Assisted fertilization

How does assisted reproduction treatment work?

There are various assisted fertilization techniques which should be used according to the couple’s problem and which all have precise steps to follow. Let’s see in detail what they are.

Depending on the couple’s needs, various assisted fertilization techniques ( MAP ) can be used, such as: intrauterine insemination ( IUI ), in vitro fertilization and embryo transfer ( IVF ), intracytoplasmic sperm injection ( ICSI ).

The team of a PMA center must be made up of specialist doctors in gynecology, andrology and medical genetics, as well as specialist biologists and biotechnologists with proven experience in the embryological sector.

The path of PMA

1. Reservation and acceptance

During the acceptance phase, the patient is asked to present the previous health documentation, so that it can be consulted during the diagnostic activities.

An appointment is made for the 3 initial specialist visits (gynecological, andrological and genetic counseling).

2. Gynecological visit

During the first visit, after an accurate evaluation of the anamnesis, of the diagnostic tests carried out previously and of any previous treatments of ART, blood chemistry and instrumental tests are prescribed, if indicated, to complete the diagnostic procedure.

A pelvic ultrasound is also performed to evaluate the patient’s uterus, appendages, and ovarian reserve.

3. Andrological visit

During the first visit, after a careful evaluation of the anamnesis and the diagnostic tests carried out previously, blood chemistry and instrumental tests are prescribed, if indicated, to complete the diagnostic procedure.

Furthermore, the quality of the spermatozoa is evaluated by means of analysis of the seminal fluid in order to direct the couple towards the most suitable ART technique for their clinical situation.

4. Genetic counseling

During the first visit, the couple’s genetic and family history is collected, the genetic pathology is framed and the risk of recurrence is explained. Furthermore, the couple is informed of the different possibilities and techniques of pre-implantation diagnosis, with evaluation of the benefits, disadvantages and success rates.

Finally, in the case of pre-implantation diagnosis, the couple and, where required by the chosen technique, the family members of the couple are taken a blood sample for DNA analysis, in order to allow for the personalized development of the diagnosis.

5. Analysis view

When the couple has carried out the required examinations, it is checked again and the therapeutic procedure is decided on that occasion.

The procedure and any risks of the technique are then explained, the couple’s questions are answered and informed consent is given. Then, the personalized and appropriate therapeutic protocol for the treatment is explained to the couple.

6. Ovarian stimulation

Treatment requires the use of drugs aimed at obtaining multiple follicular growth. Depending on the stimulation protocol used, the duration of the entire ovarian stimulation cycle varies from 10 to 20 days. The different protocols are chosen based on the characteristics of the ovarian reserve, age and clinical history of the patient.

In the agonist protocol, the patient starts the administration of a GnRH agonist (GnRH-a) from the 21st day of the menstrual cycle to transiently block the pituitary secretion of FSH and LH, synchronize the growth of follicles and prevent spontaneous ovulation. On the 3rd-5th day of the following cycle, the patient begins true ovarian stimulation with gonadotropins.

Follicular growth is controlled by serial ultrasound investigations for a total of 3-5 times and blood samples for hormonal dosages. These controls make it possible to modulate the drug dosage on each individual patient based on the response obtained.

In the antagonist protocol , the patient starts ovarian stimulation with gonadotropins and ultrasound-hormonal monitoring directly from the 2nd-4th day of the cycle.

When the size of the largest follicles has reached 14-15 mm, the patient starts another drug (GnRH antagonist) to reduce the risk of spontaneous ovulation.

Once 2 or more follicles with a diameter greater than 17-18 mm are obtained, ovulation is induced by administering 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 follicular wall.

7. Oocyte retrieval

The oocyte retrieval (pick-up) takes place trans-vaginally under ultrasound control under local anesthesia or neuroleptoanalgesia (mild sedation) at the request of the patient or as per medical indication. Intraoperative antibiotic prophylaxis is administered during this procedure. All the follicles present, within certain diameters (>16 mm), are aspirated and the follicular liquid obtained is immediately checked under the microscope for the search for oocytes.

8. Evaluation of the oocyte

The oocyte is evaluated according to two criteria: nuclear maturity and morphological aspect.

The oocyte is considered mature (from a nuclear point of view), and therefore usable for in vitro insemination , if it has reached the stage of metaphase II.

