Strategies for fertility preservation
and importance of stem cell research
Dr. ir. Hanane Derradji
Radiobiology Unit
[email protected]
OPERA Workshop: Drawing lessons from stem cells
and animal models outside the radiation field
Mol, 13-06-2014
Copyright 2014
SCK•CEN
Content
 Central dogma of mammalian reproductive biology
 Fertility preservation strategies
 Germline stem cell theory
Oncofertility
Oncofertility
=
Oncology
 Fertility preservation: is a new discipline
which refers to strategies that promote or
retain the fertility of cancer patients
undergoing chemo/radiotherapy.
+
Fertility
Unbalanced options for fertility preservation
between men and women
In 2005
 Fertility preservation was an option for men having cancer
 No fertility options were available for young women having cancer
Anatomical differences
2nd polar body
Spermatozoa
Fertilized oocyte
Spermatozoa
- Smallest cell of the body (70 µm)
- Available at any time (puberty till
death)
- Available in large numbers (1000/sec)
- Easy to access
Oocytes
- Largest cells of the body (20mm)
- Available once a month
- Available in a very small amount
(1/month)
- Difficult to access
Unmet need for young women cancer
patients
 Three main gaps:
1. Information gap
2. Data gap
3. Option gap
Oncofertility consortium (Prof. Woodruff, 2005)
Ovary
Mouse ovary
Primordial follicle
Secondary follicle
pre-antral follicles
Blastocysts ready to implant
Central dogma of mammalian reproductive
biology (Pearl and Schoppe, 1921)
Hassold, etal., Environ Mol Mutagen 1996. 28: 167-175
The women’s ovarian stockpile is limited
No possible activation in vitro
Damaged cells will never be replaced !
Effects of cancer treatments
 Hair loss
 Nausea
 Dry and sensitive
skin
 Lost and fragile nails
 Swollen hands/feets
Visible effects
Radio-chemotherapy-induce gonadal toxicity
Non visible effects
Radio-chemotherapy
Radiotherapy
(low
abdomen)
and/or
chemotherapy of cancer patients affect the
ovarian pool and lead to germ cell death,
Premature Ovarian Failure (POF) and infertility
Need for fertility preservation strategies
Cryopreservation of embryos
Clinically a well-established technique
Pregnancy rates 30-40%
Requires: ovarian stimulation, oocytes retrieval
and IVF (2-5 weeks)
Delay the initiation of cancer treatment
Not appropriate for children and pre-pubertal girls
Not suitable for women without partner
Hormonal treatment is undesirable for estrogen-sensitive
tumors (breast cancer)
Ethical issue : disposal of the embryo if the patient die.
Cryopreservation of oocytes
Pregnancy rate is lower than the embryo
storage
Requires ovarian stimulation and oocyte
retrieval
Delay cancer therapy
Hormonal treatment is undesirable for
estrogen-sensitive tumors (breast cancer)
Available for women without partner
Cryopreservation of ovarian tissue
Still in experimental phase : “few cases of success”
Storage of hundreds of immature oocytes
No need for ovarian stimulation
No delay in initiating cancer treatment
Suitable for pre-pubertal girls
Ovarian cortical tissue
prepared
for cryopreservation
Limitations :
» Blood malignancies : recurrence of the disease
» Ischemia : only few primordial follicles survive
ischemia due to re-implantation
Surgery
 Oophoropexy: ovarian transposition
before radiotherapy
Oophoropexy
 Limitations
Irradiation should be delayed to allow the incision to heal
The ovary can migrate back to its original
Not reliable and risk of cyst formation and injury to the
vasculature
Applicable only for patients treated with radiotherapy
Experimental: Fertoprotective molecules
Irradiation
Chemotherapy
Cell stress
Fertoprotective molecules
S1P, FTY720
Fragmented
oocyte
Intact oocyte
In vitro Follicle Maturation (IFM)
Alginate beads (Woodruff ‘s Lab, 2009)
- No reintroduction of cancer cells into the patients
- Collection of many mature oocytes (Woodruff, 2007)
In vitro Follicle Maturation (IFM)
 3D culture of macaque follicles:
 3D culture of secondary follicle in alginate beads to small
antral stage
 3D culture of baboon follicles
 3D culture of pre-antral follicle to small antral follicles in
soft hydrogel
 Demonstration of fertilization and early embryonic
development (blastocyst stage) in vitro (Peluffo, 2012)
Identification of optimal conditions for nonhuman primate follicle
culture prior to human application
Central dogma of mammalian reproductive
biology
 Women are born with a finite, non-renewing pool of germ cells,
all of which are arrested in meiosis I (oocytes) and are
enclosed by somatic cells in structures referred to as follicles
(Pearl and Schoppe, 1921)
 Whether or not germ cells were fixed early in life
Debate raised in 1921
Challenged by Allen, 1923 : formation of oocytes
throughout reproductive life
Dogma confirmed by Solomon Zuckerman, 1951 : oocytes
are not produced throughout life-span in most mammals.
Evidence for the presence of Germline Stem
Cells in the mammalian ovary (Johnson et al, 2004)
 Discordance in non-atretic follicle numbers versus atresia over time :
Existence of proliferative germ cells that sustain oocyte and follicle
production in the postnatal mouse ovary
Johnson, Tilly et al., March-2004, Nature
Evidence for the presence of Germline Stem
Cells in in the mouse (Johnson et al, 2004)
 Postnatal female germ-cell proliferation
 Histological analysis: juvenil and young adult
ovaries revealed the presence of large ovoid cells
in the surface epithelial layer covering the ovary
 Immunohistochemical: staining for Mouse Vasa
Homologue (MVH, a germline-specific marker)
confirmed that these large ovoid cells were of a
germline lineage
 BrdU staining: Proliferative potential of ovoid cells
Presence of mitotically-active germ cells in juvenile and
adult mouse ovaries
Purification of female germ cells
in the mouse (Zou et al, 2009)
 Neonatal or adult derived FGSC could reconstitute
ovarian function in adult female mice rendered sterile
by Busulfan and cyclophosphamide
 Transplantation of FGSC/GFP+ cells in ovaries of
chemo-ablated wild type female mice : GFP-positive
oocytes within follicles of all maturational stages
 Mating trial: offspring containing the GFP transgene
Existence of GSC in adult mammalian females able to generate
oocytes that can be fertilized and yield viable offspring
Purification of active germ cells in adult women
(White and Tilly, 2012)
 Described and validated a fluorescence cell sorting based protocol to isolate
mitotically active germ cells from mouse adult ovaries and human ovarian
cortex
 These cells could generate follicles/oocytes in vivo (xenotransplantation in
immunodeficient mice)
Ovaries of reproductive-age women possess rare mitotically
active germ cells
Conclusions
 Animals are a useful/helpful model in the development of
core of technologies that can be applied later on to humans
 New evidences that challenge the validity of the basic dogma
in reproductive biology that persisted for more than 50 years
 Clinical implications to postpone premature ovarian failure
Thank you for your attention
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