In Vitro Fertilization

History, Concepts and Issues

Renz L. Salumbre
MS Zoology Program
Graduate School
UPLB
Generalities
 Definition

“In Vitro” meaning “in glass”

Fusion of gametes outside the womb

Fertilization in a laboratory setting

Laboratory simulation

Test tube babies

 Definition

Retrieval of more than one secondary

oocyte from women who have received
injections of follicle stimulating hormone
and fertilizing these oocytes in vitro
(Jansen, 2005)
 Definition
A method of assisted reproduction that
involves combining an egg with sperm in
a laboratory dish. If the egg fertilizes and
begins cell division, the resulting embryo
is transferred into the woman’s uterus
where it will hopefully implant in the
uterine lining and further develop. IVF
may be performed in conjunction with
medications that stimulate the ovaries to
produce multiple eggs in order to
increase the chances of successful
fertilization and implantation. IVF
bypasses the fallopian tubes and is often
the treatment choice for women who
have badlydamaged or absent tubes
 Purpose

Enhancing productivity of food animals

Conservation of endangered animals

Specimens for embryological research

Treatment for infertility

 Early History
Internal physiology was not yet fully
understood

Rabbits and Hamsters were commonly

used as laboratory specimens

Most researchers failed to provide sound

evidence

Most researchers misinterpreted their

results
 The Golden Age

Discovery of sperm capacitation

In experiments, most sperms were

capacitated in vivo before insemination

First use of a spermatozoa capacitated in

vitro was reported in 1977
 The Golden Age

In 1969, human eggs were fertilized in

vitro

Sperm penetration

Polar body emission

Pronuclei emission
 First IVF Baby
Collaboration between
Robert Edwards, a
physiologist, and Patrick
Steptoe, a gynecologist

First attempt produced

an ectopic pregnancy

Initially employed a
“natural” IVF

In July, 1978 the First

IVF Baby, Louise Brown
was born
 Modern Age

Fertility Clinics

Assisted Reproduction Technology

Research

Stem Cell

Human Admixed Embryos

Methods
 Methods

Ovarian Stimulation

Egg Retrieval

Insemination

Embryo Culture

Selection

Embryo Transfer
Ovarian Stimulation
Process is for 10 days but usually 8-14
days

Injection of hormones

gonadotropins e.g. FSH and FSH

analogues

GnRH agonists/antagonists

Clomid

hMG

Stimulate egg production per menstrual

cycle
 Egg Retrieval

HCG is given as an LH analogue

The removal of eggs from the ovaries

Follicular aspiration or Transvaginal

ultrasound aspiration

prior to HCG medication

Laparoscopic surgery may be employed

 Insemination

Sperm is separated from the semen

Removal of seminal fluids and inactive

cells

Added to a culture of eggs

Incubated
 Embryo Culture

Fertilization

Incubation period of 18 hours

Confirmation of formation of pronuclei

2-4 cell after 2 days of fertilization

6-10 cell after 3 days of fertilization

Blastocyst after 5 days of fertilization

 Culture Media

For in vitro culture of

follicles

male germinal cells

embryos

In the form of lyophilizate

 Culture Media
Contains hormones and growth factors in
equilibrium concentrations

Growth factors

hepatic growth factor

Transformation growth factor (a)

granulocyte-macrophage colony
stimulating factor

epidermal growth factor / heparin-

binding EGF
 Culture Media
Growth and Differentiating Factors
(GDF-9)

Insulin-like Growth Factors (IGF-1/IGF-

2)

Corticoids preferable hydrocortisone

In hydrosoluble salt

in the form of hydrocortisone

hemisuccinate

Coenzymes (NAD/NADH or
NADP/NADPH)
 Culture Media
IVF Kits include Upgraded B9 CCD
culture medium
Mineral salts: KCl, NaCl, MgSO4, NaHCO3, Na2HPO4, KH2PO4

Essential Amino Acids including Glutamic Acid, Glycine,

Taurine, Cystein, Glutamin

Metabolic Derivatives: Glucose, Pyruvate, Lactate,

Acetate

Vitamins: B group and C

Purine and Pyrimidine Bases

Antibiotics: penicillin G, streptomycin

 Selection

Oocyte and Embryo Quality

number of cells

evenness of growth

degree of fragmentation

6-10 cell stage are preferable

Blastocyst stage may also be transferred

 Embryo Transfer

3 days after fertilization

Eggs are soaked in liquid

Cervical cleansing

Abdominal ultrasound

Eggs are transferred back to the uterus

Positive pregnancy if eggs attached to

the uterine wall
 Embryo Transfer
3-day transfer

less costly and time-consuming

usually employed

risk of having many embryos

implanted

Blastocyst transfer

much more effective and few embryos

require

mimics natural process

costly
8-Cell Embryo for Transfer
Blastocyst for Embryo Transfer
Variant Methods
 Intracytoplasmic
Sperm Injection
ICSI is a recent micromanipulation technique

