In vitro fertilisation

In vitro fertilisation (IVF) is a process by which egg cells are fertilised by sperm outside the body: in vitro. IVF is a major treatment in infertility when other methods of assisted reproductive technology have failed. The process involves hormonally controlling the ovulatory process, removing ova (eggs) from the woman's ovaries and letting sperm fertilise them in a fluid medium. The fertilised egg (zygote) is then transferred to the patient's uterus with the intent to establish a successful pregnancy. The first successful birth of a "test tube baby", Louise Brown, occurred in 1978. Robert G. Edwards, the doctor who developed the treatment, was awarded the Nobel Prize in Physiology or Medicine in 2010. Before that, there was a transient biochemical pregnancy reported by Australian Foxton School researchers in 1953 and an ectopic pregnancy reported by Steptoe and Edwards in 1976. At the same time, Subash Mukhopadyay, a relatively unknown physician from Kolkata, India was performing experiments on his own with primitive instruments and a house hold refrigerator and this resulted in a test tube baby, later named as "Durga" (alias Kanupriya Agarwal) who was born on October 3, 1978.

The term in vitro, from the Latin root meaning in glass, is used, because early biological experiments involving cultivation of tissues outside the living organism from which they came, were carried out in glass containers such as beakers, test tubes, or petri dishes. Today, the term in vitro is used to refer to any biological procedure that is performed outside the organism it would normally be occurring in, to distinguish it from an in vivo procedure, where the tissue remains inside the living organism within which it is normally found. A colloquial term for babies conceived as the result of IVF, "test tube babies", refers to the tube-shaped containers of glass or plastic resin, called test tubes, that are commonly used in chemistry labs and biology labs. However, in vitro fertilisation is usually performed in the shallower containers called Petri dishes. One IVF method, Autologous Endometrial Coculture, is actually performed on organic material, but is still considered in vitro.

Indications

IVF may be used to overcome female infertility in the woman due to problems of the fallopian tube, making fertilisation in vivo difficult. It may also assist in male infertility, where there is defect sperm quality, and in such cases intracytoplasmic sperm injection (ICSI) may be used, where a sperm cell is injected directly into the egg cell. This is used when sperm have difficulty penetrating the egg, and in these cases the partner's or a donor's sperm may be used. ICSI is also used when sperm numbers are very low. ICSI results in success rates equal to those of IVF.

For IVF to be successful it typically requires healthy ova, sperm that can fertilise, and a uterus that can maintain a pregnancy. Due to the costs of the procedure, IVF is generally attempted only after less expensive options have failed.

IVF can also be used with egg donation or surrogacy where the woman providing the egg isn't the same who will carry the pregnancy to term. This means that IVF can be used for females who have already gone through menopause. The donated oocyte can be fertilised in a crucible. If the fertilisation is successful, the embryo will be transferred into the uterus, within which it may implant.

IVF can also be combined with preimplantation genetic diagnosis (PGD) to rule out presence of genetic disorders. A similar but more general test has been developed called Preimplantation Genetic Haplotyping (PGH).

Method

Theoretically, in vitro fertilisation could be performed by collecting contents from a woman's fallopian tubes or uterus after natural ovulation, mix it with semen from a man and reinsert into the uterus. However, without additional techniques, the chances of pregnancy would be extremely small. Such additional techniques that are routinely used in IVF include ovarian hyperstimulation to retrieve multiple eggs, ultrasound-guided transvaginal oocyte retrieval directly from the ovaries, egg and sperm preparation, as well as culture and selection of resultant embryos before embryo transfer back into the uterus.

Ovarian hyperstimulation

There are two main protocols for stimulating the ovaries for IVF treatment. The long protocol involves downregulation (suppression or exhaustion) of the pituitary ovarian axis by the prolonged use of a GnRH agonist. Subsequent ovarian hyperstimulation, typically using follicle stimulating hormone (FSH), starts once the process of downregulation is complete, generally after 10 to 14 days.

The short protocol skips the downregulation part, 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 antagonists that are used just during the last days of stimulation to block the natural surge of luteinising hormone (LH) and allow the physician to start the ovulation process by using medication, usually injectable human chorionic gonadotropins.

Ovarian stimulation carries the risk of excessive stimulation. This complication is life-threatening and ovarian stimulation using gonadotrophins must only be carried out under strict medical supervision

Egg retrieval

Further information: Transvaginal oocyte retrieval

When the ovarian follicles have reached a certain degree of development, final maturation is induced, generally by an injection of human chorionic gonadotropin (hCG). Commonly, this is known as the "trigger shot. hCG acts as an analogue of luteinising hormone, and ovulation would occur between 38 and 40 hours after a single HCG injection, but the egg retrieval is performed at a time usually between 34 and 36 hours after hCG injection, that is, just prior to when the follicles would rupture. This avails for scheduling the egg retrieval procedure at a time where the eggs are fully mature. HCG injection confers a risk of ovarian hyperstimulation syndrome. Using a GnRH agonist instead of hCG eliminates the risk of ovarian hyperstimulation syndrome, but with a delivery rate of approximately 6% less than with hCG.

The eggs are retrieved from the patient using a transvaginal technique called transvaginal oocyte retrieval, 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 anaesthesia.

Egg and sperm preparation

In the laboratory, the identified eggs are stripped of surrounding cells and prepared for fertilisation. An oocyte selection may be performed prior to fertilisation to select eggs with optimal chances of successful pregnancy. In the meantime, semen is prepared for fertilisation by removing inactive cells and seminal fluid in a process called sperm washing. 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.

Fertilisation

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 fertilised by that time and the fertilised 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 fertilised egg is passed to a special growth medium and left for about 48 hours until the egg consists of six to eight cells.

In gamete intrafallopian transfer, eggs are removed from the woman and placed in one of the fallopian tubes, along with the man's sperm. This allows fertilisation to take place inside the woman's body. Therefore, this variation is actually an in vivo fertilisation, not an in vitro fertilisation.

Embryo culture

Typically, embryos are cultured until having reached the 6–8 cell stage three days after retrieval. In many Canadian, 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.

Culture of embryos can either be performed in an artificial culture medium or in an autologous endometrial coculture (on top of a layer of cells from the woman's own uterine lining). With artificial culture medium, there can either be the same culture medium throughout the period, or a sequential system can be used, in which the embryo is sequentially placed in different media. For example, when culturing to the blastocyst stage, one medium may be used for culture to day 3, and a second medium is used for culture thereafter. Single or sequential medium are equally effective for the culture of human embryos to the blastocyst stage Artificial embryo culture media basically contain glucose, pyruvate, and energy-providing components, but addition of amino acids, nucleotides, vitamins, and cholesterol improve the performance of embryonic growth and development. Methods to permit dynamic embryo culture with fluid flow and embryo movement are also available. A new method in development uses the uterus as an incubator and the naturally occurring intra-uterine fluids as culture medium by encapsulating the embryos in permeable intra-uterine vessel.