Treatment and Laboratory Methods

Minimal ovarian stimulation is intended for women who do not want to undergo classic IVF stimulation. Clomiphene citrate tablets (Clostilbegyt or Letrozole) are used for stimulation in combination with low doses of injectable preparations known from classic IVF cycles. Egg fertilization is performed using the ICSI method. The probability of pregnancy is lower than with classic IVF, but there is a lower risk of OHSS (hyperstimulation) and lower costs for IVF stimulation.

In practice, minimal stimulation is most often used in the following cases:

1. Previous stimulation was complicated by ovarian hyperstimulation syndrome.
2. The woman is concerned about hormonal stimulation with higher doses.
3. In women with polycystic ovary syndrome (PCO syndrome).
4. In case of repeated failure of infertility treatment with the classic IVF method.
5. When it is necessary to minimize the costs of treatment.

The first successful attempt for artificial fertilization was performed in 1978 when thanks to Dr. R. Edwards and Dr. P. Steptoe was the first child born as a result of the IVF/ET method (Louise Brown, Great Britain). According to the latest estimates by experts, approximately 500,000 to 800,000 children are born worldwide each year thanks to IVF (and other assisted reproduction methods). Assisted reproduction methods are a complex of procedures when the egg and sperm are manipulated outside of the female body.

After hormonal preparation aimed at maturing a larger number of eggs, they are transferred by aspiration directly from the ovaries into a special culture medium just before ovulation, and depending on the preferred fertilization method, they are further manipulated in the laboratory. After fertilization, the fertilized eggs are cultured in special media for two to six days. Usually one embryo is then transferred back into the woman’s uterus, the other high-quality embryos can be frozen.

Preparation for IVF:

Hormonal stimulation:

Multiple growth of follicles increases the chances for achieving a bigger amount of quality eggs and consequently more embryos suitable for transfer or freezing. This is achieved by hormonal stimulation under constant doctor’s control.

Egg retrieval:

The egg retrieval is performed under short general anesthesia guided by trans-vaginal ultrasound and it takes about 5-10 minutes. The procedure is done with a special thin needle, which goes via vaginal whole straight to an ovary. From there are the eggs suctioned. The patient is discharged about 2 hours post procedure.

The sperm collection and preparation:

A sperm sample is collected from a partner on the day of egg retrieval in special room. Sexual abstinence is recommended 2-3 days prior the collection in aim to get the best possible quality sample. Following its laboratory preparation eggs are fertilized by partner’s sperm in a special solution (for for example Swim up, Microfluid Sperm Sorting Chip). Fertilization happens spontaneously, the sperm moves towards the egg and penetrates its shell. The common cultivation of eggs and sperm lasts 16-20 hours. The second, more widespread method of egg fertilization is the ICSI method, where the sperm is injected directly into the egg by an embryologist with a thin needle. The ICSI method has a higher fertilization success rate (80-90%). After about 18 hours from the ICSI/IVF procedure, the embryologist determines the success of fertilization, which is characterized by the presence of two pronucleus and two polocyts.

If the man is in any doubt about the ability to give a sample in the day of egg retrieval, he can use the option of cryopreservation in advance.

For men who have no sperm in the ejaculate is MESA/TESE one of the options to get the sperm in IVF cycle.

Embryo cultivation:

Embryos are cultivated in special media for 2-6 days. After this time they are transferred in to the uterus. Surplus embryos can be frozen and stored for potential use in the future.

Is a procedure when the embryos are brought back in to the womb cavity using a special thin catheter. Embryos are transferred together with a small volume of the media. This procedure is quick and in most cases painless, does not require GA. The number of embryos transferred is depending mostly on the age of the patient, number of previous failed IVF cycles and on wishes of the patient. Mostly 1 embryo is transferred. The patient is leaving the Clinic in about 1 hour post ET. Pregnancy test is done in 2 weeks.

Cultivation to the blastocyst stage

By using the special solutions it is possible to extend the time of cultivation of embryos for up to 5 or 6 days (blastocyst stage, expanded blastocysts). The benefit of this procedure is the option to choose the best quality embryos for transfer and increase the chances for pregnancy. Use of the long cultivation is suitable especially when the number of mature eggs is sufficient (6 and more). Determination of an optimum date for ET is very individual and can be different in repeated cycles.

