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Embryology Laboratory

Embryology Laboratory

All procedures, starting from egg collection (OPU) to embryo transfer, are carried out in the Embryology Laboratory.
Embryology Laboratory

All procedures, starting from egg collection (OPU) to embryo transfer, are carried out in the Embryology Laboratory, where an environment equivalent to the uterus is provided outside the female body (in vitro). The embryo formed as a result of fertilization of the egg with sperm has a certain developmental potential depending on the quality of both gamete cells. This potential is determined entirely by the gamete cells, and it is not possible to achieve a positive change with any external intervention after fertilization.

Liv Hospital Embryology Laboratory


Liv Hospital's Embryology Laboratory is a unit that provides services with the latest technology and expertise in in vitro fertilization treatment processes. The laboratory is equipped with a team experienced in reproductive endocrinology, genetics and embryology, and is a leader in solutions for patients' reproductive health. Liv Hospital's Embryology Laboratory is known as a reliable partner for personalized in vitro fertilization treatment in an environment that complies with high standards of safety and ethics.

Laboratory Quality is Important


The importance of the Embryology Laboratory emerges at this point. Because if the laboratory does not have the appropriate personnel, equipment and environmental conditions, it is likely to negatively affect this potential. Therefore, it is vital for success for couples to be conscious about laboratory quality when choosing a center.

Routine Procedures Applied in the Embryology Laboratory


  • Egg collection (OPU)
  • Denudation (Egg preparation)
  • fertilization
  • embryo culture
  • Assisted hatching (AHA)
  • Embryo biopsy (Preimplantation genetic diagnosis-screening)
  • embryo transfer
  • embryo freezing

Egg Collection-OPU (Oocyte Pick-up)


The task of the embryology laboratory in the egg collection process is to collect the eggs in the follicular fluids aspirated by the physician (entered into the follicle with a needle and withdrawn) under the microscope in the sterile working cabinet. Oocytes (egg cells) arriving at the laboratory are collected at body temperature and placed in culture fluids and kept for 3 hours until the denudation process, which will separate the surrounding cells.

Egg cells may not emerge from all of the follicles from which the egg was collected and whose growth was observed with ultrasound until that day. This may be due to differences between the number of follicles indicated to the patient during follow-up and the number of eggs collected in the laboratory.

Denudation (Egg Preparation)


The eggs collected in the OPU process are surrounded by thousands of cumulus cells, defined as COC (Cumulus oocyte complex), which support the development and vitality of the egg during the development phase and the process until fertilization. If microinjection (ICSI) is planned, cumulus cells are separated from the eggs through a process defined as denudation in order to identify mature eggs and egg quality. In the microscopic evaluation, mature and immature eggs are detected, they are placed in culture liquids where they will be kept until the microinjection process, and they are placed in incubators that provide the necessary environmental conditions. Of the collected eggs, only the cells that have completed their maturity (Metaphase 2 - M2) are treated. For this reason, not every egg cell collected can be processed.



In embryology laboratories, two different methods called IVF and ICSI are used to ensure the fertilization of the egg with sperm:

IVF (In vitro Fertilization)


It is the first method used to ensure fertilization in in vitro fertilization treatments and was first successfully applied in England in 1978 by physiologist Robert Edwards. In this method, sperm prepared by the andrology laboratory are brought together in the same culture environment with eggs (COC) whose cumulus has not been cleared in the embryology laboratory, and the sperm is expected to naturally fertilize the egg.

Experience Required


In IVF application, there is a natural selection since only sperm with fertilizing capacity can enter the egg. However, this may also lead to a high rate of fertilization failure in cases where the fertilization potential of sperm is not adequately evaluated. Therefore, choosing the right laboratory for IVF application is very important. In IVF application, fertilization failure may also occur due to egg-related reasons.

