In Vitro Fertilization and Preimplantation Genetic Diagnosis Adrianna Vlachos, MD DBA Camp 2015

In Vitro Fertilization and Pre-Implantation Genetic Diagnosis Goal: Child unaffected by a genetic (hereditary) illness defined by a known genetic mutation Secondary goal: “Creating” a child unaffected by the genetic illness who is a transplant donor for the affected child

In Vitro Fertilization and Pre-Implantation Genetic Diagnosis IVF Hormonal therapy to the mother to get many eggs for ovulation Number of eggs depends on age of mother Side effects of these medications to mother Eggs harvested (under anesthesia) Eggs are fertilized with sperm outside of mother (“in the test tube”)

In Vitro Fertilization and Pre-Implantation Genetic Diagnosis PGD Fertilized egg reaches 8-cell stage One cell taken for RP/HLA testing “RP neg, HLA matched” fertilized egg implanted in mother (1/8 chance) Hormonal therapy to continue pregnancy, usually until 10-12 weeks CVS (10 wks) or Amnio (18 wks) for confirmation

Pre-natal vs Pre-implantation diagnosis Dr. I. Souter, MGH Fertility Center

Pre-natal Diagnosis Amniocentesis Chorionic Villus Sampling (CVS)

Pre-implantation Diagnosis Introduced initially in 1990 Biopsy of a single cell per embryo followed by its genetic diagnosis through different techniques the subsequent replacement to the patient of those embryos classified by genetic diagnosis as unaffected

PGD Indications Primary Goal Procedure is offered to couples: With known single gene disorders that can be detected by PGD - DBA With known chromosomal abnormalities that can be detected by PGD requesting sex selection for X-linked disorders – DBA

PGD Indications Secondary Goal Requesting PGD for HLA-typing (to allow selection of embryos that are histocompatible with live siblings)

Single Gene Disorders

PGD Process Ovulation Induction Retrieval Fertilization Embryo Bx on Day-3 Genetic Analysis Embryo Transfer

Ovulation induction

Oocyte Retrieval

Fertilization Conventional Insemination Intracytoplasmic Sperm Injection (ICSI)

Embryo Culture

Day 3/Cleavage Stage Embryo

Cleavage Stage Biopsy

Genetic Analysis/PCR DNA amplification Mutation Characterization sequence harboring the mutation Mutation Characterization FISH PCR HLA Matching

Embryo Transfer

Early Pregnancy

Risks Embryo damage Oocyte and Embryo Biopsy are invasive procedures Misdiagnosis False negative result False positive result The chance for NO result The chance for mosaicism IVF Risks Not Achieving Pregnancy There may not be any normal embryos available for transfer. The embryos may not implant and develop even if they do not have the defect. The workup for PGD is expensive and labor intensive PGD can only detect a specific genetic disease in an embryo. It cannot detect many genetic disorders at a time and cannot guarantee that the fetus will not have an unrelated birth defect. . The Risk of Embryo Biopsy While PGD is a relatively new procedure in IVF, the micromanipulation or biopsy techniques required to perform the procedure have been in use for many years. The risk of accidental damage to an embryo during removal of the cell(s) is very low -- 0.6%. Concerns have been expressed regarding the development and viability of the embryo after biopsy Studies have shown that biopsy at the 8 cell stage has no detrimental effects on further growth The balance between potential biopsy damage and beneficial effects of PGD seems to be positive. The other risks include those common to all IVF treatments - and these include: OHSS ( ovarian hyperstimulation syndrome), ectopic pregnancy and multiple pregnancy Misdiagnosis Mosaicism is defined as the embryo having cells with different chromosome make up. Typically, all cells of the embryo have the same chromosomal make up as they originate from the same fertilized egg. However , it is possible for cells of the same embryo to have differing numbers of chromosomes. . If we analyze a cell that has normal chromosomal content, but another cell has an extra chromosome, we erroneously diagnosed that embryo as being chromosomally normal. Due to the chance of misdiagnosis as well as the presence of anueploidy for which we do not test, we recommend prenatal testing as stated earlier. What Is The Reliability of PGD? There is a small error rate with PGD due to the complexity of testing a single cell in the laboratory.. It must be stressed that this technology is still considered experimental. For this reason, prenatal testing by chorionic villus sampling or amniocentesis is required by the PGD laboratory to confirm their diagnosis. PGD can only detect a specific genetic disease in an embryo. It cannot detect many genetic disorders at a time and cannot guarantee that the fetus will not have an unrelated birth defect. Patients opting for PGD have to be aware of the limitations Conventional prenatal diagnosis still needs to be recommended to patients to confirm PGD results by amniocentesis or CVS. they have to go through IVF even though they may be fertile, the workup of new diagnosis for PGD is labour intensive, expensive and may take some time, Single cell PCR is a technically challenging procedure as it is at risk of contamination and allele dropout (ADO). Since only 1 cell is amplified, if another cell or piece of DNA enters the tube, it will also be amplified. Therefore ICSI is used to ensure that there are no excess sperm present (paternal contamination) and all of the cumulus cells have to be carefully removed (maternal contamination). Stringent conditions have to be employed to ensure that no other contamination results No part of the future fetus will be lacking because one or two cells are removed form the embryo approximately 2 days after fertilization. All the cells of the embryo remain totipotent until after the fourth day which means having all the potential into growing into a whole fetus. The accuracy of the PGD for translocation is 90%.

Summary Before PGD is performed, genetic counseling must be provided to ensure that patients fully understand the risk for having an affected child the impact of the disease the available options the multiple technical limitations including the possibility of an erroneous result Prenatal diagnostic testing is strongly encouraged to confirm the results of PGD

Conclusions It is a personal choice!!! This requires identification of your family’s DBA gene. Obstacles Practical Religious Ethical Financial There are other options available Adoption Sperm/egg donation Just having a baby