A additional technique during which a laser hole is made into the shell (zona pellucida) of a 2-4 day old embryo. The embryo then uses it to leave the shell. It is recommended to use this technique for embryos with thicker shell or in cases of repeated implantation fail (in previous cycles the embryo did not implant spontaneously).

Freezing of embryos is a method thanks to which we are able to preserve surplus quality embryos for as long time as needed (max. for 25 years). They are kept in liquidized nitrogen and ready for later transfer. Cryopreservation in used when there is enough of quality embryos which have not been transferred in given cycle (for example for the risk of multiple pregnancy). In case of serious Ovarian hyper-stimulation syndrome or not suitable conditions for transfer (other illness of the patient, low lining etc.) all of the embryos are frozen. Despite of standardization of the procedure not all of the embryos will survive the defrosting in a condition suitable for transfer (1-2% of embryos).

The success rate of the transfer of frozen embryos-defrosted embryos (cryo-embryo transfer) is lower than with fresh embryos. For the lady this is more convenient as she does not need to repeated hormonal therapy and egg retrieval. Based on current knowledge there is not know increased risk of abnormalities or congenital developmental defects caused by this method.

PGT preimplantation genetic testing (PGT-A. PGT-SR, PGT-M) is a diagnostic method which, in connection with the IVF cycle, allows one to test some genetic properties of the embryos prior to their transfer into the uterus. 5-10 cells are gently removed from a 5-day or 6-day-old embryo and subjected to genetic analysis. The embryos are frozen on the day of cell collection. An embryo with a normal genetic status is then prepared for KET (cryo embryo transfer). Embryos usually survive this procedure without any harm.

Preimplantation genetic testing of aneuploidies (PGT-A) makes it possible to detect, in particular, acquired or inherited variations in the number or structure of the chromosomes. These abnormalities are often associated with miscarriages.

Preimplantation genetic testing for monogenic diseases (PGT-M) can detect changes (mutations) in individual genes associated with a specific inherited disease that has already occurred in the family.

The PGT method is especially recommended for couples where:

• The female partner’s age is older than 35 years, which increases the probability of giving birth to a child with an abnormal number of chromosomes (eg Down’s syndrome).
• A miscarriage or birth of a fetus with a chromosomal disorder has already occurred.
• There are repeated failures in IVF treatment or repeated miscarriages in early pregnancy.
• Chromosome rearrangement was detected cytogenetically in one of the partners. Although the individual itself is not clearly affected, this rearrangement may result in the formation of germ cells (eggs or sperm) with abnormal genetic makeup and the transmission of the defect to offspring.
• There is a so-called gender-related disease in the family (the disease only affects male individuals, but is genetically transmitted by women – such as hemophilia).

One of the partners has undergone or is undergoing some type of chemotherapy or radiotherapy.

Preimplantation genetic testing cannot fully guarantee the selection of an embryo that has no defect. This is due to the principle of the method, where it is possible to examine only a certain spectrum of abnormalities by which the embryos of a given pair are most endangered. Nor can it guarantee the success of an IVF program, ie the implantation of an embryo after transfer to the uterus and the onset of pregnancy. It cannot guarantee the birth of a healthy child. All this is influenced by a large number of other factors.

Molecular diagnostic test for the examination of endometrial receptivity (the state of the uterine lining).

Endometrial receptivity is a state when the endometrium (uterine lining) is ready for implantation (nesting) of the embryo. This optimal situation should be between the 17th and 21st days for every fertile woman.

The endometrium is usually assessed by ultrasound examination, which shows its height and structure. The ERA test offers wider possibilities for assessing the endometrium, thereby increasing the likelihood of successful pregnancy.

The examination is suitable for women with repeated embryo implantation failure.

The test examines 248 genes that are involved in endometrial receptivity. The most suitable time to perform the test is the cycle preceding the cycle in which embryo transfer is planned.

The doctor can evaluate whether the endometrium is properly receptive for embryo transfer on given days or whether the so-called “implantation window” has shifted.

A revolution in the male infertility was the news in the 1992 about first pregnancies achieved by injecting single sperm in to the egg through its shell (zona pellucida). This method helps countless couples who’s cases were hopeless and were forced to use a donor sperm only few years before.

The method of ,,intracytoplasmatic“ injection of sperm (ICSI) is a elaborate micromanipulation technique when the sperm is aspirated in to a thin sharp glass capillary and inserted in to an egg through its shell.

