In vitro

Classic indications for IVF treatment include blocked Fallopian tubes, moderate male factor, and ineffectiveness of previously used treatments. This method was developed by a team of British doctors and embryologists led by Edwards and Steptoe. As a result of their work, on 25 July 1978 in England Luise Brown was born – the first human child born as a result of IVF treatment and transfer of the developing embryo into the uterine cavity. The effectiveness of the first trials, conducted in the natural cycle, was relatively low. Only the introduction of ovulation stimulation and soon after controlled ovarian hyperstimulation, significantly increased it. Thanks to this, IVF treatment has gained worldwide recognition. After nine years, on 12 November 1987, at the Institute of Obstetrics and Gynaecology in Białystok, the first child conceived as a result of in vitro fertilization performed in Poland was born. Currently, IVF is performed throughout the world, and indications for its use include almost all causes of infertility.



Indications for IVF treatment according to the latest recommendations of the Polish Society of Reproductive Medicine include:

  1. Fallopian tube factor*
    1.  in patients with permanent damage to fallopian tubes
    2.  in patients not qualifying for surgery
    3.  in patients with impaired function of fallopian tubes with retained patency, or after microsurgical surgery and 2 years without pregnancy; It is recommended to wait when there are no other factors that may affect the chance of pregnancy (abnormal semen, woman’s age > 35 years, duration of infertility <3 years, endometriosis, ovulation disorders)
  2. Unexplained infertility**
    1. lasting longer than 3 years
    2.  if the patient is over 35 years of age – sooner
  3. Male factor
    1. total number of mobile sperm <1 million – ICSI recommended
    2. number of mobile sperm is 1 – 10 million – in case of infertility lasting longer than 2 years**
    3. sperm count > 10 million – as in unexplained infertility
  4. Endometriosis
    1. degree I, II, as in unexplained infertility
    2. degree III, IV, as in fallopian tube factor
  5. Hormonal Disorders**
    1. 12 stimulation cycles without effect
  6. Unsuccessful attempts at intrauterine insemination
    1. maximum of 6 trials <35 years of age
    2. a maximum of 4 trials > 35 years of age
      *It is advisable to remove the fallopian tube in case of hydrops
      ** 4-6 intrauterine inseminations should be considered before IVF/ICSI treatment
  7. Currently, IVF treatment is also used in fertile couples, where:
    1. the male partner is HIV or HCV positive and the female partner is not infected
    2. the couple is a carrier of genetic modifications that cause severe, irreversible changes in the offspring, and pre-implantation diagnostics avoids a difficult decision to terminate a pregnancy
    3. female partner begins cancer treatment and treatment is likely to irreversibly damage the ovaries
    4. ovulation induction results in a large number of developing follicles and this situation poses a high risk of multiple pregnancy.

Course of treatment

IVF treatment can be divided into several stages.

– Pharmacological ovarian stimulation


The first treatment consists of pharmacological ovarian stimulation for the growth and maturation of Graaf follicles. Most infertility researchers believe that this process is a key component of treatment. The end result of treatment largely depends on the success of ovulation induction. In the most commonly used stimulation regimens, GnRH analogues are administered simultaneously with gonadotropins. The stimulation process requires an ultrasound assessment (assessment of the number and diameter of growing follicles), and determination of serum estradiol (E2) concentrations as the index for the endocrine function of developing Graaf follicles. When the dominant pre-ovulation follicles reach the size of > 18 mm and the average E2 concentration of 150-200 pg/follicle, the intramuscular administration of 10,000 units of hCG – human choriogonadotropin simulates an exogenous luteinizing hormone (LH).

– ovarian puncture


Egg collection takes place 34-36 hours after hCG administration, by puncture under ultrasonography. For this purpose, transvaginal probes (5-7.5 MHz) are used with a guide used for procedures performed via the posterior vaginal vault. The procedure involves searching for eggs in the resulting follicle fluid and transferring them to the culture medium. The ovary is surrounded by the oocyte-corona radiata-cumulus complex (OCCC). OCCC morphology, in assessing the degree of the dispersion of the cumulus oophorus and the corona radiata, is the basis for determining egg quality and maturity. Genetically immature ovaries, those in the germinal vesicle (GVBD) and germinal vesicle breakdown (GVBD) phases, are not capable of being fertilized.

