In the period from egg collection (OPU) to embryo transfer, all applications 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 development potential depending on the quality of both gamete cells. This potential is entirely determined by the gamete cells and it is not possible to achieve a positive change with any external intervention after fertilization.
The importance of the Embryology Laboratory is emerging at this point. Because if the laboratory does not have appropriate personnel, equipment and environmental conditions, this potential is likely to adversely affect. Therefore, it is vital for couples to be conscious about laboratory quality in central elections.
Routine procedures applied in embryology laboratory
- Egg collection (OPU)
- Denudation (Egg preparation)
- 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 inside the microscope under the microscope in the sterile working chamber of the aspirated follicle fluids which are aspirated by the physician. The oocytes (egg cells) that come to the laboratory are collected at the body temperature and are kept in the culture liquids for 3 hours until the denudation process to remove the cells.
Eggs may not be removed from the whole of the follicles that are collected by eggs and are observed to grow with ultrasound. This may be due to differences in the number of follicles indicated in the laboratory and the number of eggs collected in the laboratory.
Denudation (Egg Preparation)Eggs collected in the OPU process are surrounded by thousands of cumulus cells supporting the development and viability of the egg, which is defined as COC (Cumulus oocyte complex) in the development stage and up to the process of fertilization. If microinjection (ICSI) is planned, the cumulus cells are separated from the egg by a process defined as denudation in order to identify mature eggs and egg quality.
In the microscopic evaluation, mature and non-mature eggs are detected and taken to the culture liquids where they are kept until the microinjection process and removed to the incubators that provide the necessary environmental conditions. The collected eggs are treated only with cells that have completed maturity (Metaphase 2 - M2). Therefore, it may not be possible to process each collected egg cell.
FertilizationIn embryology laboratories, two different methods, called IVF and ICSI, are used to ensure that the egg is fertilized with sperm.
IVF (In vitro Fertilization)It was the first method used to provide fertilization in IVF treatments and was first applied successfully by Physiologist Robert Edwards in the UK in 1978. In this method, the sperm prepared by the andrology laboratory are brought together in the embryology laboratory in the same culture medium as the cumulated eggs (COC) and the sperm is expected to naturally fertilize the egg.
Since IVF application is only possible to enter the eggs with fertilization capacity, it is natural selection. However, this may lead to a high rate of fertilization failure in cases where the fertilization potentials of the sperm are not adequately evaluated. Therefore, choosing the right laboratory for the IVF process is very important. Failure to fertilize in IVF may occur due to egg-related reasons. Therefore, the common approach is to use the ICSI method in some of the eggs in IVF planned and in large numbers of eggs collected.
The common practice in IVF procedure is the insemination of 50ına100,000 spermatozoa per oocyte into the culture medium where the oocyte is present. Less sperm use can lead to fertilization failure, and more sperm use can lead to fertilization of more than one sperm. Since fertilized eggs can 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 patients with sperm parameters (number, mobility), severe sperm morphological defects or sperm obtained in the testicular way. First, in 1990, It was successfully implemented by Gianpiero D. Palermo in Brussels.
In order for the ICSI process to be carried out, it is imperative that the personnel who have certain equipment in the laboratory and that will implement them should be trained and have sufficient skills in this regard. The pipettes used for the ICSI process, called the micropipette, are used to hold the egg (holding pipette) and the other to inject the sperm into the egg (microinjection pipette). In addition, this device should be kept on an anti-vibration table in order to prevent vibrations that may be harmful during the process.
For a successful ICSI implementation, the following requirements must be met and regular control of variables is essential:
Performing the transaction on an antivibration table
For the evaluation of oocyte morphology and most importantly, for the selection of the most smooth sperm morphologically for the microinjection, the inverted microscope's image settings are adequate and the embryologist has enough information about these settings.
The temperature of the heated glass surface in which the microscope is placed with the sperm and eggs in which the ICSI is applied is suitable (to provide 37 C in the container)
Select the correct pipette to minimize the risk of damage to eggs and sperm during the procedure and to place the pipettes at the right angles to the micromanipulator (35 degrees to the microscope axis) and parallel to each other.