Therefore, the common approach is to use the ICSI method on some of the eggs in cases where IVF is planned and a large number of eggs are collected. The common practice in the IVF process is the insemination of 50 thousand to 100 thousand forward motile sperm per oocyte into the culture medium where the oocyte is located. Using fewer sperm may lead to fertilization failure, while using more sperm may cause more than one sperm to fertilize the egg, which is defined as polyspermia. Since eggs fertilized in this way will lead to genetically abnormal embryo development, developing embryos cannot be used for transfer.

ICSI-Microinjection (Intra Cytoplasmic Sperm Injection)


It is a method developed to be used in cases where fertilization is not possible with IVF application, sperm parameters (number, motility) are low, there are severe sperm morphological defects, or sperm is obtained via the testicular route. First, in 1990, Prof. It was successfully implemented in Brussels by Gianpiero D. Palermo.

Technical Equipment Required


In order to perform the ICSI procedure, it is necessary that there be certain equipment in the laboratory and the personnel who will perform it must be trained and have sufficient skills. The pipettes used in the ICSI process, called micropipettes, one of which is used to hold the egg (holding pipette) and the other to inject the sperm into the egg (microinjection pipette), are controlled with the help of a special device called a micromanipulator mounted on a microscope. In addition, this entire mechanism should be placed on an antivibration table to prevent potentially harmful vibrations during the process.

For a successful ICSI application, the following conditions must be met and regular control of variables is essential:

  • The procedure must be performed on an antivibration table.
  • In order to evaluate oocyte morphology and, most importantly, to select the most morphologically correct sperm for microinjection, the image settings of the inverted microscope must be at a sufficient level and the embryologist must have sufficient knowledge about these settings.
  • The temperature of the heated glass surface of the microscope, where the container containing the sperm and eggs to be applied to ICSI is placed, must be appropriate (to ensure 37 C in the container).
  • To minimize the risk of damage to eggs and sperm during the process, choosing the right pipette and placing the pipettes in the micromanipulator at correct angles (35 degrees to the microscope axis) and parallel to each other.
  • If a hydraulic micromanipulator system is used; Repeating system maintenance and checks before each operation
  • The most important thing is that the embryologist who will perform the procedure has sufficient knowledge, skills and experience in all these matters.
  • ICSI procedure is a very sensitive microsurgery procedure, and if the above-mentioned conditions are not met at a sufficient level, it is possible to observe a high rate of fertilization failure, degeneration of eggs (damage to the point of losing their viability), and problems in embryo development and quality after the procedure.

If the man applying for treatment has the following indications, ICSI method should be preferred for fertilization purposes:

  • Routine semen analysis parameters are insufficient for IVF
  • Severe sperm morphological defects (globozoospermia, megalo-pinhead sperm, tail defect-fibrous sheath dysplasias, total immotile sperm)
  • Minimal morphological defects that may prevent sperm from binding to the zona pellucida (acrosomal deformity, irregular acrosomal distribution)
  • Conditions that prevent sperm from being obtained through ejaculate, obstructive and non-obstructive azoospermia conditions that require the use of testicular sperm.
  • History of low IVF/ICSI fertilization or total fertilization failure in previous attempts
  • Only eggs determined to be mature are used in the ICSI procedure. Under the equipment and conditions mentioned for the procedure, the fastest moving and morphologically correct sperm are selected, one per egg, and taken with a microinjection pipette and transferred into a solution that slows down sperm motility, where the sperm tail is broken with a pipette blow. The purpose of this process is to damage the layer surrounding the sperm tail (fibrous sheath) and the cell membrane inside, and to ensure the exit of protein structures (phospholipase C), which is defined as the oocyte activation factor in the sperm cytoplasm and which enables the oocyte to detect the entry of sperm at the molecular level and initiate chain reactions that will ensure fertilization, into the oocyte cytoplasm. . At the end of the process, the eggs are transferred into fresh culture liquid and incubated until fertilization control.

