Infertility is a common and serious problem

in reproduction now-a-days. Animal culture

and tissue techniques provide a solution to
this problem through the techniques of IVF
and Embryo Transfer (ET).
 DEFINITION
Invitro fertilization( IVF) refers to the union
between egg cell and sperm outside body in a
culture vessel involving their collection and
fusion under appropriate conditions in vitro
to give rise to zygotes which are cultured in
vitro to obtain young embryos.
 The implantation of young embryos
developed in vitro or obtained from the
uterus of females into the womb of selected
females is termed as Embryo Transplantation
or Embryo Transfer.
STEPS INVOLVED-
 Collection of oocytes
 Collection of sperms
 IVF of the oocytes
 Implantation of the resulting zygotes in the

uterus.
COLLECTION OF OOCYTES
 Collected from females desirable of having
baby.
 Cannot be collected from females with non-

functional ovaries.
 Can be collected during a natural or induced

menstrual cycle.
 Time for this is determined by monitoring

rise in the level of Luteinizing Hormone(LH) in

urine or blood.
 Ovulation may be stimulated by administration of
Clomiphene or Human menopausal Gonadotropin
(hMG).
 Follicle development maybe arrested at the
optimum stage by administering Human chorionic
gonadotropin (hCG) so that ova are not released.
 Recovery of oocytes can be done most
conveniently by a laparoscopic instrument that
allows the visualization of ovary through a
monitor, aspiration (suction) of the follicular fluid
containing the oocytes and the necessary surgical
manipulation of ovary using sensors, laproscopic
scissors and an aspirating apparatus inserted into
the abdomen of the female via a suitable tube.
Fig. Collection of oocytes from ovary
COLLECTION OF SPERMS
 Collected about 60-90 minutes prior to
fertilization, liquefied and centrifuged.
 Resuspended in culture medium, and

incubated for 30-60 mins at 37⁰ C.

 The most active sperms are located in the

surface of the medium.

 If sperm collection from the prospective

father is not possible due to Oligospermia or

Azoospermia, semen is collected from a
suitable donor.
Fig. Procedure of collection, Centrifugation
and Suspension of sperms in culture medium
 FERTILIZATION OF THE
OOCYTES BY SPERMS IN VITRO
 Oocytes are identified by microscopic
examination of the follicular fluid aspirated
during laparoscopy, and are incubated for
10-15 hours depending upon the expected
time of maturation.
 Following mediums maybe used for serving

the purpose-
1. Modification of Ham’s F10 medium
2. Earl’s solution
3. Modified Whitten’s medium
4. Whittingham’s T6 medium
 All these media has the following in common-
 Utilizes a bicarbonate/CO2 buffer system to

keep PH in the range of 7.2-7.4.

 Osmolarity range of 275-290 osmol/kg.
  Temperature setting of 37.0-37.5°C

  Cultured under paraffin oil, which prevents

evaporation of the medium preserving a constant

Osmolarity and minimizes fluctuations of pH and
temperature.
 Media must have 99% water,.
 Albumin or synthetic serum in concentrations of 

5 to 20% w/w or v/v as protein source.

 Carbohydrates- Pyruvate, lactate and high conc.

of glucose.
 Amino acids- all 20 amino acids.
 Salt solutions- NaCl, KCl, KH₂PO₄ etc.
 Antibiotics- Penicillin, Streptomycin etc.
 Chelators like EDTA to prevent abnormal

glycosis.
 Fertilization is started by adding 10,000-
50,000 motile sperms to about 100 µl to 1 ml
culture medium in which the oocytes is being
incubated.
 The oocytes is examined after 12-13 hrs for

detection of –
1. Number of pronuclei and polar bodies
2. Granulation of the oocytes and
3. Shape of the oocytes
A normally fertilized oocyte (actually a zygote
now) contains two pronuclei and two
polar bodies. Any zygote other than this and
having abnormalities and granulation of any
kind are rejected.

