in the family. This is because most of us are probable carriers of small changes in DNA called variants or mutations which we are not aware of since we are apparently normal. As these changes are present on only one copy of a pair of chromosomes while the corresponding chromosome has the normal gene, we are carriers. A common example is Thalassemia. The aim of these genetic screening test is to detect carrier status of a variant in the same gene or different genes of a similar disease group such as blindness or deafness. In case both partners happen to be such carriers, there is a 25% chance in every pregnancy that their child will inherit 2 copies of this variant, one from each parent, and will be affected. Previously, the only option available was to carry out a Prenatal diagnosis at around 11 weeks gestation at the earliest. If the fetus was affected, the couple could consider a medical termination of pregnancy.
With the option of PGT of embryos during an ICSI cycle, at our centre, many couples have been treated successfully. Though they do not have any issues of infertility, they can opt for IVF where a single sperm is injected into each ovum/egg to obtain embryos in the laboratory. Day 5 embryos are biopsied and tested, while the embryos are frozen. Unaffected embryos can then be selected for transfer. This eliminates the trauma of medical termination of an affected natural pregnancy in the second trimester. Before implantation, the normal/unaffected embryos are further screened to check for abnormalities of all the 23 pairs of chromosomes, including Trisomy 21 or Down syndrome. Single embryo transfer of the normal embryo is preferred. The other normal embryos are frozen for later use. We have had success with PGT for many rare as well as common disorders.
Genetic Counseling OPD: With prior appointment taken directly with the counsellor.
We have 2 well-experienced genetic counsellors and a specialist genetic counsellor for hematological disorders who guide couples with a history of known genetic disorders.
Honorary Consultant Geneticist: Dr. Prochi F. Madon, Ph.D. (>40 years of experience in the field of Genetics)
HOD, Genetics Lab: Dr. Arundhati S. Athalye, Ph.D. (>20 years of experience in the field of Genetics)
Genetic Specialist for Hematological Disorders: Dr. Sona B. Nair, Ph.D. (>20 years of experience in the field of Genetic diagnosis of hematological disorders)
Contact for appointments: +919820006336 (Dr. Prochi Madon, Direct) / +91-22-66573343
