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IVF is the process of fertilizing the female ova with sperm of her partner in a specific laboratory
(outside her body)

Ovarian stimulation and 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 egg 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 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

Egg retrieval is the procedure by which eggs are retrieved from the ovaries. It is performed in a special room at the Center under sterilized operating room conditions, approximately 35-36 hours after the final injection for induction of final oocyte maturation. Egg retrieval is performed by a gynecologist transvaginally, under continuous ultrasound monitoring. The woman is usually under intravenous analgesia, administered by an anesthesiologist. The ovarian follicles are punctured successively using a special needle that is inserted transvaginally. The follicle content (follicular fluid) is delivered directly to the embryology lab, where an embryologist identifies the eggs and prepares them for fertilization on special dishes in a nourishing culture media. All ovarian follicles do not necessarily contain eggs. The process is relatively painless and the couple may return to their activities soon after the retrieval procedure.

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 previously, and 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 sample ahead of time so that they may be used when necessary. Following sperm retrieval, the most mobile and morphologically healthy sperm are selected by 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 an urologist/associate of the Center, or by fine needle aspiration (FNA).

Testicular biopsy

In cases of aspermia or azoospermia (no sperm or immobile sperm) in ejaculation, there is the option of retrieving sperm directly from testicular tissue with the testicular biopsy procedure. Since sperm come from testicular tissue, the couple must commence the process of IVF in conjunction with the process of intracytoplasmic sperm injection (ICSI). The reason for this is the inability of sperm derived from testicular tissue to fertilize eggs without any external help.

The technique of testicular biopsy is performed by a specialized urologist/associate of the Center, under general anesthesia. It can be done on the same day as egg retrieval or at an earlier time. The patient may return home a few hours after the conclusion of the biopsy.

If the quality of the testicular tissue permits it and mature sperm have been found that can be used in one IVF cycle, then there is also the option to freeze this tissue for future use. Nevertheless, it is better to use a fresh rather than a frozen tissue sample, because the sperm derived from the biopsy is very sensitive to the whole freezing process and may not survive the thawing.

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.

Intracytoplasmic Sperm Injection (ICSI)

Intracytoplasmic sperm injection or ICSI is indicated in couples with poor sperm numbers or function and in cases of previous failed cycles or low fertilisation rates after classic IVF.

Couples go through the same preparatory processes as with IVF, (ovulation induction and egg collection). The only thing different from a classic IVF cycle is the way of fertilization. With this procedure a single sperm is selected and injected into each of the eggs collected (Pict.5). One of the major advantages of ICSI is that very few sperm are required and the ability of the sperm to penetrate the egg is no longer important as this penetration is bypassed by the whole technique. This makes it an excellent tool for cases where low sperm number, motility and morphology are present but also in cases where sperm has been retrieved surgically from the testis (TESE). The fertilised embryos are allowed to develop as for standard IVF treatment prior to embryo transfer
However, ICSI can only be carried out on mature eggs. Unfortunately, egg maturity can only be truly identified under the microscope and it is, therefore, possible that following egg collection, none of the eggs are suitable for ICSI. This situation is fortunately rare.
It is also important to remember that whilst ICSI is a technique used in the laboratory to help fertilisation to occur, it does not guarantee it. Furthermore, a small percentage of eggs will be damaged by the injection process and the damage is evident at the time of the injection procedure. These eggs can no longer be used.

Fertilization check

The morning after the egg collection the eggs are checked for the presence of fertilization. The first signs of fertilization are shown by the presence of two pronuclei within the egg.  Anything else is considered abnormal and is removed from culture.

It is important to be aware that there is always a possibility that a low proportion or even none of the eggs fertilize. If this occurs the couple will be seen by the clinician to discuss their future options.

Embryo culture

The following days, the embryologist evaluates the progress of the fertilized eggs (now called embryos) in culture. The determination of the day those embryos are going to be placed back in the uterus depends on their division in cells (blastomeres), their morphology and the picture of the woman’s endometrium on the day of egg collection. On the second day after egg collection, the embryos should have divided in 2-4 cells and on the third day in 5-8 cells. Generally the less fragmentation an embryo has the better its morphology and chances of implantation are. The best embryos are selected to be transferred back to the uterus

Blastocyst culture

The term blastocyst refers to the stage of development of the embryo after 5-6 days of culture.  At this stage, the embryo is a hollow sphere, consists of 60-200 cells and its two different cell components are the “trophectoderm cells”, which become the placenta after implantation and the “inner cell mass”, which forms eventually the baby.

The decision for an extended culture at the blastocyst stage depends on the number and the quality of the embryos on the 3rd day of culture, but also on the maturity of the  endometrium.  The most common reason for having a blastocyst culture is the easier selection of the best and strongest embryos for transfer, which also give a higher chance of pregnancy.

Assisted hatching

In order for an embryo to implant into the lining of the womb and a pregnancy to occur, whether it is after IVF or after natural conception, it is necessary for the embryo to push out from or “hatch” out of its outer coating called the “zona pellucida”. This occurs naturally on day 5 or day 6 after fertilization at the blastocyst stage.

However, some times this outer coating is thicker and harder than normal. This may mean that the embryo could fail to hatch and would therefore be unable to implant. In cases like that, the embryologists perform a technique called “assisted hatching”. With this technique,  hatching is assisted by making a small hole in the zona pellucida of each embryo.  Using the laser equipment instead of other methods (i.e. acid, mechanical) ensures the accuracy of the procedure with the minimum danger to the integrity of the embryo itself.

Embryo transfer

Embryo transfer is the procedure during which the fertilized and divided embryos are placed  in the uterine cavity.  It takes place 2-5 days after the egg collection.  It is generally a painless procedure  and takes up a few minutes. It is done with the help of a thin, soft, elastic catheter. During the procedure, the embryologist aspirates the selected embryos within the catheter and the doctor places them in the uterine cavity.

A maximum number of two to three embryos are usually transferred. This number depends on the quality of the embryos, the woman’s age and the number of previous IVF attempts. Internationally, it is becoming more common to transfer fewer embryos due to the increased chance of multiple pregnancies and the complications associated with that.

Confirmation of pregnancy

The pregnancy test is done twelve days after the embryo transfer by checking the levels of the hormone β-hCG in the blood. This hormone is only produced by the body when pregnant.  If the pregnancy test is positive it is repeated 2-3 days late.
The multiplication of the hormone attests that a normal pregnancy is beginning. fifteen days later, the first ultrasound scan is done, to confirm the clinical pregnancy. On that scan the doctor ensures the intrauterine pregnancy,  looks for the number of sacs and checks the embryo or embryos heart beat.

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