From a morphological point of view, the oocyte cytoplasm can present different types of polymorphisms: granularity, presence of organelles or vesicles, accumulation of cisternae of the smooth cytoplasmic reticulum, presence of areas of the cytoplasm with the absence of organelles and/or vacuoles. In some cases, in the presence of oocytes with particular morphological characteristics, Laser-ICSI is performed.

This technique allows a small part of the outer shell of the oocyte (zona pellucida) to be thinned using a laser to reduce the pressure exerted during the injection.

In this way, the mechanical stress due to the injection technique is reduced, which can damage the most fragile oocytes, compromising their survival.

9. Collection and preparation of semen

On the morning of the oocyte retrieval surgery, the male partner collects the semen . The seminal fluid sample is then prepared in the laboratory with techniques aimed at promoting the fertilizing capacity of the spermatozoa.

In case of absence of sperm in the ejaculate (azoospermia) or in case of failed ejaculation, sperm can be collected from the testicle and/or epididymis by surgical recovery procedures (TESE, TESA/PESA).

10. Sperm evaluation

Sperm cells are normally viewed and selected at 400 times magnification.

In some cases, to perform a more accurate selection, the IMSI (Intracytoplasmic morphologically selected sperm injection) technique may be indicated, which consists in evaluating the quality of individual spermatozoa at very high magnification where it is possible to identify morphological anomalies such as vacuoles and nuclear defects.

11. ICSI – Introcytoplasmic Sperm Injection

This technique consists in mechanically removing all the oocyte barriers, made up of cumulus and corona radiata cells, and introducing a single selected sperm directly into the oocyte cytoplasm.

This procedure requires a laboratory instrument called a micromanipulator. ICSI offers the enormous advantage of being able to observe and select gametes (oocytes and sperm) before using them. Embryos obtained with ICSI are grown in vitro.

12. Embryo culture

Following the fusion between the sperm and the oocyte, a cascade of events is triggered which leads to the formation of the embryo. The signs of successful fertilization are expressed 18-20 hours after insemination of the oocytes.

In fact, inside the fertilized cell (zygote) we observe the presence of two nuclei which respectively carry the genetic information, one of maternal origin and the other of paternal origin. After a further period of in vitro culture (24-48 hours), the number of embryos that have formed and the embryonic quality are evaluated.

The classification of embryos at the 2-8 cell stage is based on 4 criteria:

  • number of cells present in the embryo (growth rate)
  • cell symmetry
  • presence of anuclear fragments in the perivitelline space of the embryo
  • identification of the nucleus (or of any multinucleations) present in each cell

The quality of an embryo is therefore given by a set of parameters which must be carefully evaluated during the various stages of development.

The accurate evaluation of the embryos plays a fundamental diagnostic role in the treatment and must therefore be considered of primary importance.

The quality of the in vitro culture of the oocytes/zygotes/embryos is also of fundamental importance.

It is carried out in our laboratory in optimal pH and temperature conditions through the use of particular culture media and an adequate number of incubators suitable for providing the best conditions for embryonic development and growth.

13. Embryo biopsy for PGT – OPTIONAL

The biopsy is carried out according to the methods agreed with the couple and on the basis of the specific diagnostic needs on the 5th day of in vitro growth.

It consists in taking a few cells from the embryo without compromising its normal development.

Once the biopsy has been performed, the embryos are frozen by vitrification until the diagnostic result is obtained, after which the suitable embryos are transferred to the uterus.

14. Embryo transfer

The embryos obtained are transferred to the uterus via a thin catheter and in very rare exceptions by trans-myometrial route.

This procedure is painless and therefore does not require anesthesia.

From the day following the oocyte retrieval, the patient begins the administration of natural progesterone vaginally to support the luteal phase.

Dr Kathryn Barlow

Kathryn Barlow is an OB/GYN doctor, which is the medical specialty that deals with the care of women's reproductive health, including pregnancy and childbirth.

Obstetricians provide care to women during pregnancy, labor, and delivery, while gynecologists focus on the health of the female reproductive system, including the ovaries, uterus, vagina, and breasts. OB/GYN doctors are trained to provide medical and surgical care for a wide range of conditions related to women's reproductive health.

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