What happens to your egg and sperm inside the embryology laboratory?

embryology laboratory

The journey of egg and sperm from Egg collection to embryo vitrification or transfer.

The Embryologist’s Perspective

I consider the 6 days (In the laboratory) as the most important in the IVF journey. This is the only stage where we are able to take a look at the gametes. This is an important stage because everything is timed to perfection. The microscopic work of selecting the gametes to grading the embryos is carried out in a controlled environment. Patients are often curious and anxious about the color, size, and weight of their gametes. Be informed that all gametes look the same irrespective of what the parents look like. There are no VIP gametes or embryos. They are all treated equally, with equal care, without any discrimination. While we are hard at work in our labs, for the patient, it is a period of wait and watch, and it will be good for them to not be stressed about the number of eggs or the quality of sperms. Be sure that the clinician will keep you updated.

Deciding to have a family is one of the most momentous decisions of your life. It is probably the most exciting and emotionally-charged time of your life. Now that you have decided to embark on this journey with the help of technology, it would be good to be armed with some basic knowledge about the steps involved in this process. You will be required to visit the clinic on day zero which is the day of egg aspiration and on the day of embryo transfer (ET). During the days in between egg aspiration and egg transfer, the embryos are taken care of by your embryologist. The day of ET could be any day between day 2 to day 6 depending on individual cases.

From the start of the menstrual cycle, which is the second or third day of your periods, the patient has to be at the clinic for a scan and injections. When the clinician feels the follicle has reached the desired size, a special injection called the ‘Trigger’ is given; 36-38 hours post which egg retrieval is planned. Any delay in the procedure may result in the rupturing of the follicles and we may lose the eggs. So be on time ☺

The days in the laboratory are termed as : 

  • Day 0 – Egg retrieval
  • Day 1 – fertilization check
  • Day 2 – Cell stage, Cleavage stage, single cell line
  • Day 3 –  Cell stage, Cleavage stage, single cell line
  • Day 4 – Compacted Morula or Morula
  • Day 5/Day 6 – Blastocyst stage

Day 0 – Egg Retrieval

On this day, you and your partner will be required to come to the clinic at least one hour before the scheduled egg-retrieval time. It is important to arrive well ahead of the scheduled time to ensure that the procedure starts on time to avoid losing the follicles. Do account for any roadblocks on the way to the clinic. Remember you have undergone the ordeal of the last 10 days just to reach this stage. On this day, the eggs are retrieved under anesthesia from the ovaries of the female partner and the sperms are simultaneously collected from the male partner.

As mentioned earlier, two departments are involved in the process. The clinician will retrieve the eggs from the ovaries and handover the follicular fluid containing the eggs to the embryologist. Each tube is scanned for eggs and they are collected in a separate dish at a temperature of 37c. The dish is placed in an incubator to ensure that the eggs recuperate at an optimum temperature and carbon dioxide level (essential to maintain PH). The next step is the fertilization of the egg by the sperm and the method depends on the treatment of choice. In conventional IVF, eggs are incubated overnight with a calculated amount of sperm. The sperm on its own penetrates and activates the egg. If the egg is mature, fertilization is achieved. ICSI requires an additional step which is the selection of mature eggs and the injection of only those that are mature with selected sperm. The injected eggs are placed in the incubator bringing an end to day zero.

Eggs retrieved can be mature or immature. Mature eggs are called MII and immature eggs are labelled MI or GV. Only mature eggs that have the correct number of chromosomes should be used. Immature eggs that don’t have the normal number of 23 chromosomes should be discarded. In order to differentiate between mature and immature eggs, the eggs are stripped off of a protective layer called the cumulus. This is achieved with the help of an enzyme. Once, the embryologist is sure of the number of mature eggs, a single sperm is injected into a single egg with the aid of a gadget called the micromanipulator. The process of selection of the sperm to be injected is equally critical to the success of the whole procedure, and it is something the embryologist does based on the morphology (outer appearance) of the sperm. It is important to understand that sperms can be selected only on the basis of appearance and any internal damage cannot be seen under the microscope. For that, there are more advanced tests like DFI and MACS.

The job of the embryologist entails a lot of responsibility, and they have to be extremely meticulous and cautious about handling details such as the labeling of dishes.

Patient: “Can you please label my egg and my husband’s sperm properly? I am pretty paranoid.”

Embryologist: “Ma’am, it is impossible to label a human egg and sperm, as they are microscopic.”

Patient: “Then how do you ensure that you are not mixing the wrong gametes?”

Embryologist: “Only one couple’s gametes are handled at one time and all the dishes are well-labeled and double checked.”

