Laboratory Procedures in IVF Treatment

Routine Sperm Analysis

-The ejaculate (semen) sample taken from the patient in one or two separate containers (split)1 is kept in a cabinet (oven) at 37C for 15-30 minutes (liquefaction time; the liquefaction time required for the sample to be examined). Then, the number of sperm in the sample, their mobility and morphological (shape) characteristics are evaluated.

-After the evaluation, the semen sample is put into the washing process. This two-stage process takes approximately 35-40 minutes. (The purpose of the process is to purify the sperm cells in the semen sample taken from the patient from other seminal plasma cells (waste cells) and to increase sperm movements and make them ready for fertilization.)

Vaccination

-Following the washing process, the prepared sample is kept in special cabinets (incubators) containing 37C temperature and 6% CO2 (carbon dioxide - an environment similar to an artificial body environment) for 30 minutes in order to increase sperm movements. At the end of this period, the sperm are injected into the woman's uterus with a syringe. Thanks to this process, the path that the sperm must travel is shortened.

-Pregnancy rates in insemination vary between 15-25% depending on the cause of infertility of the couple, the age of the woman, the number of follicles (cells in which the egg develops) formed as a result of the stimulation protocol (drug treatment) and sperm parameters.

IVF-Microinjection

The decision to apply microinjection or in vitro fertilization to the patient is determined based on the cause of infertility, the number of oocytes (eggs) collected, and sperm characteristics. While the procedures to be applied to the patient in in vitro fertilization and microinjection applications are similar, the procedures performed in the IVF laboratory differ. In in vitro fertilization, the eggs taken from the prospective mother and the sperm taken from the prospective father are placed in a special culture fluid together and kept in special cabinets called incubators for 14-16 hours, and at the end of this period, the sperm cells are expected to fertilize the eggs. In microinjection, unlike in vitro fertilization, a single sperm cell is mechanically placed into each egg. Thus, instruments called micromanipulators assist in the task to be performed by the sperm. The majority of studies conducted to date have shown that similar pregnancy and implantation (the number of embryos that can attach to the mother's uterus per transferred embryo) rates can be achieved after in vitro fertilization and microinjection.

IVF

-After egg collection, the eggs are incubated in special culture solutions in special incubators containing 37C temperature and 6% CO2 for 3-5 hours to complete their maturation.

-In this process, the semen sample is washed and made ready for processing and kept in incubators at 37C and 6% CO2 until the fertilization process is performed.

-At the end of the period, the prepared sperm sample is placed in the medium where oocytes (eggs) are cultured in a concentration determined according to their number and mobility and is incubated at 37C and 6% CO2 for approximately 14-16 hours for the fertilization process to take place.

-A day later, the eggs are cleaned of surrounding cells and checked for fertilization. Observation of the male and female nuclei in the egg is an indication of fertilization.

Microinjection

-After egg collection, the oocytes are kept in incubators at 37C and 6% CO2 for 2-3 hours to complete their mastication in culture solutions.

-During this time, a semen sample is prepared.

-At the end of the period, the cells around the egg are cleaned under a microscope and their maturity is checked. According to general world statistics, 70-75% of the collected eggs should be mature. Only mature eggs should be processed.

-Oocytes are kept in incubators at 37C and 6% CO2 for 1-2 hours after the cells around them are cleaned and microinjection is performed. The most obvious difference between microinjection and in vitro fertilization is that a single sperm is mechanically placed into a single oocyte using special micromanipulators under a microscope. Subsequent fertilization and embryo development stages proceed in a similar manner.

-Within 24 hours of fertilization, eggs can reach the 2-4 cell stage, at the 72nd hour the 6-8 cell stage, at the 96th hour the multicellular stage called morula, and at the 5-6th day the blastocyst stage, which is the last stage of the embryo before it attaches to the uterus. From the 24th hour onwards, fertilized eggs are divided into groups according to their quality by examining their morphological structures under a microscope. In this classification, the cell stage that the embryo should be at that day (cleavage speed), the equality of the cells to each other, the percentage of intracellular atypical structures called fragmentation, which have been shown to negatively affect the further development of the embryo, and the brightness and homogeneity of the intracellular appearance are examined. According to these evaluations, the best quality embryos are those that have reached the division stage that they should have been at that day, have equal cell sizes and are homogeneous in appearance, and do not contain any fragmentation. (G1 embryo) G2 embryo: Unlike G1, G3 embryos contain less than 20% fragmentation; G4 embryos contain unequal cells and 20-50% fragmentation; G4 embryos contain unequal cells and more than 50% fragmentation. Studies have shown that the probability of pregnancy is higher with the transfer of G1-G2 embryos than with the transfer of G3-G4 embryos due to the higher developmental potential of these embryos. However, the ratio between G1 and G2 and between G3 and G4 are similar. When the embryos reach the blastocyst stage, they are reclassified among themselves according to their quality. In this classification, the development speed of the blastocyst and the structure of the inner cell layer are examined. According to this classification, good quality blastocysts are called BG1-BG2 (the only difference between BG1 and BG2 is that BG1 blastocysts develop faster); bad quality blastocysts are called BG3. BG1-BG2 blastocysts have a higher probability of causing pregnancy than BG3 blastocysts. The difference between BG1 and BG2 is similar.