– Egg fertilization and embryo development


in vitro

Sperm used during in vitro fertilization are prepared using the ascending migration or the discontinuous Percoll gradient centrifugation method. Before insemination, the ova are incubated for about 3 hours, which allows them to complete the maturation process. Approximately 100,000 sperm are added to the oocytes placed in the culture plate, and after 19 hours of incubation, the number of pronuclei (PN) is evaluated. Their presence testifies to a successful fertilization. Correctly fertilized eggs will have two pronuclei, and in the perivitelline space there should also be two polar bodies present. Fertilized cells are transferred to a new surface. Approximately 5-10% of cells are fertilized incorrectly, as evidenced by the presence of more than two pronuclei. They should be eliminated from further cultivation. About 28-32 hours after fertilization the first division of the embryos occurs and two blastomeres are visible. On the second day the embryos consist of 3-5 cells, after 48-72 hours they reach the stage of morula-blastocyst. The embryos may be transferred to the uterine cavity at 2, 3 or 5 days after fertilization. In the morphological classification of embryos, the rate of cell division, the symmetry of blastomeres and the extent of their fragmentation are evaluated. The greatest development potential is shown by fast-dividing embryos, containing equal blastomeres, showing no fragmentation. The chance of getting pregnant decreases with increased morphological abnormalities in the embryo.

– embryo transfer


In the past, anywhere from one to four, and sometimes even more embryos would be transferred to the uterine cavity, depending on the adopted norms of a given country and clinic. It has now become standard to transfer a single embryo to women up to the age of 35, and up to two embryos to older women. This standard is also recommended by the Polish Society of Reproductive Medicine, and followed by us at KRIOBANK. The trans-cervical transfer of embryos to the uterus is done by using special catheters. Embryos not used for transfer are cryopreserved at the early stage of embryonic development (4-6 blastomeres), or after the blastocyst stage in prolonged culture.

-supplementation of the luteal phase


Experts agree that, as a result of the IVF treatment, luteal failure occurs as a consequence of defects in corpus luteum production. Among the reasons for this are the aspiration of granular cells during ovariectomy, as well as the low LH concentration resulting from GnRH agonists. Hence the accepted practice is supplementation with gestagens during the second phase of the cycle. During the second phase of the cycle progesterone is administered intramuscularly at a dose of 25 – 100 mg per day, or intravagally at a dose of 90-600 mg per day. Another type of supplementation is oral dydrogesterone (Duphaston) or estradiol valerate with 17β-hydroxyprogesterone caproate (Gravibinon) administered intramuscularly. Supplementation of the second phase of the cycle is also possible with hCG. In controlled studies, the highest efficacy was demonstrated by the intramuscular use of progesterone and hCG, as well as intravaginal progesterone supplementation.

Intracytoplasmic sperm injection - ICSI

The Intracytoplasmic sperm injection (ICSI), introduced more than 10 years ago, involves the direct microsurgical introduction of male gametes into the cytoplasm of the egg. This allows to bypass most of the stages of natural gamet interaction, and thus eliminate some of the causes of infertility associated with the male factor. The most common indications are low sperm count (oligospermia), impaired motility (asthenospermia), and abnormal teratozoospermia (OAT – oligoasthenoteratozoospermia). Also, ICSI is the only method in the absence of spermatozoa (azoospermia), both obstructive and non-obstructive, as well as ejaculatory disorders and necrospermia.


