The oocyte

From GV to MII
Julius Hreinsson

Laboratory director
Reproductive Medical Centre
Uppsala University Hospital

Overview
• Ovarian reserve, oocyte maturation
• Follicle growth from the oocytes point of view
• Nuclear vs. cytoplasmic maturation
• Optimisation of methods
• IVM - In Vitro Maturation

Ovary
• Cortex, central medulla
• Produces germ cells and hormones
 Ovary
• Primordial follicles in a dense stroma
• Grow inwards towards the medulla when follicle
maturation commences

Primordial Primary Secondary

Follicle growth
• Long process, >100 days (estimated 220 days), the
initial stages are independent of gonadotrophins
• After antrum formation the follicles become dependent
on FSH for continued development
• The oocyte communicates with surrounding granulosa
cells and partially controls its own development
• The ”health” of the follicle is reflected in the
developmental potential of the oocyte
– Blood flow, growth factors

Ovarian reserve

• Oocyte numbers
decrease with age
• Independent of IVF
treatment
• Approximately
A i l 1000
follicles disappear every
months, 999 become
atretic, 1 matures

Infertilitet 2005, Landgren & Hamberger

 When does the fertile period start?

• 1.st attempt at
pregnancy at
the age of 38
years

Infertilitet 2005, Landgren & Hamberger

Ovarian reserve
• Primordial follicles, changes with age

Follicle growth
• A continuous trickle of primordial follicles are released
from dormancy throughout reproductive life
• The oocyte plays an active and dominant role in
directing follicle growth – bidirectional communication
• Captain of the follicle instead of a passenger
 Follicle growth
• Pre-antral follicle growth occurs independent of
endocrine stimuli
• Early stages show oocyte growth, limited granulosa cell
proliferation
• GDF-9
• BMP-15
• Kit-L

Hutt, Albertini, RBM Online, 2007

Growth factors in follicle growth

• GDF-9: Stimulates granulosa cell growth and cumulus
cell mucification, reduces apoptosis
• BMP-15: Stimulates granulosa cell growth
– Both are very important at early and later stages of oocyte and
follicle growth
• Kit-Ligand: Produced by pre-antral granulosa cells,
promotes oocyte growth
• Granulosa cells transfer important metabolites to the
oocyte

Follicle growth
• Later stages show granulosa cell proliferation and
cytoplasmic maturation of the oocyte
• FSH

Hutt, Albertini, RBM Online, 2007

 Follicle growth
• Signals regulating growth initiation of primordial follicles
are still unknown - not conclusively confirmed
– Probably involve AMH (anti-Müllerian hormone) which inhibits
initiation
– c-kit and kit-ligand which stimulate growth initiation
• At the antral stage
stage, the follicles become dependent on
gonadotrophins for growth and development
• That is why neither FSH stimulation nor birth control pills
affect ovarian reserve of follicles
• Granulosa cells inhibit meiotic progression through
transfer of meiosis-arresting signals via gap junctions
• High levels of c-AMP in the oocyte maintain meiotic arrest

Follicle growth
• Trans-zonal
projections between
cumulus cells and
oolemma
• Bi-directional
communication

Hutt, Albertini, RBM Online, 2007

Follicle and oocyte growth

• Telfer& McLaughlin, RBM Online 2007

 Oocyte growth
• Nuclear maturation – ability to resume meiosis. Can be
initiated by removal from the antral follicle
• Cytoplasmic maturation - relocation of organelles, synthesis
of proteins, modification of mRNAs
• Imprinting processes
• Of these complex processes, only GV-breakdown/polar
body extrusion are visible in the light microscope. Complex
processes occur on the molecular level – microscopic
evaluation gives limited information on oocyte status
• High oocyte quality is a prerequisite for good embryo quality
and foetal viability

Final oocyte maturation

• Germinal vesicle (GV), the visible nucleus disappears,
1.st polar body is formed, granulosa cells withdraw from
the cell membrane of the oocyte
• Flow of cAMP and nutrients to the oocyte stops,
inhibition of maturation ceases

GV MI MII
sathembryoart.com

Oocyte capacity and fertilisation

Oocyte secreted factors facilitate

cumulus expansion: GDF 9, BMP 15
Synthesis of ZP proteins
p
Sperm binding,
g, g
gamete fusion
Oocyte activation
Sperm processing
Formation and migration of pronuclei
Important stages in fertilization
Swain & Pool HR Update 2008
 How to optimize the maturation period?
• Optimal time period from aspiration to ICSI
• Selection of sperm and injection with physiological
substances
• Should we use MI oocytes for ICSI?
• What happens if vital processes are disrupted? IVM

Temperature sensitivity
• Microtubules are temperature
sensitive – start to decompose
at temperatures under 35°C
• Reformation after re-heating is
not effective
• Aneuploidy
• At temperatures under 30°C
the oocyte is destroyed
• Work on a heating plate,
continuous temperature
control

