In Vitro Fertilisation – IVF

Today, IVF is the ultimate choice in all cases of infertility that did not have successful outcome by using other assisted reproduction protocols.

Furthermore, in problems of male infertility, IVF using the technique of Intra-cytoplasmic Sperm Injection (ICSI) combined with the testicular biopsy methods of Testicular Sperm Extraction – Fine Needle Aspiration (TESE – FNA) is the only option for dealing with the problem.

Indications for IVF

 Male factor

  • Sperm parameter disorders

Female factors

  • Fallopian tubes patency problems
  • Ovulation disorders
  • Endometriosis
  • Unexplained infertility

Preliminary couples testing

Before embarking on an IVF attempt, it is necessary for the couple to undergo the following tests:


  • Reproductive hormone and thyroid screening (FSH, LH, E2, Prolactin, TSH, PRG, Testo, DHEA-S, Τ3, Τ4)
  • AMH – Anti-Mullerian hormone (indirect indicator of the level of ovarian sufficiency in eggs, and assessment of the effectiveness of hormonal ovarian stimulation)
  • Anti-TPO, anti-TG
  • Hysterosalpingography (HSG)
  • Gynaecological ultrasound (assessment of the anatomy of the uterus and ovaries)
  • Embryo transfer test (in cases where the cervical canal is narrow or deformed, cervical dilatation is recommended)
  • Hysteroscopy (where indicated)
  • Complete blood and urine panel
  • Blood type – Rhesus
  • Haemoglobin electrophoresis to exclude thalassaemia, etc.
  • Infectious diseases (Hepatitis Β and C, HIV, Syphilis)
  • Prenatal testing (Mycoplasma, Chlamydia, CMV, Toxoplasma, Herpes II, Rubella)


  • Sperm analysis test (spermogramme) and culture
  • Complete blood panel
  • Blood type – Rhesus
  • Haemoglobin electrophoresis to exclude thalassaemia
  • Infectious diseases (Hepatitis Β and C, HIV, Syphilis)

In case of non-obstructive azoospermia or severe oligospermia, with a sperm concentration less than 5 million per ml, additional diagnostic tests are recommended:

  • Cystic fibrosis screening
  • Testing for Y chromosome microdeletion (YCM)
  • Karyotype to exclude numerical or structural abnormalities

Ovarian stimulation protocols in IVF 

Natural cycle

Mild ovarian stimulation – induced ovulation

Controlled ovarian stimulation for multiple ovulation

Long agonist protocol

Short antagonist protocol

IVM (in vitro maturation)

Stages of IVF:

  • Controlled ovarian stimulation with hormones
  • Egg retrieval, processing of sperm sample
  • Egg fertilisation
  • Embryo culture
  • Endometrial embryo transfer
  • Embryo cryopreservation

Controlled ovarian stimulation for multiple ovulation

Since birth, every woman has all the eggs in her ovaries that she will use throughout her life; there are about 1 million per ovary. This number is gradually reduced and at the age of menarche (start of menstrual cycles), it is about 450,000. Ultimately, only 1 or 2 of these will reach ovulation during the final maturation stage. The remaining eggs will become atretic follicles and will be absorbed by the body.

The purpose of controlled ovarian stimulation is to achieve maturation of more than one or two eggs per cycle. This requires the administration of gonadotropin hormones (FSH and LH) combined with GnRH agonists (long protocol) or GnRH antagonists (short protocol) in injectable form, hypodermically or intramuscularly. An important factor that affects ovarian response to the administered treatment is the woman’s age.

Hormonal stimulation using gonadotropins usually lasts 9-12 days, for the purpose of producing several mature eggs. Administration begins in the 2nd or the 3rd day of the cycle, after the woman is first submitted to a comprehensive blood and ultrasound screening of the endometrium and the ovaries, in order to exclude the presence of ovarian cysts. The gonadotropin dosage is customised daily and every 3-4 days a new blood panel and ovarian ultrasound are performed, so that the administered dosage can be adjusted depending on ovarian responsiveness and test findings. Follicles are considered mature when they exceed 18 mm in diameter and chorionic gonadotropin (hCG) is administered to induce final maturation of the follicles which, 32-36 hours later, will lead to spontaneous ovulation.

To prevent the premature rupture of the follicles and loss of the eggs due to ovarian stimulation, agonists and antagonists are administered that inhibit hormone secretion from the pituitary gland (FSH, LH) and permit the gradual growth of follicles until final maturation without the risk of premature luteinisation and follicle loss. The difference is that GnRH agonists are administered daily starting on the 20th day of the cycle previous to the stimulation and continue until hCG is administered (long protocol), whereas GnRH antagonists are administered on the 6th day after starting stimulation with gonadotropins, and until the day when hCG is administered (short protocol). The two protocols appear to yield the same pregnancy rates per attempt; the agonist protocol has the advantage of fewer injections administered, and helps to avert ovarian hyperstimulation syndrome.

Egg retrieval

Egg aspiration does not require general anaesthesia. The anaesthesiologist administers a mild anaesthetic intravenously, and the gynaecologist administers local anaesthesia with xylocaine in the cervical area. Eggs are retrieved transvaginaly using a vaginal probe of an ultrasound equipped with guide through which passes a special egg retrieval needle with a diameter of 17G, which is guided into the follicle through the vaginal wall. The contents of the follicle are aspirated through this needle. The aspirated fluid is immediately checked by embryologists to find the egg.

The average time of this procedure is approximately 10-15 minutes; after that, the woman rests for about an hour and is monitored. Later, the couple is given a detailed briefing, concerning the number and quality of the eggs retrieved and the medication that the woman should take for the rest of the treatment. After the briefing, the couple can leave the clinic.

The aspirated eggs are placed in culture dishes with special culture media inside an incubator, where they remain for 4-6 hours, in order to complete their metabolic processes and to become ready for fertilisation.

Sperm collection and processing

The semen sample — is collected on the date of egg retrieval via masturbation, in a specially arranged area of the laboratory and it is submitted to specific treatment in order to improve concentration, motility, and quality. The sample is processed through centrifugation followed by density gradient separation (Percoll), in order to select the best spermatozoa in terms of viability and motility.

Alternatively, husband’s cryopreserved sperm sample collected at a prior time (only in exceptional cases), can be used.

In cases of azoospermia, a testicular biopsy is performed either with the FNA (fine needle aspiration) or the TESE (microsurgical testicular sperm extraction) method, under local analgesia and twilight anaesthesia (sedation). The biopsy is screened for immature or mature sperm that may be used to fertilise the eggs. Our laboratory was one of the first in the world to use immature forms of spermatozoa (spermatids) and achieved the birth of the 4th child in the world from spermatids. These results were published in Human Reproduction.


The fertilisation technique used depends on the characteristics of the sperm sample (concentration, motility, morphology), the number of eggs retrieved and the couple’s reproductive history. The two techniques used are classic IVF and micro-fertilisation (ICSI).

Fertilisation with the conventional method – classic IVF

This method is implemented when the values of all sperm parameters (concentration, motility, morphology) are at normal levels. In this method, eggs are cultured together with a few hundred thousand sperm overnight.