In this webinar, Carolina Andrés, Embryologist, Andrology Laboratory at Clinica Tambre talked about technical terms in embryology that you need to be aware of.
During the first consultation, the doctor will explain all the tests required for both females and males before starting any fertility treatment. One of the tests included in that is Karyotype, which is performed to analyze the pair of chromosomes present in your body. Chromosomes are structures that contain the DNA, all the information that we need to function. A human being has 23 pairs of chromosomes, however, this number can be altered, not only the number but also and also the structure. A karyotype is used to look for abnormal numbers or structures of chromosomes.
To form an embryo, we need to have gametes, which are cells (male and female) that joins with a cell of the opposite sex to form a zygote.
Male factor diagnosis
The main protagonist is the sperm, which consists of 3 important parts, the head, the midpiece and the tail. These structures are analyzed in the spermiogram. Spermiogram allows for checking sperm concentration, motility, and the aspect of sperm
Different diagnostic results that can be obtained are:
- aspermia, a complete lack of semen
- asthenozoospermia when there’s less than 32% of sperm with progressive motility
- azoospermia, when sperm cells are absent in semen
- hypospermia when we find that the volume of the ejaculate is lower than 1.5 ml
- oligozoospermia where there is less than 15 million sperm per millilitre of semen
- necrozoospermia when there is more than 58% of dead spermatozoa in the sample
- teratozoospermia when there’s more than 4% of morphologically abnormal spermatozoa in the sample
- leukocytospermia when there are more than 1 million leukocytes per millimetre, which usually indicates genitourinary infection
Apart from spermiogram, which is the basic analysis done for male factor diagnosis, another test to perform is DNA fragmentation. This test allows to detect if there are any breaks in a DNA molecule, which is a structure that contains all the information. If there are some breaks in one of the strands, it’s called simple strand fragmentation, if there are breaks in both strands, it’s called double-strand fragmentation.
Female factor diagnosis
The structure of the cell of the ovum or more scientifically called the oocyte is formed by 3 parts. Zona pellucida, which is the outer layer, the cytoplasm or ooplasm, and then the polar body.
Concerning the female diagnosis, the most important thing to do is analyze the ovarian reserve. There are 2 important tests to analyze the functional potential of the ovary, which is the antral follicle count (AFC), the doctor can see by ultrasound scan how many follicles are there. Anti-Müllerian hormone (AMH) test is done by blood, and it analyzes this hormone and will tell you what your ovarian reserve is. When the doctor has all the information and can provide a diagnosis for the couple, he will indicate the best treatment that suits this particular couple. Many treatments can be done.
Before the egg retrieval, everything is connected with the stimulation and medical terms, therefore, Carolina Andrés focused on the terminology used after the egg retrieval. On the day of the retrieval, the doctor performs an ovarian puncture to retrieve the oocytes, which are then sent to the lab for classification. It is possible to classify the oocytes into 2 different groups. The immature group stage and the mature stages. For the ICSI procedure, only oocytes within the mature stage (Metaphase II) are used. The immature oocytes (Metaphase I) are going to be discarded. Once the oocytes are classified, the microinjection will be performed, one selected sperm will be introduced into one of the oocytes, it is done on all the available oocytes. They are then put into incubators.
There are many kinds of incubators out there, one of the most popular ones is the time-lapse incubators, which have a camera inside to check the embryos at all times, they are recording their development within 24 hours. These embryos are cultured until day 5 (blastocyst stage) of their development, which helps to select better embryos, only about 50% of the embryos that fertilize are going to arrive at this stage. That means that the potential of implantation is higher if they are of good quality. After the microinjection is performed, the embryologist has to wait one day and the next day, they will check fertilization, it’s a way to know which of these oocytes has the potential to form or to develop and start dividing into embryos. On day 1, the embryologist needs to check structures that appear in the oocyte, which are the pronuclei. It’s crucial to have two of them, one coming from the mother and the other from the father. There are also non-fertilized oocytes where we don’t see any of the structure, they will be discarded. There is also a possibility to have oocytes with 1 pronucleus or more than 2, for example, 3-4. These types would also be discarded. However, sometimes oocytes with 1 pronucleus which arrive at the blastocyst stage will be considered for the genetic analysis, but only in those cases. Sometimes, in around 20% of the cases, such blastocysts can be euploid. If a genetic test (PGT) is not going to be performed, they are discarded automatically.
After the first day, embryos start developing and start dividing. On day 2, the oocytes that were fertilized the divisions must be complete and form complete cells, on day 3, it is expected for the embryos to have 8 cells or more, and on day 4, they will become a compacted morula stage. Then on day 5, it’s possible to see their development and see if they were able to form a blastocyst.
At the cleavage stage grading, the embryologist checks the number of cells, they must have an accurate number of cells. The percentage of fragmentation in the embryo is also important, cells must divide completely. The embryos are graded by fragmentation from 1 to 4. 1 being the lowest and 4 being the highest. Embryos graded 1 or 2 are of good quality, however, grades 3 and 4 are embryos of intermediate quality, and their prognosis is lower.
The blastocyst consists of the Inner Mass Cell (IMC), which leads to the formation of structures of the fetus, and then the Trophectoderm (TE), which forms the outer layer, which is going to form the placenta. There is also a quality grading system for blastocysts called Gardner Grading System. Grading 1-6 refers to the expansion of the blastocyst, so a blastocyst that is starting to expand will be graded 1, and as soon as it starts to develop and expand more, it will become a blastocyst 2 and then 3. Grade 4 and 5 are blastocysts that are starting to hatch, which means that the zona pellucida is going to be so thin that it’s going to break, which is a normal process. Finally, blastocyst graded 6 is the most expanded.
Concerning the quality of the structures, both the Inner Cell Mass and Trophectoderm are going to have a letter that goes from A to D. A and B are considered good quality, and the only difference between them is the morphological aspect, it allows the embryologist to differentiate them, for example, inner cell mass which is more compact compared to another one that’s a bit bigger, but their quality is the same. Letter C means one of the structures is not there, if there is C for the inner cell mass, that means it’s not there. Letter D means the cells are starting to be damaged and deteriorate, so they are not good. Finally, with these good-quality embryos, there are different destinations and things that can be done.
The first option would be to transfer the embryo, which means placing the embryo into the uterus with a catheter. Another option would be to vitrify embryos, in some cases patients cannot do the transfer date, or the endometrium is not prepared, or there are other reasons, so the embryos can be vitrified. The embryos are frozen and stored in tanks in nitrogen liquid at -196C. When they are needed, they are thawed and then transferred. One of the last uses would be analysing the embryos genetically, so performing Preimplantation Genetic Testing.
The PGT procedure is done regularly at the laboratory. The embryos are placed on a dish, and they are observed under the microscope, the embryologist takes 3 to 5 cells to analyse them genetically, and then sends them to the genetic laboratory for analysis. There are different kinds of preimplantation genetic testing. The most common one is PGT-A (Preimplantation Genetic Testing for Aneuploidy) to check for abnormalities in the number of chromosomes. PGT-SR checks for structural anomalies, and PGT-M testing checks monogenic/single gene defects.
PGT-A results can show a euploid embryo, which contains 23 pairs of chromosomes, there can also be an aneuploid embryo, which means that there’s a gain or loss of genetic content which affects 1 or 2 pairs of the chromosomes, and then there are also mosaic embryos. Mosaicism means that within the same embryo, there are euploid and aneuploid cells in different percentages, depending on the percentages. The genetic laboratory would give some indications concerning the transfer of these embryos.