Fertilization Control


Fertilization control is done with an inverted microscope approximately 10-18 hours after the IVF/ICSI procedure. In this control, the observation of 2 nuclei (pronucleus-PN) in an adjacent position, one belonging to the sperm and the other to the egg, and a second polar body in addition is normal fertilization, and the development stage from this stage to the first division is called zygote. If this period passes, it will not be possible to determine whether fertilization has occurred or if there has been an abnormal fertilization, as the nuclei will merge and the nuclear membrane that makes them visible will disperse.

It is also possible to observe various anomalies in fertilization control. These anomalies:

  • MonoPN: It is the case when only one pronucleus is observed. In this case, cell division and embryo development can still be observed; However, since the developing embryo will most likely have a single gamete chromosome set (haploid), it will be aneuploid (numerical chromosome abnormality). MonoPN status may also result from asynchrony in the timing of nuclear formation and deletion, in which case one may have a normal set of chromosomes (diploid). However, in this case, developmental problems are often observed in the developing embryos.
  • 3PN: It may occur because one of the gametes, which normally should be haploid, is diploid or because more than one sperm is injected during microinjection. Although embryo development is often observed, transfer is not preferred because the developing embryo will most likely be aneuploid.
  • MultiPN: 3 or more nuclei are observed. Even if the embryo develops, it is never transferred.
  • Fragmented PN: It is the observation of one or more small nuclei next to 2 normal-sized nuclei. It may improve during the fusion process of the nuclei and normal embryo development can be observed.
  • PN sizes are different: This is the case when one of the nuclei is different in size from the other. Developmental problems are often observed in developing embryos.
  • Discrete PN: It is the situation where there are varying amounts of distance between the nuclei and they are not adjacent. It is thought to be caused by problems in the microtubule structures that regulate the position of the nuclei in the cytoplasm. Since the same structures also manage cell division, problems or pauses in embryo development can often be observed.

Embryo Culture


Embryo culture can simply be defined as creating intrauterine environmental conditions (in vivo) in the laboratory (in vitro) and developing embryos in this artificial environment. These environmental conditions;

  • Incubators that provide a controlled body temperature of 37 degrees and a controlled blood level of 5-6 percent carbon dioxide.
  • Culture solutions to meet the nutritional and cellular building block needs of embryos
  • Sterile work areas that will minimize the risk of contamination during the culture process
  • Laboratory ventilation cleaned of particles and volatile organic compounds
  • It can be summarized as expert personnel who will control the provision and continuity of these conditions. You can find the details of this subject in our article titled "Quality Control".

In embryo culture, ready-made solutions specially produced for this purpose and with the necessary quality control tests (MEA-mouse embryo assay, LAL endotoxin test) are used. What these solutions have in common is that they contain energy sources (pyruvate, glucose), building blocks (amino acids), pH buffers (bicarbonate), minerals and supporting components (EDTA, albumin) that embryos will need during the development process.

In general, there are two different approaches in cultural systems. Sequential culture and monoculture. In the sequential culture environment approach, it is argued that embryos are exposed to different environmental conditions in their natural environment, in the tubes and in the uterus, and therefore the same variable conditions should be provided in the laboratory (back to nature). In the mono culture approach; It is argued that the embryo has the ability to use the same culture solution in different amounts according to its changing needs during the development process, therefore there is no need to change the solution content according to the developmental period (let the embryo choose). Both approaches yield similar success rates, depending on the performance of the laboratory using them.

Embryo development begins when the fertilized egg (zygote) undergoes the first division (20-26 hours). This developmental process is divided into two: cleavage and blastocyst period:

Cleavage Period


The development of the embryo from 2 cells (day 1 after OPU) to the stage containing 15-20 tightly combined cells (morula) (day 4 after OPU) is called the cleavage period. As the name suggests, this period is the period in which the embryo increases the number of cells through mitosis. However, beyond the obvious, metabolic activities, protein synthesis and DNA copying processes take place within the cell. One of the most important changes that occur during this period is that after the 8-cell stage, the sperm genetic structure also participates in directing embryo development (embryonic genome formation). Therefore, possible sperm-related problems mostly occur after this stage. In recent years, studies conducted with devices with continuous monitoring systems show that the timing of mitosis in this period can provide information about the advanced development potential and genetic structure of the embryo.

