Special Procedures

Embryo Freezing (Cryopreservation)

In the cycle in which IVF treatment is performed, surplus good-quality embryos remaining after the transfer of good-quality embryos to the patient can be frozen and stored for use in subsequent attempts, at the couple’s request. In Turkey, embryos may legally be stored in liquid nitrogen for 5 years. The ability to freeze and store embryos provides material and emotional advantages to the patient. If pregnancy does not occur in this procedure or ends in miscarriage, or if the couple wishes to have another child after a birth, the embryos are thawed and only embryo transfer is performed in the next IVF procedure, without the need to re-stimulate the ovaries and retrieve eggs again.

Occasionally, embryos may need to be frozen and their transfer postponed due to medical necessity. If any disease, surgery or similar situation arises in the woman during the period when the embryos would be transferred, the transfer may be postponed and the embryos frozen. If the layers lining the inner wall of the uterus do not mature sufficiently and remain thin, or if hyperstimulation syndrome (OHSS) develops due to excessive stimulation of the woman’s ovaries during treatment, the embryos may be frozen and transferred at a time when these problems have been resolved. Furthermore, when polyps, fibroids, adhesions or similar problems occupying space inside the uterus are noticed during treatment, the embryos are frozen and, after these problems are resolved by surgical treatment, embryo transfer is performed. There are 2 separate techniques for embryo freezing. These are: slow controlled freezing and vitrification, which is a newer technique. The most fundamental difference between slow controlled freezing and vitrification is the mechanism by which the structural integrity of the cell is preserved. In the slow freezing method, cell viability is ensured by forming ice crystals around the cell during gradual cooling, whereas in the vitrification technique, an attempt is made to preserve the cell by forming a glass-like solid surface around it with a sudden drop in temperature, with the help of highly concentrated freezing solutions. Embryos can be frozen at all developmental stages. Embryos are balanced with a protective fluid (cryoprotectant), placed in special plastic carriers and frozen at -196 degrees in liquid nitrogen. They are stored in special tanks with separate storage locations for each patient. When frozen embryos are to be thawed, they are removed from liquid nitrogen, separated from the protective fluid, placed in a special culture medium and put into an incubator. Embryos that look good on the same day or the next day can be transferred. Because embryos are sensitive to the freezing process, it may not be possible for all frozen embryos to have the same viability after thawing. The viability rate expected from a good freezing program is 75–80%. Clinical pregnancy and live birth rates obtained with transfer of frozen-thawed embryos are close to those obtained with fresh embryo transfers.

Freezing and Storage of Eggs

Freezing and storing eggs is a new and experimental technique compared with other IVF treatments. The most important factor affecting success is the high likelihood that eggs lose their viability during freezing and thawing. Human eggs contain a high proportion of water. During the freezing process, ice crystals may form and damage the cell, which can impair the egg’s viability and chance of fertilization. In cases of medical necessity, eggs may be frozen without being fertilized by sperm, for use in later years. These medical necessity situations are specified in the Ministry of Health Regulation. In unmarried women, eggs may be frozen before cancer treatments that carry a high risk of causing infertility, due to medical necessity. If the woman is married before cancer treatment, embryo freezing is preferred over egg freezing due to its higher success rate.

Shaving/Peeling (Assisted Hatching)

This is the procedure of partially thinning the outermost membrane (zona pellucida) of embryos to be transferred under a microscope, mechanically, with acid or with a laser. It is thought to facilitate the embryo’s attachment to the uterine wall.

It is applied in older women, in embryos with thick walls, or in patients who have had problems with implantation in earlier procedures. In our hospital, this procedure is performed with a laser device, which is a state-of-the-art technology.

IMSI (Intra Cytoplasmic Morphologically Selected Sperm Injection)

Intracytoplasmic morphologically selected sperm injection (IMSI) is a method that, compared with conventional IVF/ICSI microscopes, enables detailed examination of the morphological analysis of sperm using special magnification techniques. In standard ICSI procedures, live sperm cells can be magnified up to 400 times to examine the general morphological features of the sperm (head-neck-tail structure). However, structures that should not be present, such as organelles and vacuoles in the head of the sperm cell, and the cell nucleus cannot be examined. The magnifications used in IMSI exceed 6000, allowing the morphologically best sperm to be selected. In live sperm, intracellular structures and nuclear morphology are examined in detail. Thus, by means of microinjection performed after detailed selection of normal or nearly normal sperm, higher fertilization can be achieved. Thanks to this advanced method, it is possible to detect some intracellular structures in the sperm cell, such as vacuoles (cavities surrounded by a membrane), which are known to disrupt chromatin stabilization; sperm that do not show these defects or show the fewest defects are selected, and ICSI (microinjection) is performed with sperm that have high fertilization capacity. Applying the IMSI technique requires more time and well-trained personnel compared with conventional ICSI, and results in higher cost.

For whom should IMSI be applied?

• Especially in cases with severe male factor infertility,
• In cases of recurrent IVF failure,
• In cases of infertility of unexplained cause,
• In cases where the rate of normal sperm in the sperm sample is low; with IMSI application, implantation and pregnancy rates can be increased and the risk of early pregnancy loss can be reduced.

