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1 August 2012 content 8 Preimplantation genetic diagnosis new method of screening of 24 chromosomes with the Array CGH method...2 Maintaining fertility new opportunities in GENNET...3 Hysteroscopy without the necessity of general anaesthesia...4 Irritation of the endometrium without the necessity of general anaesthesia now as part of a cycle free of charge...4

2 Preimplantation genetic diagnosis new method of screening of 24 chromosomes with the Array CGH method We carry out a screening for aneuploidies using the Array CGH method for 24 chromosomes in our laboratories at present. The purpose of the examination is to select embryos without genetic abnormalities to be transferred into the uterus. Such abnormalities could lead to serious developmental defects, and they would also increase the risk of a miscarriage and reduce the possibility of a successful pregnancy. We use microchips for the screening of the most suitable embryos the Array CGH method. Instead of using one cell of a three-day-old embryo we use several cells of a five-day-old or a six-day-old blastocyst. Such a removal represents a lower risk and it has a higher predictive value at the same time. Furthermore, this procedure has an economic effect for the patient, because we inspect fewer embryos. Besides, we use the so-called dislocation method, which means that the embryo is transferred in the subsequent cycle after achieving optimal preparation of the endometrium. ted with extracorporeal fertilization. Apart from an IVF doctor, the couple must also consult a clinical geneticist in order to discuss the reasons, procedure, and benefits of the preimplantation genetic diagnosis. WE CARRY OUT THE FOLLOWING EXAMINATIONS IN OUR LABORATORIES: PGD with sex selection using the FISH method It concerns cases in which a geneticist indicates detection of sex. Preimplantation genetic diagnosis with sex selection is offered to couples who suffer from a genetic disease that is linked to the sex chromosome, and where it is not possible to perform PGD using the PCR method. It is carried out for genetic reasons only. Examples of X-linked diseases, for which PGD-FISH is performed, are as follows: Duchenne / Becker muscular dystrophy, hemophilia, SCID. PGD using the FISH method for translocation carriers The most common chromosomal aberrations in couples undergoing PGD are inversions, reciprocal translocations, and Robertsonian translocations. PGD offers a possibility to determine whether embryos that are derived from in vitro fertilization (IVF) contain any so-called unbalanced chromosomal changes, which result from a carriership of a structural chromosomal aberration of the parent. The purpose of PGD is to reduce the risk of miscarriage or pregnancy termination after prenatal diagnosis of an embryo with unbalanced chromosomal changes. PGD using the PCR method Preimplantation genetic diagnosis (PGD) enables analysis and elimination of genetic abnormalities in embryos before their implantation. This method is based on removing one to two cells (or even several cells) from the embryo and their genetic examination. The removal of the cells should be carried out on the third or fifth day. This method takes advantage of the fact that each cell of the embryo should contain the same genetic make-up. Individual cells in the early stages of the development of the embryo are not specialized yet and therefore, the removal of the cells does not affect a future development of the embryo. Preimplantation genetic diagnosis is dedicated for couples with a high genetic risk for their offspring. PGD can prevent a transmission of a genetic disease to another generation, without requiring a necessary prenatal diagnosis and a subsequent termination of pregnancy with a disability. PGD is always associa- Preimplantation genetic diagnosis using the PCR method for monogenic diseases is intended for couples, whose offspring face increased risks of a hereditary genetic disease. There is a necessary requirement for performing PGD, which is that the disease must be testable at the level of genes. In other words: the specific genetic change (mutation) must be known to the family as responsible for the disease. Even in case of PGD it is necessary to take into account the risk that the whole IVF cycle does not have to be completed with an embryo transfer into the uterus. The examination can fail for various reasons: because of a small number of embryos that form corrugations, due to incorrect handling of the removed embryonic cell, or due to a contradictory result of PGD. The investigation only focuses on a selected type of chromosomal disorders, and it cannot exclude the possibility that there might arise a suspicion of a different genetic disorder in case of a pregnancy. Although PGD cannot entirely replace genetic testing 4 page 2