During this period, embryo quality is determined mainly by the number of cells, the size of the cells relative to each other, and the presence and rate of fragmentation (cell particles). Additionally, possible morphological anomalies (vacuole-liquid-filled sac, granulation) may negatively affect embryo quality. Accordingly, the cleavage period embryo quality definitions are as follows:

  • Quality: Embryo with equal blastomere size and 0-5 percent fragmentation
  • Quality: Embryo with equal blastomere size, 5-10 percent fragmentation, or slightly different blastomere sizes and no fragmentation
  • Quality: Embryos with blastomeres slightly different in size, containing 10-20 percent fragmentation, or blastomeres with significantly different sizes and no fragmentation
  • Quality: Embryos with significantly different blastomere sizes, fragmentation of more than 10 percent, or the number of blastomeres cannot be counted clearly, and fragmentation of more than 20 percent

Blastocyst Period


The developmental stage that begins with the emergence of fluid-filled spaces (blastocoel) between tightly united cells in the morula stage is called blastocyst. The blastocyst period differs from the cleavage period in that the existing cells begin to differentiate for different functions. While some of the cells spread to form the walls of the embryo (trophectoderm), another group of cells forms a separate group of tightly packed cells (ICM-inner cell mass) within the blastocoel. During the fetal development phase after the attachment of the blastocyst to the uterus (implantation), trophectoderm cells form the gestational sac (gestational sac), while ICM cells form the baby (fetus).

Blastocyst stage embryo quality is determined mainly by blastocoel volume and cell density in the ICM and trophectoderm. In addition, possible morphological anomalies (vacuole, separate cells, degenerated cells) may negatively affect embryo quality. Accordingly, blastocyst period embryo quality definitions are made by a combination of evaluations under the following 3 headings.

By blastocoel volume:

  • Blastocoel begins to form, blastocoel volume is less than half of the embryo volume
  • Blastocoel volume being more than half of the embryo volume
  • Blastocoel volume covers the entire embryo volume
  • Blastocoel volume being larger than the embryo volume, thinning of the zona
  • The shingles wall breaks and the embryo begins to come out
  • Complete emergence of the embryo from the zona
  • According to ICM cell density:
  • Many tightly packed cells
  • A small number of cells forming a loose group
  • Few or no cells
  • According to trophectoderm cell density:
  • Structure consisting of many tightly adjacent cells
  • Loose structure consisting of a small number of cells
  • Structure consisting of a very small number of large cells

According to this evaluation, at the ideal blastocyst control timing (106-108th hour after IVF/ICSI), the best quality blastocyst can be considered as 4AA, and the lowest quality can be considered as 1CC. Embryo quality assessment only helps to select the embryo with the highest probability of pregnancy among the existing embryos, it does not help to detect any complications or possible genetic abnormalities that may occur after pregnancy. Advanced examinations and practices must be used to detect such risks and take possible precautions.

AHA (Assisted Hatching)


While the embryos are at the blastocyst stage, they thin the zona wall with the help of the lysine enzyme secreted from the trophectoderm cells, and they come out by tearing the zona (hatching-budding) with the help of the increasing internal fluid pressure, thanks to the trophectoderm cells changing the ion concentration in the blastocoele. The attachment of the embryo to the uterus (implantation) can only occur after the embryo has completely emerged from the zona.

Studies on this subject show that the culture of embryos in the laboratory and especially the embryo freezing and thawing process have a hardening effect on shingles and therefore make it difficult for the embryo to emerge. In addition, advanced female age and anomalies observed in the shingles layer (such as thick or irregular shingles structure, presence of extra inner membrane) are thought to cause a similar complicating effect.