Indication for ICSI is the inability of the sperm to fertilize the egg spontaneously (low count, low sperm motility), immunological causes of infertility, failing of classical IVF cycles. It is also recommended after repeated unsuccessful attempts at artificial insemination, later age of patient, low amount of matured eggs, when cryo-preserved sperm is used or when the sperm is collected by MESA/TESE method, when donors eggs are used etc. This method has a very high success rate of fertilization. The patient has to indeed undergo the normal stimulation procedure of super-ovulation and egg retrieval and the partner has to provide the sperm. In the view of collected data we can say that by fertilization of the egg using this method is not increased risk of a child with congenital developmental defect when compared to spontaneous conception (2,3%).

This is a laboratory technique (improved ICSI) which enables us to pick and transfer only single one mature sperm in to the oocyte. This increases the success rate of the infertility treatment. Naturally only matured sperm able to specifically connect to so called oocyte’s complex (hyaluronan) are able to fertilize the egg. In mature sperm was demonstrated much lower appearance of chromosomal aberrations than in immature one.

When using only the simple ICSI method the sperm is picked by its morphology and motility. This does not guarantee the sperm with optimal genetic pack will be chosen. PICSI method combines the benefits of ICSI (high probability of fertilization) with the possibility to pick the mature sperm based on its ability to connect with the hyaluronan hydrogel. PICSI is imitating the connection of the mature sperm to the oocyte’s complex which is very important for the selection of suitable sperm during the natural fertilization. This technique is possible to use only under the condition that there is enough sperm with good motility in the ejaculate.

By using a transfer medium with a high content of hyaluronan, the Clinic of Reproductive Medicine and Gynecology Zlín offers a modern procedure that increases the chance of conceiving your own child.

Transfer medium used for transferring embryos to the uterus. This medium contains all the necessary nutrients and energy sources which are needed for optimal embryo development. It also contains a higher concentration of hyaluronan, which is naturally present in follicular, oviductal and uterine fluids where it increases the viscosity of those fluids.

The transfer medium can be used for embryo transfer of all developmental stages of embryos, from two-day-old to blastocysts, both fresh and frozen.

MESA- microsurgical aspiration a of the sperm from a epididymis. A procedure performed in case of distraction of a sperm transport between epididymis and urethra. The procedure is in most case performed as an open surgery in general anesthesia. That mean via cca. 3cm long cut in the scrotum access to the ipididymis is gained and the fluid is aspirated using a pipette straight from the channels. The fluid is analyzed during the procedure in the embryology laboratory. If some live sperm is present it is used for eggs fertilization using the ICSI method.

In the case there has been no sperm obtained during MESA we proceed to TESE. This is a procedure when we can get the sperm from the epithelium if the sperm is unable to travel from the testicle in to the epididymis.

Via a small cuts in the testicles cover we get a piece of the tissue. This is proceeded in the lab. If any live sperm is present it is used for fertilization. If azoospermia(no living sperm) is repeatedly confirmed, gain of a suitable sperm is not guaranteed using these methods. The patient is informed about the outcome of a procedure straight post procedure.

The Clinic of Reproductive Medicine and Gynecology Zlín allows its patients to use a modern method of embryo cultivation in an incubator as part of infertility treatment, in which the cultured embryos are continuously monitored by a camera. The EmbryoScope device (or embryoscope) is a unique device that combines an incubator, diagnostic software with artificial intelligence, a microscope and a camera system into one unit. This specific incubator allows for the creation of ideal conditions for the growth of embryos, which it also controls, evaluates and records. Embryologists therefore have constant control over the conditions in which the embryos are located. The selection of embryos is assisted by AI, which is incorporated into the evaluation module of the Embryoscope.

Intralipid 20% infusion 

Research has shown that early pregnancy losses (spontaneous abortions, silent pregnancies) and implantation failure (embryo implantation), which manifests as infertility or repeated failures of assisted reproduction treatment, can be caused by excessive activity of NK cells (“Natural Killer”). NK cells are a specific population of white blood cells that take care of the body’s defenses, but their active form can also attack an embryo that implants in the uterine cavity and weaken it so much that it dies. It has been shown that early administration of Intralipid can prevent the activation of NK cells. Without this activation, NK cells pose no danger to the embryo.

Sperm selection method based on magnetic activation of apoptotic sperm – MACS (Magnetic – Activated Cell Sorting) System.

