What does the first guardian of the “test tube baby” do?

What does the first guardian of the “test tube baby” do?

On July 25, 1978, the world's first test-tube baby was born, opening a new chapter in assisted reproductive technology. Embryologists are the first guardians of "test-tube babies", so July 25 of each year is defined as "Embryologist Day". Prospective parents hand over sperm and eggs to embryologists, who carefully care for them and perform mysterious operations to allow the "test-tube baby" to develop healthily in a warm and large collective environment. Do you want to know what an embryologist does in a day? What mysterious operations are performed at what time? Let's take a look.

7:20 Embryo laboratory quality control

A year's plan starts with spring, and a day's plan starts with the morning. Morning is the most beautiful start of a day. In order to give the "test tube baby" a good start, the embryologist will check whether the living environment and conditions of the "test tube baby" are the most comfortable in the morning, that is, the quality control of the embryo laboratory. The quality control content mainly includes the temperature, humidity, environment (VOCs, oxygen content, particles and bacteria, etc.) of the embryo laboratory, the quality control of the incubator (temperature, humidity, CO2 and O2 content, etc.), the temperature of the operating table warm plate, the temperature of the refrigerator, the amount and temperature of the liquid nitrogen tank, the batches of reagents and consumables, etc. After 1-2 embryologists arrive at the embryo laboratory, they will test the above quality control contents. The temperature of the embryo laboratory is 24-26℃, the humidity is 40%-60%; the temperature of the carbon dioxide incubator is 37℃, the CO2 concentration is 6%, and the O2 concentration is 5%; the temperature of the operating table warm plate is controlled at 37-38℃, etc. If the above conditions are found not to be met, they should be adjusted in time to give the "test tube baby" a warm and comfortable culture environment to ensure the healthy development of the embryo.

7:40 IVF Day 1 after egg retrieval (D1) Egg peeling

IVF fertilization is to place sperm and eggs in a culture dish to combine freely. The sperm and egg combination process will digest the cumulus cells of the corona radiata in the cumulus complex, while the peripheral granulosa cells are still tightly wrapped around the egg. Embryologists need to use an egg stripping needle that is slightly larger than the diameter of the egg to repeatedly blow and wash the egg to confirm that the granulosa cells around the egg fall off, so as to facilitate the observation of the pronucleus under a microscope.

8:00 Embryo and fertilization observation

The fertilization of sperm and egg (D1), embryonic development in the cleavage stage on the third day (D3), and embryonic development in the blastocyst stage (D5 or D6) all need to be observed at specific times to facilitate the evaluation of the temporal and spatial nature of embryonic development and the selection of high-quality embryos for freezing or transplantation. 17 to 20 hours after fertilization, D1 observes the pronucleus under an inverted microscope to determine the fertilization of sperm and egg. The fertilized egg with two pronuclei and bipolar bodies is considered to be normally fertilized and transferred to G-1 culture medium for further culture. If three pronuclei appear, it is considered to be abnormal fertilization and is directly discarded; if one pronucleus appears, it will continue to be cultured during the IVF cycle to observe the subsequent embryonic development. If one pronucleus appears during the intracytoplasmic sperm injection (ICSI) cycle, it will be directly discarded. In addition to judging the fertilization of sperm and egg, the pronucleus will also be scored based on the position of the pronucleus in the egg, the relative position of the two pronuclei, the number of nucleoli in the pronucleus, and the spatial distribution. Normally fertilized eggs are cultured in G-1 until D3, and the D3 embryos are scored by the blastomere division rate, the number of blastomeres, and the degree of fragmentation. High-quality embryos are selected and either placed in G-2 culture medium to continue culturing to blastocysts, or frozen or transplanted. If the patient chooses to continue blastocyst culture at D3, the blastocyst needs to be observed at D5 or D6, and the blastocyst is scored by the degree of expansion, the number of inner cell mass cells, and the number of trophoblast cells. High-quality blastocysts are selected for cryopreservation or transplantation, and the rest of the embryos are discarded.

8:00 Collecting eggs

The G-MOPS-PLUS phosphate buffered medium for egg collection was preheated in a 37°C, CO2-free incubator 30 minutes in advance. The doctor aspirated the follicular fluid under the guidance of B-ultrasound, and the embryologist quickly transferred the follicular fluid to a culture dish and observed the cumulus complex under a stereo microscope. The cumulus complex was picked out and washed twice with G-MOPS-PLUS, and once with G-IVF, and then placed in the pre-balanced G-IVF culture medium, and cultured in an incubator at 37°C, 6% CO2, and 5% O2 for use.

8:30, 13:30 Resuscitation

After arriving at the embryo laboratory in the morning, the embryologist prepared the resuscitation fluid (including TS DS WS1 WS2) for resuscitation. TS was preheated in a 37°C, CO2-free incubator for 30 minutes, and the other three resuscitation fluids were balanced at room temperature for 30 minutes. The morning resuscitation operation began at 8:30. Most of the patients who were resuscitated in the morning in our center were resuscitated in the morning and cultured until the blastocyst transfer in the afternoon, so it is recommended that patients arrive at the center before 8:30 to complete the verification work to avoid affecting the subsequent culture and transfer. At 13:30 in the afternoon, a small number of patients resuscitated D3 cleavage stage embryos.

