Clinical Management of In Vitro Fertilization With Preimplantation Genetic Diagnosis Ilan Tur-Kaspa, M.D., Seminars in Reproductive Medicine, 2012;30(4): R4 孫怡虹 /Dr. 蔡永杰

INTRODUCTION

Preimplantation genetic diagnosis (PGD) Introduced in 1990 by Verlinsky et al in Chicago with polar body biopsy In London by Handyside et al that same year with blastomere biopsy Indications Indications: expanded rapidly  Conceive with healthy embryos tested in vitro before implantation  avoid the dilemma of whether or not to terminate a pregnancy or deliver a sick child

Preimplantation genetic diagnosis (PGD) For couples at risk of having children with heritable and debilitating genetic diseases:  A major scientific advance For couples who carry a balanced chromosomal translocation:  Significantly ↓ the risk of spontaneous miscarriage (~ <20%) & ↑ live-birth rates

Discussion in this article The safety of PGD procedures Children's outcome How to optimize ovarian stimulation and PGD success Daily routine of clinical management of IVF for PGD – Special clinical dilemmas – Protocols such as nondisclosure PGD

EMBRYO DEVELOPMENT, PREGNANCY RATES, AND CHILDREN BORN AFTER PGD Is PGD Safe?

Ovarian stimulation for IVF with PGD Embryo micromanipulation Technique used for biopsy Numbers of cells removed from the embryo  May affect embryo development, implantation rate, & the pregnancy outcome

Implantation rates of embryos After Biopsy of polar bodies ( 4 h after oocyte retrieval) + embryo  blastomere (6~8 cells, 72 h or D3 of embryo culture)  Similar (26%  25%) regardless of the number of micromanipulations performed

Embryo development 9925 embryos biopsied for PGD  28,126 non-biopsy ICSI embryos: Similar blastocysts development rates Breaching of the zona pellucida and by removing only 1 blastomere from day 3 embryos – Live-birth rate significantly ↑ – Removing 2 cells  affect embryo development significantly  should be stopped Holes created in the zona pellucida for PGD  ↑ incidents of monozygotic twins (at experienced centers)

Initial ↓ [β-hCG] with pregnancies obtained after PGD  Control: Biochemical pregnancy rate, clinical miscarriage rate, take-home infant rate: Similar in both groups May result from the blastomere biopsy – ↓ β-hCG-producing activity of the trophoblast, especially at early pregnancy – A delayed implantation May occur in biopsied embryos Or related to the type of controlled ovarian hyperstimulation (COH)  May be clinically implemented for predicting successful pregnancy outcome following PGD procedure

Reproductive specialist experienced in IVF with PGD + Skilled embryologists & PGD laboratory (including removal of only 1 cell from day 3 embryos)  May obtain high pregnancy rates ** European Society of Human Reproduction & Embryology (ESHRE)  Some centers in the United States

Children born after PGD  Natural conception or IVF/ICSI Mental & psychomotor development (age 2~4) – Similar developmental outcomes ↑ Rate of malformation or neonatal problems Adverse effects: congenital malformations, growth, neonatal intensive care admissions, behavior, or mental & psychomotor development Infertility: Independent factor affecting children's health with or without ART (  PGD, without infertility  IVF/ICSI-only: if ↓ rate of malformation or neonatal problems)

Children born after PGD  Natural conception or IVF/ICSI PGS children: showed an unexplained lower neurological optimality scores (Middelburg et al) ↑ Rate of stillbirths in multiple pregnancies following PGD (From one center that used to remove 2 cells for biopsy)

Summary PGD in experienced laboratories seems to be safe Similar clinical outcomes as with regular ART Embryo biopsy: – Performed on day 3 – Only one cell should be removed for PGD/PGS – Not add risk factors to the health of singleton children born after PGD or PGS – Need Further prospective follow-up studies

