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CONTRIBUTIONS OF ART TECHNOLOGY IN NEW
THERAPEUTIC APPROACHES
L. Gianaroli, MC Magli, AP Ferraretti
S.I.S.ME.R. Reproductive Medicine Unit - Via
Mazzini, 12 - 40138 Bologna
sismer_at_sismer.it
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Since the birth of the first baby conceived using
IVF techniques in 1978 over three million babies
have been born worldwide as the result of ART.
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- Gamete and tissue cryo-banking
- PGD HLA matching
- Stem cells
ART
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- Gamete and tissue cryo-banking
ART
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OOCYTE CRYO-BANKING FERTILITY RESTAURATION
Premature ovarian failure - POF
defined as menopause before the age of 40 years
or hypergonadotropic hypogonadism
defined as menopause consequent to chemotherapy
Depending on the extent of damage to the ovaries
Acute Ovarian Failure (AOF) loss of ovarian
function during or shortly after the end of
chemotherapy Premature menopause loss of
ovarian function that occurs years after the end
of chemotherapy (before age 40 yr)
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OOCYTE CRYO-BANKING RISK OF POF ACCORDING TO AGE
AFTER CHEMOTHERAPY

• The chemotherapeutic destruction of an already
  low follicular reserve, reduces the number of
  follicles below a certain threshold number
  required to sustain ovarian function, resulting
  in menopause.

Mattle et al, 2005
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OOCYTE CRYO-BANKING RISK OF POF ACCORDING TO
CHEMOTHERAPEUTIC AGENTS
Alkylating agents are extremely gonadotoxic
because they are not cell cycle-specific and can
damage resting primordial follicles.
Cycle-specific agents such as MTX and 5-FU do
not have any effect on ovarian reserve.
Sonmezer, M. et al. Oncologist 200611422-434
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OOCYTE CRYO-BANKING DOSE-EFFECT DAMAGE ON OVARIAN
FUNCTION
No. of primordial follicles
Dose of Cyclophosphamide (mg/kg)
Follicular damage from alkylating agents is
dose-dependent. A dose of chemotherapy strong
enough to destroy 50 of the ovarian primordial
reserve does not affect the reproductive
performance in a murine model.
Meirow et al, 1999
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OOCYTE CRYO-BANKING OVARIAN TISSUE FREEZING

• Ovary(s) removed laparoscopically, divided into
  small strips, frozen and stored
• Females, before and after puberty
• Outpatient surgical procedure
• Experimental, one possible live birth to date
• Re-implantation can restore hormone function

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OOCYTE CRYO-BANKING OVARIAN TISSUE FREEZING
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OOCYTE CRYO-BANKING OVARIAN TISSUE FREEZING
CLINICAL RESULTS

• A transient restoration of spontaneous ovarian
  follicular development and estrogen production,
  but not ovulation, was observed after
  autotransplantation of frozen/thawed ovarian
  tissue that had been harvested and banked before
  chemotherapy and radiation therapy for lymphoma
  (Redford et al., 2001).
• Embryo development was obtained after
  autotransplantation of frozen/thawed ovarian
  tissue (Oktay and Sommezer, 2004).

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OOCYTE CRYO-BANKING OVARIAN TISSUE FREEZING
CLINICAL RESULTS

• Livebirth resulted after orthotopic
  transplantation of cryopreserved ovarian tissue
  (Donnez, 2004).
• A pregnancy was obtained after transplantation of
  cryopreserved ovarian tissue and IVF in a patient
  with ovarian failure after chemotherapy (Meirow,
  2005).
• Actually, follicular growth, hormonal production
  and some pregnancies (5 as reviewed by Fabbri et
  al., 2008), spontaneous or after IVF treatments,
  have only been achieved after the
  autotransplantation technique.

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OOCYTE CRYO-BANKING OOCYTE FREEZING CLINICAL
APPLICATIONS

• Oocyte cryopreservation could be a clinical tool
  for
• Women at risk of losing ovarian function
• Women desiring fertility preservation (e.g.
  delayed maternity)
• Eliminating ethical concerns of embryo
  cryopreservation
• Solving the dilemma of abandoned frozen embryos
  in the IVF laboratory

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OOCYTE CRYO-BANKING OOCYTE FREEZING SISMER
EXPERIENCE
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OOCYTE CRYO-BANKING OOCYTE FREEZING
VITRIFICATION SISMER EXPERIENCE (2004-2008)

Implantation rate (FHB)
Spontaneous abortion rate
Clinical pregnancy rate
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- Gamete and tissue cryo-banking
- PGD HLA matching
ART
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PGD
GENETIC INVESTIGATION
Diagnosis implies looking for
specific disease mutation (including X-linked
diseases) chromosome aberration
Screening
implies looking for a genetic defect in all
members of a population at risk being the risk
dependent on the incidence and severity of the
defect
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PGD
MAIN INDICATIONS ESHRE PGD consortium data
collection 1997-2005
14.419 cycles
Goossens et al. (2008) ESHRE PGD Consortium data
collection VIII cycles from January to December
2005 with pregnancy follow-up to October 2006.
Hum Reprod 23,2629-2645.
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PGD WHY TO GO FOR IT?
Normally fertile couples whose children might
inherit - a severe disease - a
predisposition to a pathology
Normally fertile couples who wish to save a
siblings life (HLA-typing)
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PGD WHY TO GO FOR IT?
IVF aims at having a child, PGD aims at having a
healthy child and PGD/HLA testing aims at having
a healthy and helpful child.
UNESCOs report on preimplantation genetic
diagnosis (PGD) and Germ-Line Intervention, 2003.
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PGD FOR HLA - MATCHING