In terms of ovarian induction and obtaining female gametes, ICSI does not significantly differ from classical IVF. For men whose semen contains sperm, semen preparation involves classical preparatory techniques such as rinsing, ascending migration or sedimentation and percoll gradient separation. In situations where only single gametes are available, rinsing and spinning of the ejaculate sample is sufficient. In case of obstructive azoospermia, sperm are obtained by microsurgical epididymal sperm aspiration (MESA) or testicular sperm aspiration (TESA). Oocytes collected by ovarian puncture are subjected to short-term hyaluronidase activity to remove granular cells that form the cumulus oophorus and the corona radiata. This allows for an accurate assessment of the maturity of the testes and the ooplasmic state. Oocytes in the second meiotic division (metaphase II) show the presence of the first polar body in the perivitelline space, and can be directly subjected to microinjection. Eggs with a slight degree of immaturity, in the first meiotic division (metaphase I), after a short cultivation (6-12 hours) usually achieve full in vitro maturation. Oocytes in the first meiotic division do not fully mature in vitro and are not fertilized. In the essential part of the procedure sperm with the most favourable construction and mobility is identified, it is then immobilised by mechanical damage to its tail, and introduced into the interior of the injection micropipette. The holding pipette positions the oocyte in such a way that the polar body is at 12 o’clock. In this position, the insertion of the injection micropipette takes place at 3 o’clock due to the position of the carokinetic spindle of the second meiotic division, usually located under the polar body. Immediately following the penetration of the zona pellucida, the ooplasm is aspirated into the pipette, which enables the oolemma to be interrupted and the sperm is deposited in the ooplasm. The slow removal of the pipette should allow the oolemma repair process to begin. About 9-12 hours after the procedure it is usually possible to assess egg activation and the process of fertilization. Subsequent procedures are similar to those of the classic in vitro method and include observation of embryo development, selection of embryos with the greatest developmental potential and transfer to the uterus, as well as cryopreservation of excess embryos and luteal phase supplementation.


Sometimes the semen contains no sperm at all. It is important then to answer the question: do men’s testes produce sperm?

Preliminary analysis of hormone levels and assessment of the size and consistency of the testes are helpful. In most cases genetic diagnostics of both the man and the woman are necessary to assess the risk of genetic disorders in the offspring. If the results are positive, we will perform a fine needle biopsy of the testes or epididymis. It is performed under general anaesthesia, so it is painless and less burdensome for the man. He will be able to leave the clinic about an hour after the test. If the biopsy material contains live sperm, fertilization is possible by microinjection into the egg, as described above. In each case, the sperm obtained from the testes or epididymides are frozen for later use. In the absence of live sperm, the choices available are obtaining sperm from a donor, or adoption.


Intracytoplasmic Morphologically Selected Sperm Injection is a cytoplasmic injection of sperm, morphologically selected by means of high magnification and high resolution techniques. The IMSI method is based on the ICSI method, differing by the way sperm is selected for micromanipulation.

The effectiveness of IVF treatment, although very high, is still unsatisfactory in some patient groups. Therefore, new methods are being used worldwide that may have a beneficial effect on clinical outcomes. The use of non-invasive, risk-free solutions to improve performance is ideal. The IMSI method is the best example.

In order to increase the chances of fertilization during infertility treatment, preparative methods are being used increasingly more often to obtain the best quality sperm fractions. This is especially important when performing the ICSI procedure. Selection of sperm is made on the basis of their construction and motility. However, in some cases of male infertility when using normal technical equipment for ICSI, choosing the right sperm by the embryologist may be impeded. The IMSI performed before injecting the sperm into the egg, allows to view the sperm head (core) under very large magnification, which makes selection easier.

The use of IMSI also improves the chances in cases of weak ovarian response to stimulation. Obtaining a small number of oocytes poses the risk that after their fertilization we will not get a satisfactory number of normal embryos. The IMSI method of choosing the best sperm improves the chances of obtaining normal embryos, and hence the chance of pregnancy.

Clinical studies confirm that using the IMSI method improves the pregnancy rate and reduce the rate of miscarriages. This procedure is especially recommended for couples with previous ICSI failure, severe male infertility factor, and who have a history of miscarriages.

The following pictures show a comparison of sperm images with ICSI and IMSI. Thanks to the large magnification in the IMSI method, we can accurately assess the morphological structure of the sperm and select the ideal one.


It is a device that allows constant monitoring of embryo development from the moment of fertilization of the oocyte until it is transferred to the uterus or cryopreservation. The ovaries collected during ovarian puncture are fertilized in an embryo laboratory and then placed in suitable culture media and inserted into a CO2 incubator. Temperature, humidity and composition of gases provide optimum conditions for embryo development. The task of the embryologist is to evaluate the rate of embryonic division, to determine the number and symmetry of dividing cells (blastomeres), the degree of early embryo fragmentation, and the development of the embryo in the fifth embryogenesis stage (blastocyst). It should be emphasised that embryonic development is a very dynamic process and its changes are very rapid. For this reason, the usual one-time evaluation of the embryo under the microscope during 24 hours of culture does not allow to monitor for many key developmental details that determine the developmental potential of the embryo. A more frequent assessment consisting of removing incubated embryos and observing them under a microscope may adversely affect its development due to unstable culture conditions.

