Invitro Fertilization
In vitro fertilization (IVF)
• In vitro fertilization involves
– induction of superovulation,
– monitoring of follicular growth,
– oocyte retrieval and
– in vitro fertilization. This is followed by
– embryo transfer
Oocyte retrieval
• Oocyte retrieval by laparoscopy has now been
replaced by vaginal sonography.
• vaginal needle aspiration is carried out about 36
hours after hCG administration, but before
ovulation.
• The oocyte is recognizable as a single cell
surrounded by a mass of cumulus cells.
• The procedure requires intravenous analgesia
and sedation only.
Culture of Oocytes
•
After oocyte recovery, depending upon the estimated maturity of the
follicles and oocytes, they are incubated for 5-10 hours.
• For oocyte culture and in vitro fertilization the different culture media
commonly used are
– 1. modified Ham's F10 medium,
– 2. Earl's solution.
– 3. modified Whitten's Culture medium
– 4. Whittingham's T6 medium (Trounson and Counti, 1982).
• The culture medium is prepared weekly and tested by culturing mouse
embryos from 2-cell stage to blastocyst stage.
• The same medium is used for fertilization and culture techniques
Preparation of semen
• 60-90 minutes before insemination, semen is collected
by masturbation.
• I t is then liquified, centrifuged and the resultant sperm
pellet is re-suspended in culture medium.
• After re-centrifugation, the washed spermatozoa are
incubated in culture medium at 37°C for 30-60 minutes.
• A sample taken from the surface will contain the most
active spermatozoa.
In vitro fertilization
• 10,000 to 50,000 motile spermatozoa are prepared as described above.
• It is then added to 10μ l-1 ml of culture medium containing oocyte.
• After insemination, the oocyte is left for 12-13 hours.
• The oocytes are examined for two pro-nuclei and two polar bodies.
• Any abnormal oocytes having multiple pronuclei, granulation or vesiculature
of the cytoplasm or abnormal shape are not employed for embryo transfer.
• After insemination 1st cleavage occurs after 24-30 hours.
• Each subsequent division occurs within 10-12 hours thereafter.
• Absence of cleavage within 24 hours suggests abnormal oocyte and should
not be used for transfer.
Embryo transfer (ET)
• Embryos of 1- to 16-celled stages can be transferred to
the uterus leading to successful development.
• In vivo, upto 8-16-celled stage embryo stay in the
fallopian tube after which they move to the uterus.
• More advanced embryos are not suitable for transfer.
• Prolonged in vitro culture of embryo reduces viability and
thus reduces the rate of pregnancy.
• 2-4-celled stage is the best stage, though these early
embryos may not survive in the uterus.
• Embryo transfer is carried out through the cervical canal.
• The embryo is drawn into a teflon catheter (external diameter 1.27 mm,
internal diameter 1.0 mm) in 10 μl of tissue culture medium.
• The catheter is then passed down an outer teflon sheath and is
inserted into the uterine cavity
• Teflon sheath protects the catheter from vaginal contamination.
• The embryo is gently injected along the culture medium and the
catheter and the canula are withdrawn.
• Microscopic observation of catheter confirms deposition of embryo.
• Incorrect placement, excess culture medium, etc., may lead to
expulsion of embryo from uterus leading to tubal pregnancy.
Catheter
Embryo Transfer Technology
• Young embryos of cattle of superior genotype are
collected prior to their implantation in uterus, and
are implanted in the uterus of other females of
inferior genotype where they complete
development; this is called embryo transfer.
• The chief objective of embryo transfer is to obtain
several progeny per year from a single female of
superior genotype.
• A genetically superior and high productivity female serves as the
donor of embryos to be transferred.
• Healthy, young females of inferior genotype are selected to be the
recipients of embryos to be transferred; these females are called
surrogate or substitute mothers.
• The donor females are treated with appropriate doses of the selected
gonadotrophin, e.g., follicle stimulating hormone (FSH) or luteinising
hormone (LH), to increase the number ova released at the time of
ovulation; this is called superovulation.
• Under optimum treatment conditions, a single female can provide up
to 15 embryos in a single cycle.
• When the donor female is in heat, it is artificially inseminated using
semen from a genetically superior bull of top pedigree.
•
• The fertilized eggs/young embryos are collected by flushing the
uterus of donor females with a special nutrient solution 7 days after
the insemination.
• The embryos are examined under a stereoscopic microscope and
normal looking healthy embryos are selected.
• The selected embryos are incubated in a special nutrient medium at
37°C till their transfer into the surrogate mothers. Alternatively, they
may be frozen and stored in liquid nitrogen for future use.
• A single embryo is transferred into the uterus of each surrogate
mother.
• It is important that the oestrus cycles of donor and surrogate mothers
are synchronized by administering prostaglandins to provide the
optimum uterine environment for survival, establishment and normal
development of the young embryos.