• Healthy embryos are selected for transfer

avoids the need for termination of an ongoing
pregnancy in cases of an affected fetus

• HLA matching with an affected child

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PREIMPLANTATION HLA MATCHING

• One of the most recent applications in
  reproductive medicine.
• Viable option for couples with children needing
  haematopoietic stem cell (HSC) transplantation.
• Selection of embryos both free of disease and HLA
  matched with the existing child.
• PGD is used not only to avoid the birth of
  affected children, but also to conceive healthy
  children who may also be potential HLA-identical
  donors of HSC
• At delivery of the newborn, cord blood HSC can be
  used to treat the affected sibling.

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Allogeneic HSC transplantation

• Only curative option for severe cases of
  haematopoietic disorders.
• A critical factor associated with a favourable
  outcome is the use of HLA identical donors
• HSC from HLA identical siblings provide the
  higher success rate (90)
• Reduced incidence of graft rejection and other
  serious complications associated with
  transplantation.
• Transplantation using non HLA-identical donors is
  associated with higher morbidity and poorer
  survival.
• Limited availability of HLA-matched unrelated
  donor, identified from national or international
  registers.

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Indication for preimplantation HLA matching

• Severe cases of haematopoietic disorders
  requiring a HLA compatible HSC donor.
• Thalassemia
• Fanconi anaemia
• Wiskott-Aldrich syndrome
• Diamond-Blackfand Anemia
• X-linked Hyper IgM Syndrome
• X-linked adrenoleukodystrophy
• X-linked Hypohidrotic Ectodermal Dysplasia with
  immune deficiency
• Aplastic anemia
• For diseases such as Acute Lymphoid Leukemia, in
  which HLA matching becomes the primary indication.

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Patients acceptance policy for HLA program

• HSC transplantation is the best treatment option
  for the affected child (advise transplantation
  hematologist is required)
• HSC transplantation is not urgent
• The family cares unconditionally about all the
  children.

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Simplified map of the HLA region
CLASS I 3 types HLA-A, HLA-B, HLA-C.
CLASS II 3 types HLA-DP, HLA-DQ, HLA-DR.
(also MHC class II genes for HLA-DM, and TAP, and
LMP)
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Polymorphism in the MHC
The HLA Complex (Human Leukocyte Antigen) -
located on chromosome 6 - represents one of the
most polymorphic regions of human genome.
Comparative DNA sequence analysis of HLA complex
has shown the presence of a high number of
alleles in this region.
In the human population, over 1,200 HLA alleles
have been identified
492 alleles
657 alleles
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Inheritance of MHC haplotypes
Genes in the MHC are tightly LINKED and usually
inherited in a group. The combination of alleles
on a chromosome is an MHC HAPLOTYPE
all persons have 2 haplotypes (1 maternal, 1
paternal in origin)
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Minisequencing-based Preimplantation HLA matching
on single blastomeres
Father
Mother
Child
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Minisequencing-based Preimplantation HLA matching
on single blastomeres
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Overall Results and Outcome of Preimplantation
Diagnosis for Single Gene Disorders
Preimplantation HLA testing RGI Experience
04/14/2008
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- Gamete and tissue cryo-banking
- PGD HLA matching
- Stem cells
ART
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Stem Cells

• Stem Cells
• - clonogenic, self-renewing
• progenitor cells that generate one
• or more specialised cell types
• Embryonic stem cells and germline stem cells
• - pluripotential and immortal
• Organ or tissue specific stem cells
• - multipotential

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Differentiation of Stem Cells
Pluripotent stem cell
Blastocyst
Totipotent stem cell
Culture
Embryo
Embryonic stem cell (ES cell)
Embryo (gonads)
Pluripotent somatic stem cell
CNS PNS Hema. Liver Skin Mesen. etc
Primitive germ cell (germline stem cell)
Multipotent stem cells
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SOURCES OF STEM CELLS
NERVOUS, HAEMATOPOIETIC, EPITHELIAL, INTESTINAL
ADULT TISSUE
- SPECIFIC CELL TYPE - TRANSDIFFERENTIATION
UMBILICAL CORD
HIGH PROLIFERATION
VERY HIGH DIFERENTIATON POTENTIAL
EMBRYONIC STEM CELLS (ES)
INNER CELL MASS
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STEM CELLS
THERAPEUTIC APPLICATIONS
CRUCIAL FCTORS
NUMBER OF CELLS
MEDIUM
UMBILICAL CORD
LOW
ES
TISSUES
HIGH
HIGH
LOW
MEDIUM
PROLIFERATION POTENTIAL
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Applications for Embryonic Stem Cells and
their Derivatives