The first division in the zygote occurs about

24-30 hrs after insemination, but each
subsequent division takes about 10-12 hrs.
Therefore, if an oocyte fails to divide by 30
hrs after insemination, it should not be used
for implantation.
Fig. In Vitro Fertilization
EMBRYO TRANSFER
The best stage of transfer of embryos to uterus is 2-4 celled stage.

Fig. Different stages of development of a blastocyte

 TECHNIQUE
 The patient is orally administered with Valium
before embryo transfer.
 The whole process is negotiated through

cervical canal.
STEPS INVOLVED-
 The patient is placed in lithotomy (knee
chest) position.
 A sterile bivalve speculum is inserted to

visualize the cervix.

 The cervical canal and uterine cavity are

aligned.
 The embryo is drawn into a Teflon catheter in

tissue culture medium. Teflon is used due to

its low adhesiveness.
 The catheter is inserted into the uterine cavity
just short of the fundus.
 The embryo is gently inserted in culture

medium and the catheter and cannula are

gently withdrawn.
 Catheter is examined under the microscope

to ensure that the embryo has been expelled.

Fig. Steps involved in Embryo Transfer
 The babies produced using these approaches
are called test tube babies.
 The first test tube baby was born on July 25,

1978 and was named Louise Joy Brown.

 The Nobel Prize of Medicine for the year 2010

was awarded to Robert G. Edwards for this

outstanding invention.
Fig. Louise Brown with her parents Fig. Robert G. Edwards with Louise
 SUCCESS RATE
 IVF success rates are the percentage of all IVF
procedures which result in a favourable
outcome, which implies Pregnancy rate
(Number of confirmed pregnancies) or Live
birth rate (Number of live births).
 Due to advancement in reproductive

technology, the IVF success rates are

substantially better today than they were just
a few years ago.
FACTORS AFFECTING SUCCESS RATE
 Maternal age
 Duration of infertility or sub-fertility
 FSH
 Number of oocytes
 Tobacco smoking
 High body mass index
 Alcohol/caffeine intake
EXPANSIONS TO THE TECHNIQUE
 Pre-implantation genetic screening or
diagnosis (PGS or PGD)-
Preimplantation genetic screening (PGS) or
preimplantation genetic diagnosis (PGD) has
been suggested to be able to be used in IVF
to select an embryo that appears to have the
greatest chances for successful pregnancy.

 Embryo splitting- It can be used for twinning

to increase the number of available embryos.
 Intra cytoplasmic sperm injection- It  is
an invitro fertilization procedure in which a
single sperm is injected directly into an egg.

 Assisted zona hatching- Assisted zona

hatching (AZH) is a procedure of assisted
reproductive technology in which a small hole
is made in the zona pellucida, using a
micromanipulation, thereby facilitating
for zona hatching to occur. Zona hatching is
where the blastocyst gets rid of the
surrounding zona pellucida to be able to
implant in the uterus.
 ETHICAL ISSUES
Laboratory mix-ups (misidentified gametes,
transfer of wrong embryos) have occurred,
leading to legal action against the IVF provider
and complex paternity suits.
An example is the case of a woman in California
who received the embryo of another couple and
was notified of this mistake after the birth of her
son.
This has led to many authorities and individual
clinics implementing procedures to minimise the
risk of such mix-ups. 
 The Catholic Church opposes all kinds of in
vitro fertilisation because, as with contraception, it
separates the procreative purpose of the marriage act
from its unitive purpose, i.e. to produce a new life.
 Another concern is that people will screen in or out for

particular traits.
A deaf British couple have petitioned to create a deaf
baby using IVF. Medical ethicists are against this form of
Pre-implantation genetic screening or diagnosis as
“intentionally culling out blind or deaf embryos might
prevent considerable future suffering, while a policy that
allowed deaf or blind parents to select for such traits
intentionally would be far more troublesome.”
 REFERENCES
 Elements of Biotechnology by P.K. Gupta;
pg.112
 Biotechnology Expanding Horizons by B.D.

Singh; pg.282
 www.ivf-worldwide.com

 www.ivf.com
 www.advancedfertility.com
 Wikipedia