In Vitro FertilizationHistory, Concepts and Issues

Renz L. SalumbreMS Zoology Program

Graduate SchoolUPLB

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)

DefinitionA 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 BabyCollaboration 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

Patients are closely monitored

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 MediaContains 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 MediaGrowth 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 MediaIVF 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 Transfer3-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

SurrogacyTwo 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.