All sperm have a limited lifespan and are destroyed by a process called apoptosis. In sperm where the apoptosis process has already begun, there is a high probability of DNA fragmentation (breaks). Unfortunately, these breaks cannot be detected using a microscope (ICSI, PICSI methods). If apoptotic sperm is used to fertilize an egg, there is a higher probability that the embryo formed from them will not develop ideally, or will stop developing completely.

Using MACS, we are able to magnetically mark apoptotic sperm, which after the sample flows through a special column will remain trapped inside, while sperm without signs of apoptosis will be captured under the column. These “live” sperm without signs of DNA fragmentation can then be used for other treatment methods: IUI, IVF, ICSI, PICSI.

This laboratory method of sperm selection uses a special glass chip with microbarriers that sorts sperm based on the principle of natural selection.

The microbarriers mimic the natural environment of a woman’s fallopian tubes.

The selected sperm have better morphology, viability and motility.

The entire process takes place without any addition of additional reagents and significantly increases the success rate of the cycle.

PRP therapy (Platelet-Rich Plasma) is an innovative and gentle method that uses plasma obtained from your own blood. After processing, it contains a higher concentration of platelets with a large amount of growth factors that help the body regenerate.

The main benefit of PRP therapy is the growth of the endometrium and the improvement of the readiness of the uterus for embryo implantation, because the quality of the lining is essential for successful artificial insemination.

If the endometrium does not reach the required parameters, the chance of embryo implantation decreases and the risk of spontaneous abortion and other complications increases.

A great advantage of this method is also its safety – these are cells that the body knows, so you do not have to worry about adverse reactions.

HOW DOES PRP WORK?

Blood collection: First, blood is taken from a vein in the elbow, which is then processed in a special device.

Preparation of PRP: Platelet-rich plasma with a high concentration of growth factors that support tissue regeneration is separated from the blood.

Application: PRP is applied to the uterine cavity with a thin catheter. The process improves its regeneration.

WHO IS THE METHOD SUITABLE FOR?

We recommend the method to women who need to improve the quality of the uterine lining (e.g. before embryo transfer).

Advantages of PRP

• A safe procedure that does not require invasive interventions.

• Helps increase the chance of pregnancy.

• Using your own biological material minimizes the risk of allergic reactions.

• Supports the growth and quality of the uterine lining.

WHAT IS THE TREATMENT PROCEDURE?

• IVF cycle: PRP application is suitable at the beginning, in some cases even before embryo transfer, of a standard IVF cycle or KET.

• Consultation: The doctor will evaluate the situation and propose an individual treatment plan.

• PRP application: The process is quick, painless and usually takes under 1 hour.

Oocyte maturity is an essential prerequisite for its successful fertilization.

The maturity of the oocytes is standardly evaluated by an embryologist under a light microscope. An oocyte with a so-called polar body present is considered mature.

But even such an oocyte may not be genuinely mature. The presence of a dividing spindle is important, which is necessary for the correct division of chromosomes after fertilization. Now we can detect the dividing spindle non-invasively thanks to a new imaging method – polarized light microscopy and fertilization (ICSI / PICSI) of genuinely mature eggs.

This method is suitable for patients with a lower number of oocytes obtained and in patients who have failed fertilization in previous IVF cycles.

Transfer medium intended for cryoembryotransfer – frozen embryo transfer.

Contains substances, including GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), which support embryo viability, protect it from oxidative stress and improve its ability to attach to the uterus.

KET or cryoembryotransfer is the transfer of a thawed embryo into the uterine cavity. It is used for embryos that were frozen after a previous IVF cycle or IVF donor eggs cycle, for example due to an excess number of high-quality embryos, PGT genetic testing or postponing the transfer to a more suitable time.

The advantage for the woman is that repeated hormonal stimulation and egg retrieval is not necessary (it is not necessary to match the menstrual cycle of the donor with the recipient). Before the transfer itself, the uterine lining is prepared by hormons (estrogens + progesterons) so that it is as suitable as possible for the embryo to implant. 

The embryo is thawed on the day of the transfer and the embryologist assesses its quality and ability to develop further. It is then transferred into the uterus with a thin special catheter. The procedure is short, usually painless, is performed on an outpatient basis and does not require anesthesia. A pregnancy test is performed approximately 2 weeks after the transfer.

The success rate of KET is comparable to fresh embryo transfer. Current findings do not demonstrate an increased risk of congenital malformations after cryopreservation of embryos.