If the hydraulic micromanipulator system is used; repetition of system maintenance and controls before each process
Most importantly, the embryologist who will carry out the procedure has enough knowledge, skill, experience in all these subjects.
The ICSI process is a very sensitive microsurgical procedure, and if the above-mentioned conditions are not met sufficiently, a high rate of fertilization after the procedure, degeneration of the eggs (damage to the body), the development of embryo development and quality is likely.
In the case of the man presenting for treatment, ICSI method should be preferred for fertilization if the following indications are found:
Inadequate semantic analysis parameters for IVFSevere sperm morphological defects (globozoospermia, megalo-pinhead sperm, tail defect-fibrous sheath dysplasias, total immotile sperm)
Minimal morphological defects (acrosomal deformity, irregular acrosomal distribution) that may prevent the binding of sperm to the zona pellucida
Conditions that prevent sperm retrieval with ejaculate, obstructive, non-obstructive azoospermia cases requiring the use of testicular sperm
A history of low IVF / ICSI fertilization or total fertilization failure in previous trials
Only eggs that are found to be mature are used in the ICSI process. In the aforementioned equipment and conditions, the fastest and morphologically the most smooth sperm is selected and transferred to a solution which slows down the sperm motility and the sperm tail is broken by the pipette pulse. The purpose of this procedure is to provide the protein (phospholipase C) to the oocyte cytoplasm, which is defined as the oocyte activation factor in the sperm cytoplasm and damage the cell membrane in the sperm cytoplasm and initiate chain reactions that will enable the fertilization of the sperm and initiate chain reactions. . At the end of the process, the eggs are transferred into fresh culture liquid and left to incubation until fertilization control.
Fertilization ControlFertilization control is done by inverted microscope after 10-18 hours after IVF / ICSI. In this control, the observation of 2 nuclei (pronucleus-PN) and additionally a second pole body in the adjacent position of the sperm and the other in the egg are called normal fertilization, and the stage of development from this stage to the first division is called zygote. If this period is passed, it will not be possible to determine if there is fertilization or abnormal fertilization since the nuclei will be merged and the nucleus will be visible.
It is also possible to observe various anomalies in fertilization control. These anomalies:
- MonoPN: It is only one case of pronucleus. In this case, however, cell division and embryo development can be observed; however, the developing embryo will be aneuploid (with numerical chromosomal anomaly) since it will be highly likely to have a single gamete chromosome set (haploid). The MonoPN state can also be caused by the asynchronous sequence of kernel formation and deletion, and in this case may have a normal chromosome set (diploid). However, in this case, developmental problems are observed in developing embryos.
- 3PN: Normally it is caused by the fact that one of the gametes to be haploid is diploid or when more than one sperm is injected during microinjection. Although mostly embryo development is observed, the transfer of the embryos is highly unlikely since the embryo will be aneuploid.
- MultiPN: 3 or more nuclei are observed. Even if there is embryo development, it is not transferred.
- Fragmented PN: This is the case when one or more small cores are observed next to 2 cores in normal size. Cores can be recovered during the merger process and normal embryo development can be observed.
- PN sizes are different: One of the cores is different in size than the other. Developmental problems are often observed in developing embryos.
- Discrete PN: The varying amounts of spacing between the nuclei is the case of non-adjacent. It is thought that the nuclei are caused by the problems in microtubule structures that regulate their position in the cytoplasm. Since the same structures also manage cell division, problems or stagnation of embryo development may be observed.
Embryo CultureEmbryo culture can simply be defined in vitro (in vitro) and the development of embryos in this artificial environment. These environmental conditions;
- Incubators that control the body temperature of 37 degrees and blood levels of 5 to 6 percent of carbon dioxide
- Culture solutions to meet nutrient and cellular building block needs of embryos
- Sterile working areas to minimize the risk of contamination in the culture process
- Laboratory ventilation, cleaned of particles and volatile organic compounds.
Specialized staff to check the availability and continuity of these conditions summed up. Details of this topic can be found in our article "Quality Control".