How to Maximize IVF-PGD Outcome

Factors that influence pregnancy & live birth rate Patient related (outside a fertility clinic's control): – Age, infertility diagnosis, Hx of previous births, previous miscarriages, previous failed ART cycles PGD  Embryo: – Aavailable for transfer: ↓ 25 ~ 81% – 25% with recessive single-gene disorders; 50% with dominant mutations; 30 ~ 70% (depending on age) with aneuploid; 75% with unbalanced translocation – 81%: not be suitable for ET (PGD for HLA matching + recessive mutation) Genetic status  Response to ovarian stimulation

Evaluation before IVF 1.Infertility Diagnosis (other than genetic analysis) – Severe male factor, Endometriosis, Hydrosalpinx, Low ovarian reserve – Embryo development after ICSI is different To avoid DNA contamination when molecular genetics is planned  For severe male factor – Sperm quality  Aneuploidy rate in embryos – Social habits (smoking, drinking, drug)  ART result 2.Ovarian reserve: using age, antral follicle count and/or anti- Müllerian hormone, day 3 FSH levels 3.Unsuspected uterine cavity abnormality (11 ~ 22%): Polyps, Submucosal fibroids, Intrauterine adhesions

Ovarian Stimulation Is There an Optimal Number of Oocytes to Start ART? Optimal number of oocytes retrieved: 10 ~ 15  Mild ovarian stimulation  ↓ Pregnancy rates PGD results  ↓ 25 ~ 81 % embryo for transfer ↓Number of OR  associated with a fair chance for ET & pregnancy, esp. in young patients (<35 y/o)  Low number of oocytes/embryo  Canceling of cycles should be reconsidered Implantation and delivery rates: ↑↑ in the group that continued with the PGS  Canceling PGD may be abandoned in most cases

Ovarian Stimulation and Aneuploidy Inadequate stimulation and/or poor PGS/PGD techniques may indeed affect cycle outcome Exogenous FSH administration  risk of human embryonic aneuploidy The use of GnRH antagonists with COH was suggested as a more patient-friendly protocol Type of gonadotropins / GnRH analogs used for ovarian stimulation protocols / the number of oocytes retrieved  Affect embryos' aneuploidy rate: Insufficient evidence

Genetic Status of the Woman and Her Response to Controlled Ovarian Hyperstimulation Numbers of oocytes: a significant predictor of IVF- PGD cycle success (as regular ART cycles) Optimal oocyte: 15, younger: 10 ~ 15, older: 15 ~ 20 Adequately stimulation & wish to conceive with their own eggs(even 1~7 # )  OR & PGD may be continued Young, poor response to COH  No risk for OHSS  [Gn] dosage may be safely ↑ to the Max dosage Patients with fragile X, Myotonic Dystrophy, balanced translocation: ↓ ovarian response to COH; Not affect outcome of ETs

Elective Single Embryo Transfer & PGD

Elective Single Embryo Transfer (e-SET) To prevent Multiple pregnancies – Significantly ↓ twin pregnancy rates – Significantly ↓ the likelihood of live birth – ↑ Number of eSET attempts (fresh or frozen)  a cumulative LBR similar to that of DET (double-ET)  Vitrified cryo-thawed biopsied embryos + PGD  May reach a survival rate & implantation rate comparable with embryos with no biopsy  e-SET should be offered to young women undergoing PGD or PGS, (esp. when top-quality embryos available)

Clinical Expertise in IVF-PGD Treatments

Affect embryonic survival and development: – Expertise of the embryologists in performing biopsies for PGD – Type of procedure used to breach the zona pellucida – Type (polar bodies, blastomere, and/or trophectoderm) & number of cells biopsied Rate for misdiagnosis at experienced PGD laboratories: <1.0% (0.3 ~ 0.6%)