8:30, 13:30 Transplant

Both 8:30 and 13:30 can start the transplantation. Why are there two transplantation times? Is there any difference? The 8:30 transplantation includes fresh transplantation, D3 embryo transplantation, and D3 embryo thawed and blastocyst cultured transplantation. The 13:30 transplantation mainly refers to the blastocyst transplantation that has been thawed in the morning and cultured in vitro for more than 2 hours.

9:30 ICSI D0 peeling eggs

Egg collection starts at 8:00. For patients in ICSI cycles, after the cumulus complex is taken out and cultured in G-IVF for 1 to 2 hours, the egg stripping operation can be performed, which embryologists often call "egg stripping". Egg stripping is to digest the cumulus complex with a certain concentration of hyaluronidase, and repeatedly blow and wash the egg with an egg stripping needle slightly larger than the diameter of the egg to strip the cumulus cells and granulosa cells. After egg stripping, the embryologist needs to observe whether the egg contains the first polar body under a microscope. If there is a first polar body, the egg is a mature egg (MII) and is placed in a G-IVF droplet for continued culture. ICSI operation is performed at 13:30 in the afternoon. If there is no first polar body in the egg, it is an immature egg (MI, GV) and it will be discarded.

9:45 Frozen

7:40 After embryo observation, the embryologist will provide feedback on the high-quality embryos on D3, D5, and D6 to the clinician. The clinician will choose the destination of the high-quality embryos based on the individual situation and the patient's own wishes: freezing, transplantation, or blastocyst culture. The doctor will then hand the doctor's instructions back to the embryologist, who will follow the doctor's instructions to cryopreserve the high-quality embryos.

10:00 Change medium

The embryologist will give feedback to the clinician on the high-quality embryos on D3. The clinician will decide whether to continue blastocyst culture with the high-quality embryos based on the individualized conditions and wishes of the patients. The embryologist will follow the doctor's instructions for blastocyst culture and perform a medium change operation on the high-quality embryos, that is, replace the high-quality embryos from G-1 culture medium to G-2 culture medium, and culture them in an incubator at 37°C, 6% CO2, and 5% O2 for 2 to 3 days.

10:00 Biopsy

Preimplantation genetic screening/diagnosis is mainly used for patients with single gene diseases, repeated implantation failures, repeated miscarriages, and advanced age. With the advancement of biopsy and genetic analysis technology, the cycle ratio of such patients is gradually increasing. At present, the main biopsy technology used by embryologists is the blastocyst trophoblast cell biopsy technology, which uses a micromanipulator to extract 3 to 5 trophoblast cells for single-cell sequencing and analysis of chromosome number, structure, etc. After the embryo biopsy, the whole embryo is frozen, and after the biopsy results come out, normal embryos are selected for transplantation.

11:20 In vitro fertilization (IVF)

38 to 40 hours after HCG injection, it is the time for IVF fertilization by embryologists. Embryologists will mix and culture the processed eggs and sperm in a certain ratio, allowing the sperm and eggs to combine freely to complete the IVF fertilization process.

13:30 ICSI

The ICSI cycle of single sperm injection of eggs began at 13:30. A single sperm was injected into the egg through a micromanipulation system. After ICSI, the sperm-injected MII eggs were directly transferred into the G-1 culture medium and cultured in an incubator at 37°C, 6% CO2, 5% O2 until D1 pronuclear observation.

16:30 Eggs and early peeling

4-6 hours after IVF fertilization, embryologists will remove the eggs and perform early stripping. The purpose is to determine the fertilization of sperm and egg based on whether the second polar body is discharged; the second is to improve the culture environment of sperm and egg. If the patient is primary infertility and it is the first IVF cycle, 2-3 eggs will be stripped early. If 1-2 eggs have bipolar bodies, it means that the fertilization is good. If the fertilization is not good, the fertilization will be observed again at 18:00. If there is no improvement, remedial ICSI will be performed immediately.

13:30 Prepare the solution and attach a label to each patient on the culture dish

The culture medium used for egg fertilization, culture, and embryo culture should be balanced in a 37°C, 6% CO2 incubator for more than 8 hours, so the culture medium used for egg collection and embryo culture the next day should be prepared in advance. In addition to preparing the liquid used for balanced culture, the chip and verification barcode corresponding to the patient's clinic number should be attached to each culture dish for easy verification during operation.

In addition to the above work, embryologists also have to write and record medical record information, enter various quality control and embryo laboratory data, maintain laboratory instruments and equipment, etc. Embryologists who silently take care of "test tube babies" behind the scenes escort every fertilized egg and embryo, hold the "test tube baby" in their hands, and carry the starting point of life.