• ES cells for research and discovery
• Progenitor cells for drug screening
• Progenitor cells for toxicology
• Gene products (proteins), growth and
• differentiating factors, cell surface
• molecules for pharmaceutical use in
• regenerative medicine

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Embryonic Stem Cell Derivatives Applications
for Cell and Tissue Therapy

• Vehicles for the delivery of gene therapies
• - correcting genetic disease
• - new immunization strategies
• - targeting cancers

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Making ES Cells and their Derivatives Compatible
for Transplantation

• Make ES cells from all the necessary HLA
  subtypes
• subject to advice from transplant divisions
  about the
• number necessary
• Remove the cell surface expression of the
  major
• histocompatibility antigens from ES cells and
  their
• derivatives
• Make customized ES cells for each and every
  patient
• therapeutic cloning

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Embryonic Stem Cells Formed by Nuclear Transfer
of Adult Cells (Therapeutic Cloning)

• ES cells contribute to all tissues in an
  apparently normal way
• - in chimeras
• in vitro
• in teratomas
• No indication of defects seen in cloned
  foetuses/offspring
• helper cell effects
• transdifferentiation phenomenon
• May be an extremely efficient way to produce
  transplantation
• compatible cells and tissues for patients

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Therapeutic Cloning
ES cells
Compatible transplants
Nuclear transfer
Source of eggs self, mother, relative, egg bank
Cells eg skin
Problems Inefficient - may need large numbers
(50 to several hundreds) of eggs Technically
demanding - need to be available in many or all
hospitals
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Patient Specific Stem Cells
Disease phenotype
Nuclear transfer
Drug
Screening
Patients with diseases of undetermined cause,
eg. Glioblastoma Alzheimers Disease Parkinson
s Disease Motor Neurone Disease
How
Why
Blastocyst
Unaffected neurones
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Cure of Genetic Diseases Using Stem Cells
Insert HoxB4 Homeobox gene
DNA repair
Differentiate into functional hematopoietic
lineage
Mouse in a Bubble
Inject stem cells to correct Rag2 disease
Genetic Correction
Cultured skin cells
Rag2 Mouse (severe combined immuno deficiency)
ES Cells
Skin cell
or
Nuclear transfer
Cell fusion
(Rideout et al. Cell 2002)
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Allogenic Cell Therapy
Induce tolerance Immune suppression
Non-transferable embryos in frozen storage
gt 200,000 HLA types
Brain transplantation
Neuronal precursors TH dopaminergic neurone
MHC gene knockouts
Propogate cells to master ES cell bank
Select null HLA expression
Master bank of HLA typed ES cells
Direct
Neural stem cells
differentiation
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Transplantation of Cells Derived from Human
Embryonic Stem Cells

• Induction of tolerance across MHC
  histocompatibility barriers by pretransfer of ES
  cell- like cells/haematopoietic cells.
• An epithelial progenitor stem cells population
  (positive for glycoprotein MTS24) has been
  identified that is competent and sufficient
  to fully reconstitute the thymus.

(Gill et al Nature Immunology June 17 2002).
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Delivering stem cell therapy Avoiding immune
rejection by reconstructing the immune system
The thymus is the site where immune system cells
learn the difference between self and non-self
Richard Boyd and colleagues have shown that rare
progenitor cells can rebuild a thymus
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LONG-TERM ALLOGENEIC GRAFT ACCEPTANCE USING
EMBRYONIC STEM CELL-LIKE CELLS
(Fandrich et al. Nature Medicine 8 171 2002)
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Trophectoderm biopsy for developmental competence
Biopsy for genetic diagnosis
Transfer to patient
Blastocyst
8-cell
4-cell
Embryos
Inner cell mass
2-cell
Transfer to patient
ICSI
Use in regenerative medicine (eg. diabetes,
Parkinsons Disease, stroke, respiratory disease,
cardiac damage, quadriplegia)
IVF Female infertility Idiopathic infertility
few eggs
Many eggs
Immature eggs recovered in natural cycle (no
fertility drugs)
Fertility drugs given to women to make
many mature eggs
Reproductive Medicine ? Therapeutic Medicine
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EU hESC line Registry (hESCReg)

• Primary objective provide information about all
  human ESC lines derived and used in Europe
  which are available to the scientific community.
• Specific Support Action funded by VI FP European
  Commission (1.048.000 , 2007 - 2010)

Coordinated by Joeri Borstlap (BCRT - Technical
Coord.) Anna Veiga (CRMB - Scientific Coord.)
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RGIs Repository of Human Embryonic Stem Cells
As of 10/2008
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CONCLUSIONS
Therapeutic medicine
- Gamete and tissue cryo-banking
- PGD HLA matching
- Stem cells
ART
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CB Stem Cells
CB Stem Cells
Low quantity
High quantity
Direct treatment
In vitro Expansion
Complete engraftment
Locatelli, unpubblished