At present it is possible to continuously and automatically monitor and document the development of embryos without having to remove them from the incubator. This is possible thanks to the time-lapse technique. The embryoscope is currently the most advanced system for cultivation and continuous monitoring of embryo development. Inside the device is a camera that automatically captures the embryos every few minutes. The images obtained are part of a detailed film of the entire embryonic development process. The analysis of the resulting film facilitates the selection of the embryos with the best division rate and morphology, and thus having the greatest development potential. It also allows you to identify abnormal embryos. Thanks to this method it is possible to reduce the number of embryos transferred to the uterus without reducing the effectiveness of the treatment and also avoiding the risk of multiple pregnancies. Normally developing embryos that have not been transferred to the uterus are frozen for later use in future cycles. It is possible to analyse the film with the patients, which helps them to understand the problems with the effectiveness of the treatment.

This method can be used by all couples, but is especially recommended for older women and those who experienced repeated IVF failure. Our experience shows that using this method contributes to a significantly better outcome.

Nacięcie otoczki przejrzystej zarodka

Successful implantation or implantation of the embryo in the endometrium depends partially on the smooth process of its hatching from the zona pellucida (ZP). This allows direct contact of the blastocyst (embryo at the developmental stage capable of implantation) with the endometrium. The blastocyst expands and presses from the inside into the ZP, which breaks and allows the blastocyst to escape it. This phenomenon is called blastocyst hatching. Sometimes the ZP does not break, which prevents the interaction of the embryo with the endometrium and thus the implantation and development of pregnancy. Scientific studies and clinical observations show that this is caused by morphological factors, such as: the average thickness of the ZP, embryo fragmentation, or the insufficient number of blastomeres, which make up the embryo. Other factors that may hinder the blastocyst hatching process are likely to be the age of the patient and the associated zona hardening. It has also been found that the freezing and thawing of embryos may be the cause of this problem.

Assisted hatching is a laboratory technique that involves the thinning or cutting of the ZP surrounding the embryo. This treatment makes it easier for the blastocyst to hatch, thereby increasing the chance of successful implantation in the endometrium. It is done just before the transfer – this applies to both fresh and frozen embryos. The risk of damaging the embryo is very small. However, the data suggest an increased incidence of twin pregnancies after this procedure.

Indications for this procedure include:

  • Age of the woman: above 37-39 years of age
  • Women with elevated FSH levels in 1-3 days of the cycle
  • Morphology of the embryo: e.g. too thick ZP, delayed cell division or high percentage of cellular fragmentation
  • In cycles after previous failed IVF treatments
  • Before the cryotransfer

Evaluation of uterine contractions

Transfer of embryos into the uterus is the final stage of the IVF treatment. This short procedure is the culmination of the time-consuming preparations, sometimes difficult stimulation, ovarian punctures, and then the entire embryonic process. Successful implantation of embryos after transfer is mainly affected by embryo quality and uterine receptivity or “the desire of the womb to accept them”. Equally important are the quality of the procedure and the conditions for the embryo. The key component of implantation may be excessive (more than 3 contractions/3 minutes) uterine contractions. Increased uterine contractions, both spontaneous and traumatic, may reduce the chance of IVF-ET embryo implantation, resulting in the removal of embryos from the uterus after transfer.

Excessive uterine contraction is present in approximately one-third of patients treated with IVF-ET. The results of treatment (pregnancy rate) achieved in this group of patients are even 3 times lower in comparison to patients with normal activity. As it is known, uterine contractions are conditioned by the stimulation of oxytocin receptors, which creates the possibility of pharmacological intervention. It has been shown that the use of oxytocin receptor antagonists (Atosiban) reduces the contraction of the uterus and improves embryo implantation. It has also been shown that Atosiban has no embryotoxic effects and its administration during transfer is safe. Preliminary clinical findings from the study of this drug indicate an increase in the pregnancy rates in patients treated with IVF-ET. After a period of conducting preliminary studies at our centre, we introduced this procedure as a routine procedure for patients diagnosed with excessive uterine contractions.

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