Ready-made solutions are used for this purpose in embryo culture and made with the required quality control tests (MEA-mouse embryo assay, LAL endotoxin test). Common points of these solutions are the energy sources (pyruvate, glucose), building blocks (amino acids), pH buffers (bicarbonate) and minerals and supporting components (EDTA, albumin) that embryos will need during the development process.
In general, there are two different approaches in culture systems. Sequential culture and mono culture. In the sequential culture environment approach, it is argued that embryos are exposed to different environmental conditions in the natural environment, tubes and uterus and thus the same variable conditions should be provided in the laboratory (back to nature). Mono culture approach; In the embryonic development process, it is argued that the contents of the same culture solution can be used at different rates according to their changing needs, so it is not necessary to change the contents of the solution according to the developmental period (let the embryo choose-leave embryo). Both approaches give successful results in similar proportions, depending on the performance of the laboratory.
The embryo development begins with the first division of the fertilized egg (zygote) (20-26h). This process of development is divided into two:
The development of the embryo from 2 cells (one day after the OPU) to 15-20 between the tightly associated cell (morula) stage (4th day after the OPU) is called the cleavage period. This period, as the name suggests, is the period in which the embryo increases the number of cells by mitosis. However, metabolic activities, protein synthesis and DNA copying are carried out in the cell. One of the most important changes occurring in this period is that after the 8 cell stage, the sperm genetic structure is also involved in directing embryo development (embryonic genome formation). Hence, the possible problems with sperm occur mostly after this stage. In recent years, studies with devices with continuous monitoring systems show that the mitotic division timings of this period can give information about the embryonic development potential and genetic structure.
In this period, the quality of embryos is determined primarily by the cell number, the size of the cells, and the presence and proportion of fragmentation (cell particles). In addition, possible morphological anomalies (vacuole-fluid-filled sac, granulation) may adversely affect embryo quality. Accordingly, cleavage period embryo quality definitions are as follows:
Quality: 0-5 percent fragmentation embryo with equal blastomer size
Quality: Embryonic embryo with equal blastomer size, with 5-10% fragmentation or slightly different blastomere dimensions
Quality: Blastomers are slightly different in size, containing 10-20 percent fragmentation or blastomere size is significantly different, fragmentation free embryo
Quality: Embryonic embryo with more than 20 percent fragmentation, with a significant percentage of blastomer sizes, fragmentation over 10 percent or no number of blastomere
The stage of development that begins with the emergence of liquid-filled voids (blastocles) between firmly joined cells in the Morula stage is called blastocyst. The blastocyst period is separated from the period of cleavage by the beginning of the transformation of the existing cells for different tasks. While some of the cells are propagated to form the walls of the embryo (trophectoderm), another group of cells form a separate group of tightly packed cells (ICM-inner cell mass) within the blastocort. The trophectoderm cells constitute gestational sac in fetal development stage after implantation of the blastocyst to the uterus, while the ICM cells form the baby (fetus).
Blastocyst period embryo quality is mainly determined by blastocoll volume and cell density in ICM and tofectoderm. In addition, the observed morphological anomalies (vacuole, discrete cells, degenerated cells) may adversely affect embryo quality. According to this, the definitions of embryo quality in the blastocyst period are made by the combination of the following 3 headings.
By blastocol volume:
- The formation of blastocol is to be less than half of the embryo volume.
- Blastose volume more than half of the embryo volume
- Blastossal volume covers the entire volume of the embryo
- Blastosol volume greater than the volume of the embryo, the thinning of the shingles
- The shingles are broken and the embryo starts to come out
- Embryo completely out of the zone
By ICM cell density:
- Many tightly packed cells
- A small number of cells forming a loose group
- Too little or no cell
By Trofektoderm cell density:
- Structure consisting of many tightly adjacent cells
- Loose structure consisting of few cells
- Structure consisting of too many small parts
According to this evaluation, in the ideal blastocyst control timing (106-108 hours after IVF / ICSI) the highest quality blastocyst can be evaluated as 4AA and the lowest quality is 1CC. Embryo quality assesment not only helps in the selection of the embryos most likely to form a pregnancy between existing embryos, but does not help to detect any complications or possible genetic abnormalities after pregnancy. Advanced tests and applications should be used for the identification of such risks and possible measures.