3 of the fetus in later stages of the pregnancy (e.g. amniocentesis), it increases the percentage of successful implantation of the embryo into the uterus and it reduces the risk of subsequent spontaneous miscarriages. Above all, it reduces significantly the risk of a genetic barrier to the fetus. Maintaining fertility new opportunities in GENNET Recently, there has been an increasing number of people who have to undergo treatment for an oncological disease, both in their childhood and in reproductive age. Chemotherapy and radiotherapy are a potential threat to reproductive capacities of women. Current methods of assisted reproduction offer an opportunity for women and girls with oncological diseases to preserve their fertility after treatment. Modern methods of procedures of assisted reproduction (ART) offer several options how to preserve fertility in women after cancer treatment. Each method has both its advantages and limitations. Freezing of embryos Cryopreservation of embryos is the most common and most proven method of preserving fertility. It is used in IVF cycles for infertility treatment as a matter of routine. The method requires postponing the beginning of the treatment by 2 to 3 weeks because of the stimulation of the ovaries. This method is unsuitable in some cases, such as when the chemotherapy must be started immediately, or when the woman must not take any hormone therapy. It also requires a source of male gametes, therefore it is inappropriate for women with no regular partner. Despite these limitations it is the most successful method - approximately 75% of embryos survive the freezing process, with a pregnancy rate of up to 30% per cycle depending on the age of the woman and number of embryos transferred. Vitrification increases the percentage of surviving embryos up to 90%. Freezing of oocytes Freezing of oocytes is an accessible option for young women and for women without a partner. Success of this method was increased due to fast freezing - vitrification. Thanks to vitrification, survival rate of oocytes has risen to 90%, compared with 50 to 60% when using conventional methods. Removal and freezing of immature oocytes and in vitro maturation can be performed even without hormonal stimulation. Therefore, it is not necessary to postpone the start of oncological treatment. This method is relatively new and it shows promising results. Its further advantage is the fact that it is suitable for younger patients where it can replace freezing of ovarian tissue. By using the method of oocyte vitrification we currently achieve results that are comparable even with IVF treatment using fresh embryos. 4 page 3

4 Freezing of ovarian tissue Freezing of ovarian tissue includes a laparoscopic removal of ovarian tissue with a subsequent transplantation at the time of desired conception. This method has several advantages - the tissue can be removed in any phase of the menstrual cycle, and no male gametes are needed. That is why this method is suitable especially for young women and children. However, ovarian tissue can theoretically be a source of proliferation of malignant cells. Hysteroscopy without the necessity of general anesthesia We offer our patients ambulant hysteroscopy, both under general anesthesia, and without general anesthesia. We can carry out a painless hysteroscopy without general anesthesia because the GENNET Archa clinic uses a new type of hysteroscope. The advantages of the method without general anesthesia consist mainly in a simpler planning of the intervention and an easier preparation of the patient: patients do not have to enter hospital a day before intervention pre-operational tests are not necessary only premedication is used, which requires a preparation time of an hour prior to the intervention the intervention itself lasts only 5 to 10 minutes patients can observe the progress of the examination on a screen patients rest in their rooms for 30 minutes after the intervention, the length of stay in the clinic is thus much shorter patients can leave the clinic on their own, they do not have to be accompanied Irritation of the endometrium without the necessity of general anesthesia now as part of a cycle free of charge According to recent findings, which were verified by several studies, irritation of the endometrium can improve receptivity of the endometrium, or rather improve its implantation ability in some patients. Such irritation of the endometrium is performed in a goal-directed way under visual control while the hysteroscope is introduced. It is a simple ambulant procedure which lasts only a few minutes and which can also be performed without general anaesthesia, should it be more convenient for the patient. We carry out this intervention free of charge as part of an IVF cycle. 4 page 4

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