66.Fertilization of oocytes in vitro, environment and conditions

The IVF procedure

In natural conception, an egg is released from the ovary into the fallopian tube in the middle of a menstrual cycle. The egg is fertilized in the fallopian tube. The fertilized egg then begins to divide in the fallopian tube, thus becoming an early embryo. After being in the fallopian tube for several days the embryo enters the uterus where it will implant.

IVF can be thought of as a bypass of the fallopian tubes. The IVF process involves stimulating the ovaries with "fertility shots" to mature multiple eggs. Development of the eggs is monitored with blood tests and vaginal ultrasound examinations. Once the eggs are "mature," they are removed from the ovary with a very minor surgical procedure and mild anesthesia. After the eggs are retrieved, they are put into a petri dish and exposed to the partner's sperm. The eggs are examined the following morning to see if they have been successfully fertilized by the sperm. The fertilized eggs are left in the petri dish for several days during which time they begin to divide and become early embryos (as occurs in the fallopian tubes during natural conception). The embryos are then placed in the woman's uterus with a small catheter through the cervix that is generally no more uncomfortable than a Pap test. If there are extra embryos that are viable, they may be cryopreserved (frozen) for future use.

IVF laboratory procedures

The full spectrum of IVF laboratory procedures are performed at Cleveland Clinic, including:

Fertilization with intracytoplasmic sperm injection (ICSI).

ICSI is when insufficient sperm are available, sperm parameters are compromised, or when previous fertilization methods have failed. Through ICSI, even the most severe cases of male infertility – very low numbers of sperm or even no sperm in the ejaculate – can be successfully treated with IVF. In these cases, sperm often can be obtained from the testes or epididymis by a minor outpatient procedure.

Assisted hatching.

In order for an embryo to implant in the uterus it must break through the zona pellucida, a thin "shell" surrounding the embryo. The process of the embryo breaking through the zona pellucida is called "hatching." Hatching can be aided by making a tiny opening in the zona pellucida just prior to transferring the embryo to the uterus. In Cleveland Clinic's IVF program, assisted hatching is routinely performed on all embryos before they are transferred to the uterus.

Embryo freezing.

Surplus embryos not transferred to the patient's uterus may be able to be frozen ("cryopreserved") for future use by the couple. In Cleveland Clinic's IVF program, cryopreservation of embryos is carried out either on the third day after egg retrieval when the embryo is between 6 and 8 cells in size or on the fifth or sixth day after egg retrieval (the "blastocyst" stage). Embryos that have potential to become successful pregnancies in the future are cryopreserved.

Preimplantation genetic diagnosis (PGD).

In 2002, Cleveland Clinic's IVF program was the first in the region to launch a PGD program. PGD involves removing a single cell from a developing embryo at the eight-cell stage. The cell is then analyzed for chromosomal and / or specific genetic disorders. For patients with sex-linked diseases or single gene disorders such as cystic fibrosis, genetic screening of embryos is a powerful technique. In these patients, IVF coupled with embryo screening can greatly reduce the risk of offspring being affected by the disease.

Oocyte / ovarian tissue cryopreservation.

Cleveland Clinic's IVF laboratory is becoming increasingly active in the area of freezing eggs and ovarian tissue There have been very few births resulting form ovarian tissue cryopreservation and it is clearly very experimental. Egg freezing has become much more successful in the past few years but it also is still considered to be experimental. After appropriate informed consent egg freezing is currently being offered to:

Women with cancer who are about to undergo chemotherapy that can cause their ovaries to be unable to make eggs in the future

Women who are undergoing In Vitro Fertilization who do not feel comfortable freezing all or any embryos

Women in their 30's who want to delay child bearing

24-CHR screen.

Cleveland Clinic is offering a new technique for screening patient embryos and selecting those most likely to result in a pregnancy. This powerful new technique know as Trophectoderm Biopsy allows the removal of multiple cells at the blastocyst stage. Embryos can be examined for inherited genetic disorders as well as chromosomal abnormalities. Patients with recurrent losses or at risk for chromosomal abnormalities can have their embryos analyzed using the new Genesis-24 screen. By testing a patient’s embryos for chromosomal abnormalities and selecting only normal embryos for transfer, the patient's opportunity for a pregnancy with a healthy baby is increased. The Genesis-24 technique is quite powerful and allows transfer of embryos in the same IVF cycle.

67 The method of in vitro fertilization. IVF is the basic assisted reproduction technique, in which fertilization occurs in vitro. 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 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 : 1Superovulation: Normally, a female ovulates just one or more egg per cycle. With the use of fertility drugs, she may be able to produce many eggs, which can then be retrieved from the ovaries prior to ovulation to be used for artificial semination of another female unable to produce eggs or that quality of egg. 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, chorionic gonadotropin (hCG) is given. This agent would cause ovulation about 42 hours after injection. The eggs are retrieved using a transvaginal technique involving an ultrasound-guided needle piercing the vaginal wall to reach the ovaries. Through this needle follicles can be aspirated. It is common to remove between ten and thirty eggs.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. 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. 5. Embryo Transfer: Embryos are graded by the embryologist based on the number of cells, evenness of growth and degree of fragmentation.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.