In order to overcome the mentioned negativities, scientific studies show that it is beneficial to thin the shingle wall before transfer. Although mechanical, chemical and laser applications have been defined for this purpose, the most effective and widely used among them is laser application. In this application, thinning can be done by using specially manufactured devices for this process, which are mounted on an inverted microscope and provide a dose of laser beam that will cause minimum damage to the embryo, by using laser shots to the zonal layer from a point farthest from the cells. Usually, 3 consecutive shots are sufficient to thin the shingles layer by half. In case of advanced shingle anomalies, full openness may be preferred instead of thinning. Laser shots at blastocyst levels of 3 and above are not preferred at these stages, as they may damage the trophectoderm cells that are very close to the zona.

Embryo Biopsy (Preimplantation Genetic Diagnosis-Screening)


Embryo biopsy in in vitro fertilization treatments is a technique applied to select genetically normal, healthy embryos during the preimplantation period (before the embryo is transferred and attached to the uterus). Two types of genetic testing approaches are applied according to their indications.

Preimplantation genetic screening (PGS) is used to detect possible de-novo mutations (DNA abnormalities) found in men and women or in the embryos of couples with no known genetic disease in their families.

In preimplantation genetic diagnosis, it is aimed to identify embryos that do not have this disease in order to prevent the transmission of a known genetic and hereditary disease present in one of the women or men or their families to the baby. You can review the section titled "Genetics" regarding the indications in which these genetic tests are applied and the selection of application stages.

In order for both test approaches to be applied, in vitro fertilization (INV) method must first be applied and, if possible, a large number of embryos must be obtained to increase the chance of success. During the treatment process, biopsy can be performed in 3 different stages according to test indications. All biopsy procedures are performed on micromanipulator-equipped inverted microscopes and using micropipettes produced in accordance with the biopsy type.

Polar Body Biopsy-PBB(Polar Body Biopsy)


It is applied on the day of egg collection (OPU). The first and, in some cases, both polar bodies of the egg are removed by biopsy. The first polar body can be taken just before the microinjection, consecutively if both polar bodies are to be removed, or together at the latest 8 hours after the microinjection. Using an inverted microscope, a laser is used to create an opening by shooting 3-6 shots into the zona layer in an area close to the polar bodies of the eggs, and the polar bodies are aspirated by entering from there with a biopsy pipette. In order not to affect the pH of the environment during the process, it is carried out in HEPES buffered culture liquid, and at the end of the process, the eggs are transferred to fresh culture liquid and placed in the incubator.

Blastomere Biopsy


On the 3rd day of embryo development, one cell from the embryos containing 7 or more cells is taken by biopsy. Applying it to embryos containing fewer cells or removing more than one cell negatively affects advanced embryonic development. Using an inverted microscope, an opening is created by firing 3-6 shots into the zona layer from an area close to the cell detected to contain a nucleus and selected for biopsy, using a laser, and the cell is aspirated by entering it with a biopsy pipette. The procedure is performed in a special HEPES buffered liquid that does not contain calcium and magnesium, in order to weaken the bonds between the cells and thus prevent them from being damaged during the biopsy. At the end of the process, the eggs are transferred to fresh culture liquid and placed in the incubator.

Trophectoderm Biopsy


It is applied to embryos at the blastocyst stage on the 5th and in some cases the 6th day of embryo development. In order for the procedure to be performed, some of the trophectoderm cells must come out of the zona layer (hatching-budding). In order to achieve this, on the 3rd day of embryo development, an opening is created in the zona layer of the embryos planned to be biopsied by making 3-6 shots from an area far from the cells. On the 5th day, the cells coming out of this opening are held with a biopsy pipette, and a piece containing 5-6 cells is taken either mechanically or by laser shots applied to the area planned to be cut. It takes about 1 day to get results from the genetic laboratory after trophectoderm biopsy. Since keeping the blastocysts waiting during this process and transferring them late on the 6th day will reduce the chance of pregnancy, it may be preferred to freeze the embryos that underwent trophectoderm biopsy immediately after the procedure and, if a normal embryo is found, to be transferred in a thawing attempt to be planned 1-2 months later.

