A. Introduction

IVF is the basic assisted reproduction technique, in which fertilization occurs in vitro (literally, in glass). The sperm and the egg are combined in a laboratory dish, and after fertilization, the resulting embryo is then transferred to the female's uterus. The first successful IVF offspring were rabbits born in 1959. The next success came with laboratory mice in 1968. In 1978, the first IVF baby, Louise Brown, was born. Since that time, this procedure has been used with increased success rates to produce offspring from patients with various infertility problems. The first live calf was born in 1981.

B. Procedure of IVF

The basic requirements for IVF are healthy ova, sperm that can fertilize, and a uterus that can maintain a pregnancy.

Five general steps are followed for IVF :-

1. Superovulation: Treatment cycles are typically started on the third day of menstruation and consist of a regimen of fertility medications to stimulate the development of multiple follicles of the ovaries. In most patients injectable gonadotropins (usually FSH analogues) are used under close monitoring. Such monitoring frequently checks the estradiol level and, by means of gynecologic ultrasonography, follicular growth. Typically approximately 10 days of injections will be necessary. Spontaneous ovulation during the cycle is typically prevented by the use of GnRH agonists or GnRH antagonists, which block the natural surge of luteinising hormone (LH).

2. Egg Retrieval: When follicular maturation is judged to be adequate, human chorionic gonadotropin (hCG) is given. This agent, which acts as an analogue of luteinising hormone, would cause ovulation about 42 hours after injection, but a retrieval procedure takes place just prior to that, in order to recover the egg cells from the ovary. The eggs are retrieved from the patient using a transvaginal technique involving an ultrasound-guided needle piercing the vaginal wall to reach the ovaries. Through this needle follicles can be aspirated, and the follicular fluid is handed to the IVF laboratory to identify ova. It is common to remove between ten and thirty eggs. The retrieval procedure takes about 20 minutes and is usually done under conscious sedation or general anesthesia.

3. Fertilization: In the laboratory, the identified eggs are stripped of surrounding cells and prepared for fertilization. In the meantime, semen is prepared for fertilization by removing inactive cells and seminal fluid. If semen is being provided by a sperm donor, it will usually have been prepared for treatment before being frozen and quarantined, and it will be thawed ready for use. The sperm and the egg are incubated together (at a ratio of about 75,000:1) in the culture media for about 18 hours. In most cases, the egg will be fertilized by that time and the fertilized egg will show two pronuclei. In certain situations, such as low sperm count or motility, a single sperm may be injected directly into the egg using intracytoplasmic sperm injection (ICSI). The fertilized egg is passed to a special growth medium and left for about 48 hours until the egg has reached the 6-8 cell stage.

4. Selection: Laboratories have developed grading methods to judge oocyte and embryo quality. Typically, embryos that have reached the 6-8 cell stage are transferred three days after retrieval. In many American and Australian programmes, however, embryos are placed into an extended culture system with a transfer done at the blastocyst stage at around five days after retrieval, especially if many good-quality embryos are still available on day 3. Blastocyst stage transfers have been shown to result in higher pregnancy rates. In Europe, transfers after 2 days are common.

5. Embryo Transfer: Embryos are graded by the embryologist based on the number of cells, evenness of growth and degree of fragmentation. The number to be transferred depends on the number available, the age of the woman and other health and diagnostic factors. In countries such as the UK, Australia and New Zealand, a maximum of two embryos are transferred except in unusual circumstances. In the UK and according to HFEA regulations, a woman over 40 may have up to three embryos transferred, whereas in the USA, younger women may have many embryos transferred based on individual fertility diagnosis. Most clinics and country regulatory bodies seek to minimise the risk of pregnancies carrying multiples. The embryos judged to be the "best" are transferred to the patient's uterus through a thin, plastic catheter, which goes through her vagina and cervix. Several embryos may be passed into the uterus to improve chances of implantation and pregnancy

C. Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection (ICSI) is an in vitro fertilization procedure in which a single sperm is injected directly into an egg. This procedure is most commonly used to overcome male infertility problems, although it may also be used where eggs cannot easily be penetrated by sperm, and occasionally as a method of in vitro fertilization, especially that associated with sperm donation. The procedure is done under a microscope using multiple micromanipulation devices (micromanipulator, microinjectors and micropipettes). A holding pipette (on the left of picture) stabilizes the mature oocyte with gentle suction applied by a microinjector. From the opposite side a thin, hollow glass micropipette is used to collect a single sperm, having immobilized it by cutting its tail with the point of the micropipette. The micropipette is pierced through the oolemma and into the inner part of the oocyte (cytoplasm). The sperm is then released into the oocyte. The pictured oocyte has an extruded polar body at about 12 o'clock indicating its maturity. After the procedure, the oocyte will be placed into cell culture and checked on the following day for signs of fertilization. In natural fertilization sperm compete and when the first sperm penetrates the oolemma, the oolemma hardens to block the entry of any other sperm. Concern has been raised that in ICSI this sperm selection process is bypassed and the sperm is selected by the embryologist without any specific testing. However, in mid 2006 the FDA cleared a device that allows embryologists to select mature sperm for ICSI based on sperm binding to hyaluronan, the main constituent of the gel layer (cumulus oophorus) surrounding the oocyte. The device provides microscopic droplets of hyaluronan hydrogel attached to the culture dish. The embryologist places the prepared sperm on the microdot, selects and captures sperm that bind to the dot. Basic research on the maturation of sperm shows that hyaluronan-binding sperm are more mature and show fewer DNA strand breaks and significantly lower levels of aneuploidy than the sperm population from which they were selected. A brand name for one such sperm selection device is PICSI. There is some suggestion that birth defects are increased with the use of IVF in general and ICSI specifically, though results of different studies differ. In a summary position paper, the Practice Committee of the American Society of Reproductive Medicine has said it considers ICSI safe and effective therapy for male factor infertility, but may carry an increased risk for the transmission of selected genetic abnormalities to offspring, either through the procedure itself or through the increased inherent risk of such abnormalities in parents undergoing the procedure.

D. Applications of IVF:-

• Treatment of human and animal infertility
• Surrogate Pregnancy
• Livestock improvement
• Preimplantation Genetic Diagnosis (PGD)
• Harvesting of Embryonic Stem Cells
• Maintenance of genetic pool for long intervals - Embryo Cryopreservation
• Gene transfer
• Increase in survivability of Endangered Species

E. Preimplantation Genetic Diagnosis

Preimplantation genetic diagnosis (PGD or PIGD) (also known as embryo screening) refers to procedures that are performed on embryos prior to implantation, sometimes even on oocytes prior to fertilization. PGD is considered another way to prenatal diagnosis. Its main advantage is that it avoids selective pregnancy termination as the method makes it highly likely that the baby will be free of the disease under consideration. PGD thus is an adjunct to assisted reproductive technology, and requires in vitro fertilization (IVF) to obtain oocytes or embryos for evaluation. The term preimplantation genetic screening (PGS) is used to denote procedures that do not look for a specific disease but use PGD techniques to identify embryos at risk. PGD is a poorly chosen phrase because, in medicine, to "diagnose" means to identify an illness or determine its cause. An oocyte or early-stage embryo has no symptoms of disease. They are not ill. Rather, they may have a genetic condition that could lead to disease. To "screen" means to test for anatomical, physiological, or genetic conditions in the absence of symptoms of disease. So both PGD and PGS should be referred to as types of embryo screening. Generally PGD is performed on an embryo which is in the 8 cell stage. A single cell is withdrawn using micromanipulators and is screened using either PCR or FISH.

F. Advantages in Cell Culture:-

• Rapid Expansion of animal population
• Rescue of gene pool from dead/frozen animals
• Maintenance of lines with very low sperm count
• Sperm - DNA co-incubation for genetic transformation
• Genetic manipulation is easy as mating of animals and harvesting of embryos is avoided
• Large No. of zygotes can be made at a low cost
• Embryos are free from viral diseases in infected parents

G. Disadvantages of IVF:-

• Not applicable in case of malformed eggs from female
• Large Offspring Syndrome
• Embryo and Ovum damage due to manipulation
• Risk of Multiple gestation
• Ethical Issues -
i) Creation and discard of large No. of Embryos
ii) Malicious Genetic / Sex selection in Humans
iii) Tools can be used for cloning
• Some Techniques are Expensive

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