PGT (Preimplantation Genetic Diagnosis)

Whether a baby developing in the mother’s womb before birth has a genetic disease can be determined by tests performed from the 12th week of pregnancy onward. However, when these tests show a serious disease, the pregnancy must be terminated at an advanced stage (approximately the 5th month). This causes psychological and physical strain on the couple. In contrast, with preimplantation genetic diagnosis (PGT), embryos with a genetic disease can be identified before being transferred to the mother, that is, before pregnancy begins, and embryos that may have a numerical or structural chromosomal abnormality are detected. Thus, only healthy embryos can be transferred. Even if embryos appear normal in shape and structure, pregnancy may not develop or a developing pregnancy may end in miscarriage due to chromosomal abnormalities. Research has shown chromosomal abnormalities in up to 35–40% of embryos that appear to be of good quality, and up to 85–90% of poor-quality embryos. Whether the patient is suitable for PGD is evaluated by the reproductive health specialist, the genetic counselor and the physician of the relevant disease. The couple prepares for the IVF procedure. Eggs taken from the mother are fertilized with sperm taken from the father in the laboratory environment. When the divided embryos reach the 6–10 cell stage on day 3, they are first kept in a special fluid prepared for separation of the cells, and then 1–2 cells are removed from each embryo under the microscope with an appropriate technique. The cells obtained by biopsy are numbered, and after fixation, special techniques such as Fluorescence In Situ Hybridization (FISH) or Polymerase Chain Reaction (PCR), which allow chromosomes to be examined under the microscope, are applied. Embryos showing numerical or structural chromosomal abnormalities are identified and separated; healthy embryos are transferred into the uterus of the prospective mother. The cases in which this technique is applied must be selected with extreme care.

Genetic Diseases Detectable with the PGT Method

• Down syndrome (monosomy 21)
• Trisomies
• Thalassemia (Mediterranean anemia),
• Sickle cell anemia,
• Cystic fibrosis,
• Tay-Sachs disease,
• Hemophilia A and B,
• Retinitis pigmentosa,
• Alport syndrome,
• Alpha-1 antitrypsin deficiency,
• Fragile X syndrome,
• Phenylketonuria,
• Epidermolysis bullosa,
• Duchenne muscular dystrophy,
• Myotonic dystrophy,
• Fanconi anemia,
• X-linked hydrocephalus,
• Achondroplasia,
• Neurofibromatosis,
• Blood incompatibility (Rh D) disease,
• Marfan syndrome,
• Huntington’s disease.

Metabolomics

Today, embryos to be transferred are evaluated under a microscope, and the best-quality embryo with the highest likelihood of attaching to the mother’s uterus is selected according to cell count and morphological features. However, good embryo quality is not sufficient to determine whether embryos are normal in terms of genetic and metabolic activities. Assessing whether embryos are chromosomally (genetically) normal is only possible by examining one or two cells obtained by biopsy using preimplantation genetic diagnosis (PGT). However, for this method to be applied, valid reasons such as advanced maternal age, being a carrier of a genetic disease, recurrent pregnancy losses, failed procedures, or severe male factor must exist. Just as a person’s health status is learned by evaluating blood and urine tests, it is thought that information about embryos’ health status can be obtained, and the chance of establishing a pregnancy can be assessed, by examining their metabolism, that is, which substances they consume from the culture medium in which they are kept and what they secrete into that environment. The name of this newly developed method is “Metabolomics.” This method, which has not yet entered routine use and whose benefits are being investigated, has no additional advantage of increasing the embryo’s ability to attach. It has not been proven to increase pregnancy rates.

Endometrial Co-Culture (Artificial Uterus)

In couples with recurrent IVF failures and recurrent slow and/or poor embryo quality, it has been considered that culturing embryos on endometrial cells from inside the uterus, collected from the prospective mother and stored frozen before starting treatment, may be successful. On day 21 of the menstrual cycle, a small tissue sample taken from inside the uterus is processed under laboratory conditions; a culture medium is prepared with the patient’s own inactivated and filtered serum obtained from her blood, and the necessary cells are multiplied (taking 1–3 weeks). If treatment is planned for the next menstrual period, culture is continued. If a later date is planned, the cells that have developed are frozen and stored. The patient is then started on IVF treatment. When egg retrieval is performed, the stored frozen samples are thawed. An artificial uterine tissue, referred to as co-culture, is formed. After IVF is performed and fertilization is observed, the resulting embryos are grown within this tissue until transfer. Because culture fluids developed in recent years provide a very suitable medium for embryos, and co-culture applications do not significantly increase the chance of pregnancy, most centers do not prefer this method, which involves risks.

Lymphocyte Immunization

In unexplained infertility or recurrent pregnancy losses, it is thought that the mother may have an allergic rejection reaction against the father’s cells. To treat this reaction, lymphocytes taken from the father and subjected to various procedures are injected into the mother. Its effectiveness has not been demonstrated in scientifically controlled studies.

Embryo Glue (Adhesive)

This culture fluid is the fluid that holds the embryos in the catheter during transfer of embryos into the uterus and carries the embryos during transfer into the uterus. The distinctive feature of this fluid is that it contains a high proportion (0.5 mg/ml) of the substance “hyaluronan”, a glycoprotein-structured molecule. Hyaluronan is a substance that plays a role in intercellular connections and in determining the distinct properties of tissues. Although the exact role this substance plays in the embryo–endometrium relationship is not clearly known, it is thought that adding it to the transfer culture fluid increases the chance of embryos attaching within the uterus. Embryo gluing is not a technique or a procedure. An embryo without attachment potential cannot be glued to the inner wall of the uterus by any procedure or substance. As a result of studies conducted, no finding has been obtained showing that this fluid increases the chance of pregnancy.

IVM (In Vitro Maturation)

Immature eggs collected after short-term medication use or without any medication are matured in special culture fluids in the laboratory environment and then microinjected. It is an application that can be applied to a specially selected patient group and has lower success than standard IVF treatments.