In-vitro Fertilization

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 / heparinbinding EGF

Culture Media Growth and Differentiating Factors (GDF-9) Insulin-like Growth Factors (IGF-1/IGF2) 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.