The term IVF- (level 2 IVF) indicates a laboratory technique that allows female gametes obtained by trasvaginal aspiration of follicular fluid to meet in vitro with the male gametes obtained following sample preparation of seminal fluid. The embryos obtained as a result of oocyte fertilization are transferred to the uterine cavity after 2 or 3 days from gametes collection.
The main indications for IVF-ET are:
- Tubal disease
- Mild or moderate male factor infertility
- Premature reduction of ovarian reserve
- Repeated failures of intrauterine inseminations
- Immunological factor
- Idiopathic infertility
The IVF-ET technique involves several stages, each of which is of fundamental importance for the success of treatment:
- Ovarian stimulation
- Oocyte retrieval
- Collection and preparation of semen
- In-Vitro Fertilization of oocytes
- Embryo culture and transfer
1. Ovarian stimulation
The treatment requires the use of drugs to obtain multiple follicular growth. Depending on the stimulation protocol, the duration of the entire ovarian stimulation cycle varies from 10 to 20 days. The various protocols are selected based on the characteristics of the ovarian reserve, age and medical history of the patient. In the agonist protocol the patient starts the administration of a GnRH agonist (GnRH-a) from day 21 of the menstrual cycle to temporarily block the secretion of pituitary FSH and LH, synchronize the growth of follicles and prevent the spontaneous ovulation. At day 3-5 of the next cycle the patient begins ovarian stimulation proper with gonadotropins. Follicular growth is controlled by series of ultrasounds for a total of 3-5 times and blood sampling for hormonal assays. These controls make it possible to adapt the pharmacological dosage to each patient based on the response. In the antagonist protocol the patient starts ovarian stimulation with gonadotropins and ultrasound and hormonal monitoring directly from day 2-4 of the cycle. When the size of the larger follicles reaches 14-15 mm, the patient will start another drug (GnRH antagonist) to reduce the risk of spontaneous ovulation.
Once 2 or more follicles with a diameter greater than 17-18mm are obtained, ovulation is induced by administration of HCG (Human Chorionic Gonadotropin) 34-36 hours before the ultrasound-guided oocyte retrieval. This hormone contributes to the final maturation of the oocytes contained in the follicles and their detachment from the follicle wall. In selected cases monitoring can take place according to the growth of the single follicle produced spontaneously during the patient’s natural cycle (or spontaneous cycle). Assisted fertilization on spontaneous cycle can be an alternative technique for patients with a reduced ovarian reserve who produce very few follicles with hormonal stimulation or who have undergone various cycles of IVF without obtaining results. It should be noted that in these cases embryo transfer (only one embryo) occurs in 40-50% of cycles started.
2. Oocyte pickup
It is done transvaginally under ultrasound guidance and local anaesthesia or neuroleptanesthesia (bland sedation) at the request of the patient or as per medical indication. During this procedure intraoperative antibiotic prophylaxis is administered. All the follicles present within certain diameters (>16mm) are aspirated and the follicular fluid obtained is immediately checked under the microscope to search for oocytes.
3. Collection and preparation of seminal fluid
The morning of the oocyte retrieval procedure, the male partner performs the collection of seminal fluid. Our facility offers a dedicated room equipped with television and DVD to facilitate the procedure. The sample of seminal fluid is then prepared in the laboratory with techniques designed to boost the fertilizing capacity of the spermatozoa. In the case of absence of spermatozoa in seminal fluid (azoospermia) or in the case of anejaculation spermatozoa may be taken from the testis and/or from the epididymis by surgical retrieval procedures (see TESE, TESA/PESA).
4. In-Vitro Fertilization of oocytes
The collected oocytes are first classified and then inseminated. Their in-vitro fertilization can be done with the classical IVF technique or by ICSI micromanipulation. The choice of the insemination technique is evaluated by the biologists the day of the procedure. It may be different from the one decided depending on the quality and number of gametes (oocytes, sperm) and possible oocyte freezing.
In 10-20% of cases fertilization and/or cell division may not occur and it is therefore not possible to proceed with the transfer of an embryo to the uterus.
Classic In-vitro fertilization (IVF) simply consists in putting the selected spermatozoa into contact with the collected oocytes still surrounded by the cells of the outer lining (cells of the cumulus and corona radiata). It is therefore up to the spermatozoa to cross the oocyte barriers on their own.
5. Embryo culture and transfer
As a result of the fusion between the sperm and oocyte a cascade of events leading to the formation of the embryo is triggered. The signs of successful fertilization are visible 18-20 hours after the insemination of the oocytes. Inside the fertilised cell (zygote) there are two nuclei, each of which carries the genetic information of the mother and father respectively. After a further period of in-vitro culture (24-48 hours) the number of embryos to have formed and embryo quality are assessed.
The classification of the embryos at the stage of 2-8 cells is based on 4 criteria at our center:
– number of cells present in the embryo (growth rate)
– symmetry of the cells
– presence of anucleated fragments in the perivitelline space of the embryo
– identification of the nucleus (or multiple nuclei) present in each cell
The quality of an embryo is therefore given by a set of parameters that must be thoroughly evaluated during the various phases of development. The accurate assessment of the embryos plays a fundamental diagnostic role in IVF-ET and must therefore be considered of primary importance.
The quality of the in-vitro culture of oocytes/zygotes/embryos is just as important. It is carried out at our laboratory in optimal pH and temperature conditions by using special culture media and an adequate number of incubators (12 in the laboratory) that are able to provide the best conditions for embryo growth and development.
The embryos obtained are transferred to the uterus by means of a thin catheter or, in very rare cases, using the transmyometrial route.
This procedure is painless and does not therefore require anaesthesia.
The day after oocyte retrieval the patient begins the administration of natural progesterone vaginally to support the luteal phase.