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From the book
by Dr. Aniruddha Malpani, MD and Dr. Anjali Malpani, MD.
Egg collection is accomplished today by ultrasound-guided aspiration (https://www.drmalpani.com/ivfvideos/ivf-video-vaginal-ultrasound-egg-retrieval.htm).
This is a minor surgical procedure , that can be done even under intravenous sedation. We prefer doing it under general anesthesia in our clinic , because we feel this is safer, kinder for our patients, and allows us to collect more eggs. The ultrasound probe is inserted through the vagina. The probe emits high-frequency sound waves which are translated into images of the pelvic organs and displayed on a monitor , so that the mature follicles can be seen as black bubbles on the screen. The doctor guides a needle through the vagina into each mature follicle. The follicular fluid containing the egg is then sucked out through the needle into a test tube, and all the follicles are aspirated, one by one. This is a very precise procedure, which requires considerable skill, and takes about 10-40 minutes to perform, depending upon the number of eggs. On an average , we retrieve about 4-16 eggs for each patient. If there are few eggs, we flush each follicle, to ensure that each egg is retrieved.
The older method of performing egg retrieval involved a laparoscopy, and the eggs and follicular fluid were aspirated under direct vision. However, this method is rarely used today, because the vaginal-ultrasound guided method is much quicker, easier and safer.
Fig 1. Schematic of egg collection under vaginal ultrasound guidance. If you click on the picture, you can watch a video of an actual egg retrieval procedure done in our clinic
The aspirated follicular fluid is then immediately carried into the laboratory ( which is adjoining the operation theater ) where it is examined by the embryologist under a stereozoom microscope, in order to identify the egg. Each egg is surrounded by sticky cumulus cells, and is called an oocyte-cumulus complex. These are washed in medium, graded for their maturity and then transferred into the CO2 incubator The maturity of an egg determines when the sperm will be added to it (insemination). Insemination can be done immediately upon harvest, but is usually done after 2-6 hours.
Fig 2. Checking the eggs under the stereozoom microscope in the IVF lab. If you click on the picture, you can watch a video of an actual egg retrieval procedure done in our IVF laboratory
Fig 3. Mature oocyte cumulus complex, as seen under a stereozoom microscope in the IVF lab, during egg retrieval. The egg is in the center, surrounded by the cumulus cells.
On the day the eggs are harvested ( this is called Day 0) , the husband provides a semen sample. The sperm are separated from the seminal plasma in a process known as washing the sperm, and these washed sperm are used to inseminate the eggs. Some men may have considerable difficulty producing a semen sample at the appropriate time, because of the tremendous stress they are under, and the " pressure to perform". For these men, using a previously stored frozen sample can be helpful. Viagra ( sildenafil citrate) can also be used to help them to get an erection, as can using a vibrator.
A defined number of sperm ( usually 100,000 sperm/ ml) is placed with each egg in a separate dish containing IVF culture medium. The dishes are placed in a CO2 incubator with a controlled temperature that is the same as the woman's body - 37 C. The conditions in the incubator and the culture medium are designed to mimic the conditions in the fallopian tube, so that the embryos can grow happily in vitro. The culture medium , which has to be very pure, contains various ingredients such as protein, salts, buffer and antibiotics which allow optimal growth of the embryo - think of it as "chicken soup for the embryo " !
Fig 4. A view of the incubator - the heart of an IVF lab.
About 18 hours after insemination ( this is called Day 1) , the embryologist checks to see how many eggs have fertilized. This is called a pronuclear check, and normally fertilized embryos at this time are single cell , with 2 pronuclei.( Fertilization Video ) Each pronucleus appears as a clear bubble within the embryo, and the male pronucleus represents the genetic contribution of the husband , while the female pronucleus represents the contribution of the wife. When these fuse, a new life, with a unique genetic composition is formed. Abnormally fertilized embryos ( for example, those with three pronuclei), or those which have failed to fertilise, are discarded, or used for research.
Fig 5. A normal 2-PN embryo on Day 1 ( about 18 hours after egg retrieval) . This is a good quality embryo, because the two pronuclei ( the clear bubbles in the center) are touching each other; and the pronucleoli they contain are aligned properly. If you click on the picture, you can watch a video of how the sperm fertilise the egg as seen under the microscope in our IVF laboratory
Fig 6. A beautiful 8-cell embryo on Day 3 ( about 72 hours after egg retrieval) . This is a Grade A embryo, with regular, equally sized, clear blastomeres; and no fragments
There is quite a lot of suspense and anxiety till you find out from the lab how many embryos have fertilized. This is a biologic variable which we still cannot control. Sometimes, even though the eggs and sperm may look excellent , there may be a total failure of fertilization. This can be a major blow, because it means that there are no embryos to transfer. Poor fertilization rates may be because of : poor lab conditions; a sperm problem, or an egg problem. If only one patient has poor fertilization on a particular day, in a good lab, then it's usually the sperm which are held to be responsible .
The normally fertilized embryos are left in culture, where they continue to divide, and their quality graded after another 24 hours. Good quality embryos divide rapidly; and healthy embryos have 2-4 cells, of equal size, with clear cytoplasm and few fragments on Day 2 ( about 48 hours after egg retrieval) . The IVF lab is the heart of the IVF clinic today, and an IVF clinic is only as good as its lab ! Unfortunately, most patients have no idea of what happens in the lab, and they rarely get a chance to talk with the embryologist, the skilled biologist who works in the IVF lab. The embryologist is the unsung hero of IVF treatment who does all the important work behind the scenes. The dramatic improvements in pregnancy rates with IVF today are because of the important contributions embryologists have made to finding the best ways of growing and culturing embryos in vitro.
Many patients are worried that their eggs, sperms or embryos may get mixed up with someone else's. While this can happen, the probability of it happening in a well-run laboratory is very low, because good labs have quality control mechanisms to prevent such mixups from occurring.
Fig 7. Schematic of embryo transfer. The embryos are gently deposited in the uterine cavity
After 48 - 72 hours, when embryos usually consist of two to eight cells each, they are ready to be placed into the woman's uterus. This procedure is known as embryo transfer.