AHA (Assisted Hatching)In the blastocyst stage, embryos thin the zone of the shingles with the help of the lysine enzyme secreted from the trofexectin cells, and with the help of the increased internal fluid pressure, the tunicectomy cells change the ion concentration in the blastocortic contents, hitting the zona (hatching-budding). The implantation of the embryo into the uterus can only occur after the embryo has been completely removed from the zone.
Studies on this subject show that the culture of embryos in the laboratory and especially the embryo freezing process have a zoning effect and thus hinder the emergence of the embryo. In addition, it is thought that the advanced female age and the anomalies observed in the shingles layer (such as the presence of thick or irregular zona structure and the presence of extra inner membrane-inner membrane) have a similar complicating effect.
In order to overcome the aforementioned disadvantages, it is shown that the thinning of the shingles wall before transfer is beneficial. Although defined mechanical, chemical and laser applications have been defined for this purpose, it is the most effective and widely used laser application. In this embodiment, using the devices specially designed for this process, which are mounted in inverted microscopes and providing a laser beam at a minimum dose to the embryo, thinning can be performed with laser shots from the farthest point to the cells. It is usually enough to make three consecutive shots that will thin the shingles layer by half. It may also be desirable to provide complete aperture instead of thinning in the presence of highly shingles anomalies. Since laser shots at 3 and above blastocyst levels may damage the trophectoderm cells, which are very close to the zone, it is not preferred to use them at these stages.
Embryo Biopsy (Preimplantation Genetic Diagnosis-Screening)Embryo biopsy in IVF treatments is a technique applied for the selection of genetically normal, healthy embryos during preimplantation (before embryo transfer and retention of the uterus). Two types of genetic testing approaches are used according to their indications.
Preimplantation Genetic Screening (PGS-Preimplantation Genetic Screening) is used to detect possible de-novo (sperm or ovum-derived embryos) mutations (DNA abnormalities) found in the embryos of couples without a known genetic disease in men and women or their families .
Preimplantation Genetic Diagnosis (Preimplantation Genetic Diagnosis) is intended to identify embryos that do not have this disease in order to prevent the transmission of a known genetic and hereditary disease in one of the women or men or their families to the infant. You can check the section titled lar Genetics aş about the indications and the selection of the application stages of these genetic tests.
In order to be able to apply both test approaches, in order to increase the chances of success and the success of the in vitro fertilization method, several embryos should be obtained. Biopsy can be applied in 3 different stages according to test indications. All biopsies are performed using inverted microscopes equipped with micromanipulator and micropipettes produced according to biopsy type.
Polar Body Biopsy-PBB (Polar Body Biopsy)It is applied on the day of egg collection (OPU). In the first and in some cases, both pole bodies are taken by biopsy. 1. pole body may be taken consecutively immediately before the microinjection process, if both pole bodies are to be taken, or at least 8 hours after the microinjection. In inverted microscope, using a laser, openings are formed by shooting 3-6 shots to the shingles layer in a region close to the pole bodies of the eggs and aspiration of the polar bodies by entering with a biopsy pipette. The process is carried out in HEPES buffered culture fluid to prevent the pH of the medium from being affected, and at the end of the process, the eggs are transferred to the fresh culture liquid and removed into the incubator.
Blastomer BiopsyOn the 3rd day of embryo development, one cell biopsy is taken from the embryos containing 7 or more cells. Application of more than one cell to fewer embryos or adversely affect the embryonic development. An inverted microscope is used to detect nuclei using a laser and open the cell selected from the biopsy area. The process is performed in a special liquid with HEPES buffer free of calcium and magnesium to weaken the bonds between the cells and thereby prevent damage during biopsy. At the end of the process, the eggs are transferred to the fresh culture liquid and removed from the incubator.