Centers for Disease Control and Prevention (2006): – 5/426 U.S. centers performed >50 cycles with PGD – Among them >10 cycles specifically for the purpose of prevention of genetic disorders – 3 in New York, 1 in Colorado, 1 in Chicago, Illinois ESHRE & PGD International Society (PGDIS) – Minimal requirement for ART and PGD centers – Collaboration (Preferred ovarian stimulation protocol, Optimal location for the patient's monitoring, Oocyte retrieval, embryology work including embryo biopsy) – Reliable communication system for reporting PGD results

Suggestion for practice PGD ART centers should: Evaluate their experience & outcome of IVF for PGD  Decided whether to offer Monitor the stimulation locally Oocyte retrieval, embryology, biopsy, PGD testing may be performed at a more experienced center (or embryo biopsy by an experienced local or traveling embryologist, & the PGD performed at another location) Following the guidelines published by ESHRE and/or PGDIS

Special PGD Challenges for Clinical Management

Nondisclosure PGD Potential carriers of Late-onset autosomal dominant diseases: – Ex.: Huntington disease (HD) & amyotrophic lateral sclerosis (ALS) – Elect not to be tested – Wish to assure that their children will not carry the disease – Have the right not to know their genetic status

Revised nondisclosure PGD protocol Tur-Kaspa and Najeemuddin (1)Direct mutation testing  maintain nondisclosure (all medical & administrative staff in direct contact with the couple will not be aware of the genetic status of the patient) (2)General PGS  Risk of nonrelated fetal chromosomal abnormalities (thus the issue of payment for "unperformed PGD" will be avoided) (3)Aneuploidy or embryo development may cause to have no embryos for transfer, regardless of the patients' genetic status, performing sham transfers will not be needed

4)Embryo cryopreservation will be performed when possible 5)Because direct mutation testing will be performed, no unaffected embryos will be discarded 6)Avoid multiple pregnancies, the couple will decide in advance whether they wish for one or two (if possible) blastocysts to be transferred

PGD for HLA Typing “Savior siblings”: International controversy Matched Hematopoietic Stem Cell Transplantation: – Donate cord blood or bone marrow – Nonmalignant disorders Genetic diseases affecting the hematopoietic and/or the immune system: (Thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, sickle-cell disease) Acquired diseases like aplastic anemia – Malignant diseases – Malignant diseases like leukemia (↓ Post- transplant morbidity/mortality rates)

Clinical guidelines to offer PGD for HLA typing To parents of a sick child Tur-Kaspa and Najeemuddin 1.When a related matched donor is not available within weeks of the diagnosis (even for diseases with a good prognosis) 2.When HSCT is not urgent and clinically can be postponed by at least 9 to 12 months 3.If not available locally or nationally  collaboration with another center (IHR & RGI: Established clinical collaborations with >100 different centers worldwide)

Summary It’s a unique opportunity for parents to act independently to save their sick child with HSCT Clinical guidelines Will improve the care of children in need of matched HSCT Once future advances lead to improved outcomes of unrelated transplants, this rationale should be revised

The Future of PGD

PGD may now be offered (with or without disclosure): – All known single-gene disorders – Chromosomal rearrangement – HLA-matched siblings – Cancer predisposition genes – Late-onset disorders – Monogenic disorders – Translocations together with aneuploidy – Couple who carry a genetic disorder

PGS for advanced maternal age, repeated pregnancy loss, repeated IVF failures, severe male infertility  on the rise until 2007 Large-scale national IVF-PGD program  As a novel preventive medical strategy for diseases like CF  may have a profound potential in modern health- care systems PGD: should be encouraged to become an integral part of any health-care system and should be covered by medical insurances

Biopsy for PGD: – May affect implantation potential – In general seems to be safe, except when two cells are removed on day 3 – Children born after PGD have similar outcomes  Regular IVF/ICSI infants Optimization/Individualization of ovarian stimulation Collaborations with experienced centers  Allow PGD with any number of oocytes/embryos available

SET: – May be offered to young patients after PGD or PGS for 24 chromosomes – With good quality blastocysts to avoid a multiple pregnancy – Better clinical management of IVF-PGD treatments will improve its outcome

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