Aneuploidy in oocytes and embryos

• Control mechanisms for chromosome arrangement
on the metaphase plate are ineffective for the first
three days of embryo development
• Separation of chromosomes in meiosis or mitosis
may be defective
• Anomalies occur both before and after fertilization
• Mosaicism
• The oocyte is extremely sensitive
to environmental fluctuations
 Optimal time from aspiration to ICSI
• Pre-incubation of oocytes before ICSI is beneficial
(Rienzi et al., 1998; Isiklar et al., 2004)
• Most appropriate incubation time for mature oocytes
before ICSI is 5-6 hrs (Falcone et al., 2008)

Selection of sperm and injection

• Polyvinylpyrrolidone (pvp) is a plastic substance
• Hyaluronic acid (HA) is a naturally occurring
alternative which is biodegradable
• HA seems to play an important role in physiological
sperm selection
l ti - presentt in
i ECM iin cumulus
l
• Sperm which are able to bind to HA in vitro
– Plasma membrane remodelling and maturity
– Lower DNA fragmentation (5% vs 11% in pvp), less nuclear
anomalies 14,5% normal in HA vs 11% normal in pvp),
better embryo quality
– Parmegiani et al., 2009

Immature oocytes for ICSI

• MI oocytes at ICSI, exposed to gonadotrophins, still
immature
• Strassburger et al., 2009: Matured for 2, 4-8 and 24
hrs
• Nuclear maturation achieved in up to 72% of MI
oocytes at 24 hrs (30%, 62%, 72%) - tempting to use
• Lower fertilisation rates, 51% vs 71%, poorer embryo
quality
• High aneuploidy rates (40-100%), especially after
prolonged culture
• Reduced cytoplasmic competence - avoid use
 In-vitro Maturation IVM
• IVM-culture for 32-36 hours, then ICSI (IVF)
• Maturation medium with 10% patient serum, collected on
the day of OPU, FSH and hCG added to the culture
medium
• Patients at risk of OHSS,
OHSS for example PCOS - OHSS is
avoided; Younger women with good ovarian reserve;
Male factor infertility

In-vitro Maturation IVM

• Clinical pregnancy rates 20-30% per transfer (Suikkari
2007)
• In most publications somewhat lower results than
after conventional IVF / ICSI
• Varying results may be explained partly by different
patient groups and numbers of embryos for ET
• Better results in IVM cycles with in-vivo matured
oocytes (Son et al., 2008)
• Are IVM-oocytes competent to support embryo and
foetal development?

In-vitro Maturation IVM

• Reported rates of miscarriage are between 22-57%
– Lin et al. 2003 22%
– Chian et al. 2000 25%
– Buckett et al. 2007 25%
– Le Du et al. 2005 33%
– Söderström Anttila et al. 2005 36%
– Cha et al. 2005 37%
– Child et al. 2001 40%
– Mikkelsen, Lindenberg 2001 57%
• Not possible to calculate the mean, but clearly
elevated
 In-vitro Maturation IVM
• Miscarriage rates have been proposed to be PCO-
related (Buckett et al. 2007), however other groups
with even higher rates of miscarriage have not had a
majority of PCO patients

• Rates of caesarean section are also elevated

– Söderström Anttila et al. 2005 35%
– Buckett et al. 2007 39%
• Obstetric complications such as bleeding are usually
not reported

In-vitro Maturation IVM

• Higher birth weight after IVM compared to IVF or
national average - IVF is usually under the national
average except for cryopreserved embryos
– Cha et al. 2005 3252g vs. 3165g (Korean average)

– Mikkelsen et al. 2005 3720g vs. 3532g (Danish average)

vs. 3457g (IVF/ICSI,
Pinborg)

– Buckett et al. 2007 3482g vs. 3260g (controls)

vs. 3189g (IVF/ICSI)

– Söderström Anttila 2006 3550g vs. 3541g (controls)

vs. 3364g (IVF/ICSI)

In-vitro Maturation IVM

• The differences are not great, but the pattern is clear
and the number of children born is relatively high
• Same pattern is seen after IVM in farm animals
– “Large offspring syndrome”, also associated with other
complications
• How can we explain these observations?
• What mechanism might cause higher miscarriage
rates, higher rates of pregnancy interventions and
higher birth weight
 In-vitro Maturation IVM
• Genomic imprinting is achieved at different stages in
germ cell development
• During embryonic development, germ cells erase
imprinting marks and establish new imprinting
depending on their sex (Reik,
Reik Walter 2001; Tada et al 1998)
– Male germ cells re-establish their imprinting marks at the
round spermatid stage (Shamanski et al 1999)
– Oocytes complete their imprinting just before ovulation in
each ovulatory cycle (Schaefer et al 2007; Obata et al 1998)
– It is logical that imprinting defects are more likely to affect
oocytes then sperm
– Imprinting effects may be most frequently seen in placenta

In-vitro Maturation IVM

• Endometrial effects
• Growth factor deficiency - GDF 9
• IVF culture media are moving from rich to
focused/specified
• Some indications that this decreases risk for
epigenetic effects (Marees et al., 2009)
• IVM uses serum for culture in the human
• c-AMP modulators
• IVF has not needed to prove it's safety, only to show
it is not unsafe. This situation may turn around – and
it has – for IVM

Conclusions
• Knowledge of detailed mechanisms of oocyte
maturation, development and morphology allows us
to improve results of ART treatments
• Optimising oocyte competence is one of the key
issues in optimising results in ART
Thank you for your attention!