Embryo Transfer


The most important stage in the embryo transfer process is to accurately determine the embryo/embryos that have the highest pregnancy potential among the existing embryos, the transfer day, and the number of embryos to be transferred specifically for each patient, in cooperation with the physician. In our country, the number of embryos to be transferred is limited by the ART REP Regulation published by the Ministry of Health. Accordingly, in cases where the female age is under 35, 1 embryo transfer can be applied until the 3rd attempt, a maximum of 2 embryo transfer can be applied after the 3rd attempt, and in cases where the female age is 35 and over, a maximum of 2 embryo transfer can be applied. This does not make a difference for centers and patients who are aware of this issue; Because it is the medical and ethical responsibility of each center to avoid multiple pregnancies, which are much more risky in terms of loss of an already achieved pregnancy (miscarriage), many complications that may occur during pregnancy, and the possibility of premature birth, compared to a singleton pregnancy.

In the presence of appropriate laboratory conditions, it is possible to achieve live birth rates very close to the transfer of 2 embryos by transferring 1 good quality embryo. The most accurate approach is to make this comparison based on live birth rates; because, due to the complications encountered in multiple pregnancies resulting from twin pregnancies, the rate of the resulting pregnancy leading to a live birth is lower compared to singleton pregnancies.

In order to correctly select the embryos to be transferred, it is very important that all examinations and evaluations are completed and recorded daily in the embryology laboratory, starting from the sperm and egg used until the transfer day. Thus, the selection of the most accurate embryos can be achieved not only based on a single parameter such as embryo quality on the day of transfer, but also by evaluating the daily development rates, division patterns and quality of the embryos cumulatively, along with the quality of the sperm and eggs that form the embryos.

The transfer day and the number of embryos to be transferred is a very important decision that needs to include many parameters and should be made by aiming for the highest possible chance of a healthy pregnancy while keeping the possibility of multiple pregnancy low. These parameters are:

  • Sperm and egg quality
  • Number and quality of embryos available
  • female age
  • Clinical evaluation of women
  • Previous trial history, if any
  • Previous pregnancy/miscarriage history, if any
  • Family history of multiple pregnancy, if any
  • Whether Pgt has been applied or not
  • Patient expectation

There is an important parameter related to embryonic development that should be taken into account in the decision regarding the transfer day. Participation of sperm DNA in the management of embryo development (embryonic genome formation) occurs on the 3rd day of development (transition to the 8-cell stage). Therefore, a possible negativity that may arise from damage to the sperm DNA structure; It only emerges starting from this stage.

On average, 30-35 percent of zygotes and 55-60 percent of embryos developing on day 3 can reach the blastocyst stage on day 5. In addition to egg quality, sperm plays a major role in this decline. For this reason, especially in cases where there are more quality embryos than the planned number for transfer on the 3rd day, it is best to wait until the 4th or 5th day to select the correct embryo. On the other hand, in cases where there are already a number of quality embryos planned to be transferred on the 3rd day, it is unnecessary to wait for later days. Waiting for the 4th or 5th day is important in order to prevent an unnecessary transfer that will not yield results in cases where there are few and low quality embryos on the 3rd day and it is doubtful that they will continue their further development.

When the patient and the physician are ready for the transfer process, the selected embryos are loaded into the catheter specially produced for this process by the embryologist and delivered to the physician to be transferred. The ideal method during this loading is to load the embryo with very little culture liquid (approximately 5 microliters) in two small air spaces, as can be seen in the figure below. In this way, it is possible to release the embryos into the uterus in a controlled manner during the transfer and to ensure that the embryo is released by monitoring the air bubbles on ultrasound. Air gaps also prevent the embryo from sticking to the catheter. In addition, by keeping the amount of culture fluid transferred with the embryo to a minimum, the displacement of the embryo by the flow of fluid after the transfer is largely prevented.

Embryo Freezing


To get information about the embryo freezing process, you can review the section titled "Freezing Procedures".