Trofektoderm BiopsyIt is applied to embryos at the blastocyst stage in the 5th and in some cases on the 6th day of embryo development. In order to be able to perform the procedure, a part of the trophectoderm cells must be out of the shingles layer (hatching). In order to achieve this, on the 3rd day of embryo development, the shingles of the embryos which are planned to be biopsied are created by shooting 3-6 shots away from the cells. On day 5, cells coming out of this opening are taken with a biopsy pipette and a piece containing 5-6 cells is taken by laser shots to be mechanically or to be cut. Trofektoderm biopsy after the biology laboratory results take about 1 day. Since the blastocysts are kept in this process and transfer of late in the 6th day will decrease the chance of pregnancy, it is preferable to transfer the embryos performed after trofectoderm biopsy immediately after the procedure and in the case of normal embryo transfer in a trial attempt which will be planned after 1-2 months.
Embryo TransferThe most important step in the embryo transfer process is to accurately determine the number of embryos to be transferred to each patient, in particular the embryo / embryos having the highest pregnancy-forming potential of the embryos present, and the day of transfer. The number of embryos to be transferred in our country is limited by the regulation published by Ministry of Health. According to this, 1 embryo transfer up to the 3rd attempt in cases with female age less than 35, no more than 2 embryo transfers after the third trial, and no more than 2 embryo transfers in cases with female age 35 or above. This does not make a difference in terms of conscious centers and patients; Because, compared to singleton pregnancies, the loss of a pregnancy obtained (low), many complications that may occur during the pregnancy period and avoiding multiple pregnancies which are much more risky in terms of the possibility of premature birth are the medical and ethical responsibility of each center.
With the transfer of one embryo of good quality obtained in the presence of appropriate laboratory conditions, it is possible to reach live birth rates very close to 2 embryo transfers. This comparison is the right approach to live birth rates; because of the complications encountered in multiple pregnancies that occur as a result of twin pregnancy, the rate of reaching a live birth is lower compared to singleton pregnancies.
In order to select the embryos to be transferred correctly, it is very important to complete and record all examinations and evaluations from the sperm and eggs used in the embryology laboratory until the day of transfer. Thus, not only depending on a single parameter such as embryo quality on the day of transfer, but also the quality of the sperm and eggs forming the embryos, the daily development rates, division patterns and qualities of the embryos can also be evaluated cumulatively and the selection of the most accurate embryos can be ensured.
The number of embryos to be transferred and the number of embryos to be transferred is a very important decision that should be included by targeting many parameters and keeping the possibility of multiple pregnancies at the highest possible level while keeping the chance of a healthy pregnancy low. These parameters are:
- Sperm and egg quality
- Number and quality of existing embryos
- Female age
- Clinical evaluation of women
- Previous experiment history
- Previous pregnancy / low history
- Family history of multiple pregnancy if available
- Whether Pgt is applied
- Patient expectation
There is an important parameter for embryonic development which must be taken into consideration in the decision on the day of transfer. The incorporation of sperm DNA into the management of embryo development (embryonic genome formation) takes place on the 3rd day of development (transition to 8 cell stages). Thus, a possible negativity caused by damage to the sperm DNA structure; however, it starts from this stage. On average, 30-35 percent of zygotes and 55-60 percent of embryos developing on day 3 may reach the 5th day blastocyst stage. In this decrease, the role of sperm in addition to egg quality is great. Therefore, on days 3, especially in cases with higher quality embryos than the number planned for transfer, it is best to expect the 4th or 5th day to select the right embryo. On the other hand, it is unnecessary to expect advanced days in cases with good quality embryos already planned on the 3rd day. Waiting for day 4 or 5 is important in order to avoid unnecessary transfer, which in the case of suspected cases with a small number of embryos on the 3rd day and with low quality embryos and will continue to develop further.
When the patient and the physician are ready in the transfer process, the selected embryos are delivered by the embryologist into the catheter specially produced for this procedure and delivered to the physician to be transferred. The ideal method during this loading is to load the embryo with very little culture fluid (about 5 microliters) in the two small air voids, as seen in the figure below. This allows both embryos to be released to the uterus in a controlled manner and air bubbles are monitored on ultrasound to ensure that the embryo is released. Air gaps are also prevented from adhering the embryo to the catheter. In addition, the amount of culture fluid transferred with the embryo is kept to a minimum and the transfer of the fluid with the flow of the embryo after transfer is also largely prevented.