“In Vitro Fertilization (IVF) is the procedure by which a woman’s eggs are fertilized with her partner’s sperm in a special lab (outside her body).

Stages of IVF
Ovarian stimulation & monitoring
In IVF, we employ a controlled treatment regime (stimulation protocol) to attempt to intervene in the female reproductive cycle and regulate ovarian stimulation, so that the ovaries will produce more than one egg and, consequently, we can retrieve more than one eggs on the appropriate day.
The ovarian stimulation medication protocols used are typically three: the long protocol, the short protocol and the antagonist protocol. The medication used in all three protocols is the same; the difference lies in the dosage used and the duration of the treatment.
The choice of the protocol to be used depends on the couple’s history, the woman’s age, the responsiveness of her ovaries in previous attempts, and the clinical image presented by her biochemical and hormone test results. During ovarian stimulation, adjustments may be made depending on the ovaries’ response.
During ovarian stimulation, the woman is submitted to frequent ultrasound checks. Given that the developing eggs secrete increasing quantities of estrogens, frequent hormone blood tests are also required. Each time, the medication dose to continue the stimulation is decided along with the date for retesting, in order to assess egg growth, avoid potential complications (overstimulation), and determine the most appropriate time for egg retrieval and embryo transfer.
A very small percentage of women who commence treatment for IVF may be required to suspend it, if the ovarian response to the treatment is not the anticipated. In specific, difficult cases, screening and monitoring is more thorough and specialized.

Egg retrieval
During ovarian stimulation, the woman is submitted to frequent ultrasound checks. Given that the developing eggs secrete increasing quantities of estrogens, frequent hormone blood tests are also required. Each time, the medication dose to continue the stimulation is decided along with the date for retesting, in order to assess egg growth, avoid potential complications (overstimulation), and determine the most appropriate time for egg retrieval and embryo transfer.
A very small percentage of women who commence treatment for IVF may be required to suspend it, if the ovarian response to the treatment is not the anticipated. In specific, difficult cases, screening and monitoring is more thorough and specialized.

Sperm preparation
Either at the same time as egg retrieval or directly before it, the partner’s sperm is retrieved (by masturbation). It is important to have abstained from sexual contact for 2-5 days before – not longer. In special cases, where the male partner cannot be present on the day of egg retrieval or there are difficulties in ejaculation, the Center can freeze one or more sperm samples ahead of time so that they may be used when necessary. Following sperm retrieval, the most mobile and morphologically healthy sperm is selected through a special process and preserved in sterile culture conditions in a nourishing culture media, until they are placed together with the eggs in order to fertilize them.
In cases of obstructive azoospermia (inability of sperm ejaculation), retrograde ejaculation or anejaculation, sperm is retrieved surgically, usually by a urologist/associate of the Center, or by fine needle aspiration (FNA).
Testicular biopsy

Standard IVF
After egg retrieval, an embryologist places a specific number of mobile sperm on each culture dish that contains the eggs, without any other intervention. The sperm come into contact with the egg on their own and one of them penetrates and fertilizes it.

Confirmation of pregnancy
Twelve days after embryo transfer, the presence of human chorionic gonadotropin (β-hCG), the pregnancy hormone, is tested in the blood. If the test is positive, it is repeated after 2-3 days. The increase of the initial value of this hormone confirms the smooth progression of the pregnancy. Fifteen days after the positive pregnancy test, a transvaginal ultrasound is performed that confirms the clinical gestation. The endometrial pregnancy is ascertained, as well as the number of gestational sacs, the presence of an embryo or embryos is confirmed and their heart function is checked.

Blastocyst culture
The term blastocyst refers to the embryo’s development stage after 5-6 days of culture. It is a hollow sphere and in this stage the embryo consists of 60-200 cells, which form two discrete groups: the trophoblast cells (a peripheral cell mass), which will form the placenta, and the embryoblast (an inner mass of cells), which will form the embryo.
The decision for continued culture at the blastocyst stage depends on the number and quality of embryos on the 3rd culture day, and on the maturity of the endometrium. The main advantage of cultivating embryos at this stage is the option to select the fastest-growing embryos, which statistically give the highest rates of success.