Single sperm is injected into the oocyte’s cytoplasm using

a pipette

Addresses male infertility

azoospermia

oligospermia

asthenospermia

teratospermia

Hypospadias and reproductive problems in children

conceived thru ICSI
Oocyte injected during ICSI
Zygote Intrafallopian
Transfer
A highly invasive and very expensive
ART procedure

Fertilized eggs are implanted into

fallopian tubes by laparoscopy.
Afterwards, it can pass down into the
uterus to undergo normal pregnancy

Risk of having multifoetal pregnancy

ZIFT
Gamete Intrafallopian
Transfer
Eggs are taken from the woman and
combined with wash sperm

Immediately placed in the fallopian tube

(ampullary region)

Process develops in a natural

physiological manner

Bypasses egg pick-up of fallopian tube

Allegedly, the only artificial conception

method accepted by the Roman Catholic
Church
Tubal Embryo Transfer

Embryos are transferred to the fallopian

tube

Used in conjunction with ZIFT

For patients who have difficult

transcervical intrauterine transfer
Issues
 Surrogacy
Two types

Traditional

surrogate is genetically related to

the child

Gestational
• surrogate is not genetically related
to the child
• Legal and psychological problems
 Assisted Hatching
• Improves implantation and specifically
for patients with poor IVF results
• Also lessens the number of embryo
being transferred
• A micromanipulation technique that
creates a small hole in the zona
pellucida of the embryo
• Risk of damaging the embryo
Prevention of Genetic
Diseases

• Preimplantation Genetic Diagnosis (PGD)

• Embryo screening
• Searches for unhealthy embryo
• Limits development of genetic
diseases
 Cryopreservation

• Unused eggs are stored for future use

• Excess embryos which are not implanted
are preserved to be further used when
prior IVFs are unsuccessful
 Sex Selection

• Preconception gender selection through

sperm separation
• Albumin gradients
• Percoll gradients
• Sephadex columns
• Swim-up techniques
• Flow cytometry
Ethical Considerations
• Objection to artificial conception
• Conception must be an act of the
consummation of marriage
• Rights of the child to be born
• Unregulated experimentation on human
embryos
• Does not give importance on human life
• “Playing” God
 Financial
Consideration

• In vitro Fertilization costs $12,000

• Gestational Surrogates cost $10,000
• Availability in third-world countries
 Biological Risks
• Ovarian Hyperstimulation Syndrome
• Infection and damaging of other
structures during laparoscopy
• Multiple Pregnancy
• Underweight Infants by IVF
• Increased likelihood of having a
handicap child
• Impediment of normal process may lead
to complications of normal physiological
processes
 Biological Risk
• Large Offspring Syndrome
• Survivor’s Syndrome
• Angelman Syndrome
• Beckwith-Wiedeman Syndrome
• Retinoblastoma
• Chimerism
•
References
American Society for Reproductive Medicine. Patient’s Fact Sheet : Intracytoplasmic Sperm Injection.
http://www.asrm.org. Created August, 2001.

• American Society for Reproductive Medicine. (2006). Third party reproduction: A guide for patients.
http://www.asrm.org.

• Bavister, B.D. Early history of in vitro fertilization. Reproduction 2002; 124, 181-196.

• Bellini, C (interview). In vitro children and the risks they face. http://www.zenit.org/article-10293?|
=english. Created June 6, 2006.

• Choay, P. & S. Weinman. Cellular culture medium, particularly for in vitro fertilization, or for the culture of
follicles, male germ cells or embryos. (August 26, 2003). United States Patent.

• Ethics Committee of the American Society for Reproductive Medicine. Preconception gender selection for
nonmedical reasons. Fertility and Sterility 2001; 75; 5: 861-864.

• Jansen, R.P.S. Benefits and challenges brought by improved results from in vitro fertilization. Internal
Medicine Journal 2005; 35: 108-117.

• http://www.sharedjourney.com

• Ratzinger, J. Instruction on respect for human life in its origin and on the dignity of procreation: Replies
to certain questions of the day. Congregation for the Doctrine of the Faith. February, 1987.
http://www.vatican.va

• Wikipedia.org. In vitro fertilisation. http://en.wikipedia.org/wiki/In_vitro_fertilization. Retrieved August 19,

2008.