PHY3181 Lecture Notes - Lecture 10: Aneuploidy, Pronucleus, Cell Potency
30 Jun 2018
School
Department
Course
Professor
Lecture 16: Insight into IVF
• IVF uses mature eggs that haven’t
ovulated looking for eggs in the
follicular fluid
• Several ultrasounds will occur before
this to know how many follicles we are
expecting
• Number follicles does not equal the
number of oocytes retrieved we
only want the large follicles
• Average oocyte number is 8-10
o Very different for each patient
o PCOS = more eggs
o Older = less eggs
• Conditions are tightly controlled –
under 32degrees at egg collection can
adversely affect the metaphase
spindle
• Once the follicular fluid has been
aspirated the oocytes are removed
from the fluid in an IVF chamber and
placed into culture media
• Tries to bypass the processes in the
fallopian tubes and put the embyro
back at around day 5
The normal fertilisation implantation
process that occurs naturally
In detail process of IVF
1. Egg retrieval (day 0): the follicles o the
ovary are aspirated, follicular fluid is
searched for eggs
2. Eggs are incubated at 37deg, with CO2
and O2
3. Insemination: by microinjections or
standard insemination (3-6hours after
egg retrieval)
4. Day 1: fertilisation check occurs (16-18
hours after insemination)
5. Day 3: embryo divides into 8 cells
into culture to support further
development
6. Day 5: embryo contains around 60
cells ICM and trophectoderm
present (2 cell types)
Option 1: can then freeze using vitrification
later used in FET (frozen embryo transfer)
• Use a strong dose of cryoprotectors
• Freeze very rapidly, room T to -196 deg
in less than 1 sec
• Uses liquid nitrogen
• Can leave indefinitely
Option 2: embryo transfer (usually a single
embryo is transferred)
Throughout this process there are strict
conditions maintained in the lab look for
volatile organic compounds ect, monitor the
amount of O2 (less than 10%). High O2 is
detrimental to embryo can result in larger
babies, poorer outcomes for patients. Note
that the growth media contains anti-oxidants.
Light exposure is minimised. Slow, gentle
pipetting is used.
There are differences in the environments in
the oviduct and uterus
The fallopian tubes and uterus secrete
compounds at different concentrations – try to
mimic the relative environment in the lab for
these two distinct environments.
• High concentration of pyruvate and
lactate I the oviduct
• Hypoxic environment in the uterus
• The uterus has a high availability of
amino acids
*************************************
There are differences in the physiology of the
mammalian embryo for development from
the zygote to the blastocyst stage
• At the pre-compaction stage and the
post-compaction stage – there are
different needs, these two stages have
different mitochondria
• Pre-compaction: immature
mitochondria, pyruvate metabolism
• Post-compaction: mature
mitochondria, use glucose
Between day 1 and day 3 the embryo
undergoes three cell divisions up until day2
the maternal genome is used.
On day 2 to day 3 there is a switch to the
embryonic genome. If this switch does not
occur the embyro will still at the 4 cell stage at
day 3 (when it should form 8)
Pre-compaction stage:
• Low biosynthetic activity
• Maternal genome
• Totipotent
• No cell cell communication
Post-compaction stage:
• High biosynthetic activity
• Embryonic genome
• Differentiation into ICM and
trophectoderm
• Complex systems for maintenance of
cellular homeostasis
*************************************
Oocyte maturation
• Only mature oocytes will fertilise
only inject or fertilise mature eggs
• Majority of the eggs will be mature (at
metaphase II) in the mature egg you
can see the polar body (assume
cytoplasmic maturity). Note that the
nuclear and cytoplasmic maturation
do not always align
Note that for microinjection we have to
remove the support cells
Immature egg can see the germinal vesicle
Immature egg there is no germinal center,
but no polar body
Mature egg at metaphase II can see the
polar body
*************************************
Sperm parameters and preparation
Document Summary
The normal process that occurs naturally fertilisation implantation. In detail process of ivf: egg retrieval (day 0): the follicles o the ovary are aspirated, follicular fluid is searched for eggs, eggs are incubated at 37deg, with co2 and o2. Icm and trophectoderm cells present (2 cell types) Option 2: embryo transfer (usually a single embryo is transferred) Throughout this process there are strict conditions maintained in the lab look for volatile organic compounds ect, monitor the amount of o2 (less than 10%). High o2 is detrimental to embryo can result in larger babies, poorer outcomes for patients. There are differences in the environments in the oviduct and uterus fallopian tubes and uterus secrete. Between day 1 and day 3 the embryo undergoes three cell divisions up until day2 the maternal genome is used. On day 2 to day 3 there is a switch to the embryonic genome.