Embryo culture
Over the next few days, the embryologist monitors the smooth growth of fertilized eggs, now called embryos. Assessment and selection of embryos for continued culture and embryo transfer is based on two morphological criteria: the division (or cleavage) of the emb¬ryo into cells (blastomeres) and the morphological appearance of these cells. On the second day after egg retrieval, embryos must be divided into 2-4 cells, and on the third day they must reach the stage of 5-8 cells. The best morphology is presented by embryos without fragmentation, with a regular and uniform shape of blastomeres.
In addition to embryo morphology, the day for embryo transfer is also determined by the morphology of the endometrium and the perfusion of the woman’s uterus on the day of egg retrieval.

Assisted hatching
In order for the embryo to become implanted in the woman’s uterus so that the pregnancy may commence, it is necessary for the embryo to hatch, i.e. to breach its protective layer, known as the zona pellucida. This normally happens in the blastocyst stage, on the 5th or 6th day after fertilization.
However, sometimes the zona pellucida is tougher or thicker than normal, and as a result the embryo cannot hatch and become implanted in the uterus. In these cases, the embryologist employees a technique known as “assisted hatching”. With this technique, hatching is artificially assisted by making an opening on each embryo’s outer zone. This is accomplished using special laser equipment. The use of laser is superior to other techniques (use of acids or mechanical opening), due to its great precision combined with the least possibility of damage to the embryo during the procedure.

Embryo transfer
Embryo transfer is the process of placing the fertilized and cleaved embryos in the woman’s uterus. It is performed 2-5 days after egg retrieval. It is a simple and painless procedure, does not require anesthesia and takes only a few minutes. Using a fine and flexible catheter and a small syringe, the embryos are selected by the embryologist and aspired in a small quantity of culture fluid. The physician inserts the catheter through the vagina and cervix into the uterine cavity where the embryos are deposited with the assistance of ultrasound monitoring, where necessary.
Usually, two to three embryos are transferred. The number of embryos transferred depends on their quality (according to the morphological criteria mentioned previously), the woman’s age and the number of attempts the couple has undergone. Internationally, there is a noted trend to reduce the number of embryos transferred, in order to reduce multiple births and the perinatal complications these entail.

Pre-implantation screening
Indications: Genetic diseases of the parents
• When both parents are carriers of a recessive disease
• Detection of chromosomal anomalies
• Women over 40 years of age
• Couples with a history of multiple failed IVF attempts
• When one parent is a carrier of a dominant or sex-linked disease
• Couples with offspring or pregnancies of embryos with chromosomal disorders
• Couples with a history of miscarriages
• Giving birth to HLA-compatible offspring
Pre-implantation genetic testing (PGT) is used for the identification
of single gene disorders in embryos, such as:
• Single gene disorders
• Thalassemia
• Sex-linked diseases
• Duchenne’s muscular dystrophy
• Cystic fibrosis
• Huntington’s chorea
• Hemophilia
• Sex-linked mental retardation
Obviously, PGT can only be performed in the context of IVF. The couple’s embryos are submitted to a biopsy and the cells retrieved from each embryo are submitted to genetic screening. Only normal embryos are implanted in the woman’s uterus.

Pre-implantation genetic diagnosis
In recent years, it has become possible to test embryos before their transfer into the uterus, with the technique of pre-implantation diagnosis. This is now feasible thanks to the knowledge we have acquired at the molecular level about the mechanisms that produce genetic diseases. The ability for extended embryo culture in the laboratory and the development of the technique of embryo biopsy have been instrumental in the development of the above techniques.
Pre-implantation genetic testing (PGT) aim to eliminate the risk for couples who have an increased chance of transmitting hereditary diseases to their offspring, as well as to avoid the significant psychological burden from an induced termination of pregnancy, when a serious issue is diagnosed with the traditional prenatal screening method (during the pregnancy). Additionally, pre-implantation genetic testing helps eliminate multiple unsuccessful IVF efforts and repeated miscarriages. “