-Embryo transfer can be done between days 2-6 of embryo development. Although studies conducted to date are still contradictory, pregnancy rates are similar according to the day of transfer.

Blastocyst Transfer

Blastocyst is the final developmental stage that the fertilized egg reaches before attaching to the mother's uterus. The diameter of the embryo gradually increases and the outer sheath thins as it reaches the blastocyst stage. The blastocyst, hatched from its outer sheath, attaches to the inner layer of the uterus and creates pregnancy. In today's culture environments, only an average of 45% of embryos can reach this stage, and only 15-20% of these blastocysts can continue their development until the hatched stage. These rates decrease even further when the embryos in the cleavage stage are of poor quality. For these reasons, blastocyst transfer can only increase the chance of pregnancy if it is applied to selective patient groups under current conditions. (The most accepted indications are: Patients who have had at least two unsuccessful IVF-ICSI cycles and whose embryo development needs to be monitored more carefully until the final stage, patients with a high risk of multiple pregnancy, young patients with a large number of embryos and patients who can select embryos with high developmental potential (blastocysts) and require fewer embryo transfers.)

Embryo Freezing

It involves the freezing of embryos by gradually cooling them with a special device after they are balanced in protective culture solutions (cryoprotectants) and storing them in liquid nitrogen (-196C). Depending on the embryo quality and the patient's age, the number of embryos to be transferred may vary between 2-4. If the patient has more good quality embryos after the transfer (or without any transfer in that cycle due to negative clinical findings), these embryos can be frozen for transfer in the repeat cycle. Since the best quality embryos will be minimally damaged by the freezing process (viability rate in embryos after freezing and thawing; 75%-90%), only good quality embryos are frozen. If poor quality embryos are frozen, their probability of survival is quite low. (20%-25%) The freezing process can be done between the 2nd and 6th days after the selection of the embryos to be transferred, or it can be done on the day fertilization occurs. According to world statistics, pregnancy rates after the transfer of frozen embryos after thawing vary between 25%-50%. These rates vary depending on clinical and laboratory procedures, patient age, cause of infertility, and viability rate per thawed embryo.

Testicular Biopsy

- In patients with no sperm cells found in their ejaculate (azoospermia) or with all or at least 80% of the ejaculated sperm being dead, sperm cells are searched for in the testicles or epididymis (ducts), which are the source of sperm production.

-If the cause of azoospermia is a blockage in the ducts (epididymis) (obstructive azoospermia), the presence of sperm is investigated in the fluid aspirated from the ducts under a microscope in the laboratory. (MESA - microepididymal sperm aspiration or a different method, PESA - percutaneous epididymal sperm aspiration). If no sperm cells are found in the ducts, a testicular biopsy is performed.

-In cases where sperm cannot be found in PESA/MESA or in patients whose azoospermia is directly related to a production disorder in the testicles (ovaries) (non-obstructive azoospermia), the presence of sperm cells is investigated by taking a sample from the testicle instead of the fluid taken from the ducts (epididymis).

-The tissue piece taken during the testicular biopsy is examined under a microscope in the laboratory.

-If a tissue sample has been taken, it is completely separated in the laboratory to reveal possible sperm cells.

-At the end of the examination, the sample is washed. This process consists of two stages and can take between 35-45 minutes depending on the sperm count.

-Testicular biopsy can be performed on the day the spouse's eggs are collected, or it can be performed 24 hours before. If it is performed 24 hours before, the sample prepared by washing in the IVF laboratory can be incubated in incubators at 37C and 6% CO2 (the aim is to create an artificial body environment) until the microinjection process. Since testicular sperm are immature cells, they may not have gained the ability to move yet. Therefore, taking a tissue piece 24 hours before and keeping the washed sample in incubators can be beneficial in terms of starting sperm movement activity.

Assisted Hatching

The indications for assisted hatching accepted in assisted reproductive techniques are: Advanced age (35 years and above), at least one failed IVF-ICSI cycle, high basal hormonal values, the outer embryo sheath being thicker than normal, and poor embryo quality. When the method is applied selectively in these patients, it can increase pregnancy rates. Studies have shown that it has no effect on pregnancy rates when applied to patients not included in these indication groups. An embryo that can complete its normal development should be able to shed its outer sheath before attaching to the inner layer of the uterus.

The aim of assisted hatching is to thin the outer shell of the embryo a little before transfer, thus facilitating its attachment to the uterus and increasing the chance of pregnancy. This procedure can be applied at different stages of embryo development (2-5 days). Scientific studies have shown that assisted hatching can negatively affect further cell development in 2-cell embryos that have just completed their first division. Therefore, assisted hatching is not applied to 2-cell embryos. Assisted hatching can be applied with different methods in the IVF laboratory. For example: using laser beams, melting with acidic solutions or cutting with sharp pipettes.

Scientific studies have reported similar pregnancy rates for different methods. Today, laser-assisted hatching is the fastest and most practical method and requires minimal user experience, making it the most widely used method.