Now that it is clear that both female and male gametes are required for fertilization to occur, listed below are the options for procuring gametes excluding the ideal case which is that of the female partner’s egg and the corresponding male partner’s sperm:

  • Donor egg + partner sperm
  • Own egg + donor sperm
  • Donor egg + donor sperm

Medical science presents the above options where the ideal case does not exist. However, it should be borne in mind that the use of donor eggs or sperm involves stringent ART laws which should be discussed at length with the doctor. Also, if you opt for donor egg and/or donor sperm, only the source of the gamete will be different on Day 0. The rest of the procedure remains unchanged.

Day 1 Fertilization Check

On this day, embryologists will start their routine procedures with the fertilization check or the activation of oocyte by the sperm. The fertilization check is done 16-19 hours after the ICSI procedure. That is the time taken for the activation of oocyte by the sperm. Checking for fertilization is a crucial step.

  • Only the oocytes which are fertilized will progress to become embryos.
  • Fertilization signs disappear after 19-20 hours.
  • Fertilization can be normal (marked by eggs with two pronuclei) or abnormal (eggs with one, three, or multiple pronuclei).
  • It is important to separate normal from abnormal eggs.
  • If you miss the fertilization check, it is impossible to tell the normal from the abnormal eggs the next day.
  • The fertilized oocytes after the fertilization check are immediately kept inside the incubator and not checked or touched again for 24 hours.


Do not panic, have faith. Your embryologist wants positive results as much as you do.

Embryologist: “Out of 10 mature eggs you have 7 which are activated or fertilized by the sperm.”

Patient: “Just 7!? But the doctor told me that 15 eggs were retrieved.”

Embryologist: “All eggs retrieved are not always mature, only the eggs which are mature can be injected with sperms. Having said that, not all eggs will have the potential to get activated by the sperm. Hence, depending on the quality of the eggs and sperms, the fertilization number will vary.”

Patient: “So, this is normal?”

Embryologist: “Yes, absolutely! In fact, 70% fertilization rate is considered good.”

All decisions are taken with good scientific backing.

Embryologist: “We have three normal, fertilized and two abnormal, fertilized eggs for you, which means we have three potential embryos.”

Patient: “Why three? Did you not just say five? Why do we need to discard the abnormal fertilized eggs?”

Embryologist: “Abnormal eggs don’t show the usual two pronuclei (PN) or two circles. They either have one or three PN or circles which is not normal and should be discarded.”

Please be wary of clinics which don’t do fertilization checks. If the fertilization check is missed, it is impossible to tell a normal fertilized embryo from an abnormal one, as very often they grow into beautiful embryos. But being abnormal, they are not to be transferred.

Day 2 – Cell Stage, Cleavage Stage, Single Cell Line

On this day, embryos are checked 23-26 hours after the fertilization check. The activated egg from the previous day starts to divide and multiply and is now called an embryo. The ideal cell stage on Day 2 should be between 2-6 cells. From this stage, slight variations can be observed and accordingly the embryos are graded. Grading is the process of classifying the embryos into different categories.

A – Best embryo and has the highest potential to give a positive pregnancy

B – Good embryo and has good potential

C – Below average embryo with low potential

D – Worst embryo and has the lowest potential to give a positive pregnancy

All these four categories comprise of embryos with live cells. The grades are checked only once per day according to the timeline.

On Day 2, we know that (1) an average of 95% of fertilized eggs will form embryos and (2) we have three options—embryo transfer, vitrification, or culture to Day 3. At this stage, this is all the information the lab can provide and it doesn’t help to keep inquiring.

Patient: “I want to know the grade of the embryo.”

Embryologist: “I thought we had informed you about the grades in the morning.”

Patient: “I was told in the morning I have two B and one A and one C; I want to know how my embryos are doing now?”

Embryologist: “Well, the embryos are checked only once a day as it is not advisable to disturb them again and again.”

Patient: “Oh! So, when can I know about their status?”

Embryologist: “Tomorrow, which will be Day 3 for the embryos, until then rest assured they are fine and in the incubator.”


Day 3: Cell Stage, Cleavage Stage, Single Cell Line 

Day-2- and Day-3-embryos are referred to as cleavage-stage embryos. The reason for this designation is that the cells in the embryo are dividing (or cleaving), but the embryo itself is not growing in size. For example, think of a pizza that is being sliced to create more pieces of it, but in the process, the size of the pizza does not increase. This is what a cleavage-stage embryo is like. The genetic material replicates and the cells divide, but the volume of the embryo does not differ from the volume of the unfertilized egg. On this day, 24 hours after the Day-2-check, the embryos should be around 6-8 cells. This is the day, the embryos are moved into a new petri dish with media (solution to support growth) that is similar to uterine fluid for their next stage of growth. The embryologists grade the embryos, but they still do not know how many will continue to grow to the blastocyst stage (Day 5 and 6). Some clinics perform embryo transfers and freezing on Day 3 and some clinics wait until Day 5. This depends on the number and the quality of embryos that you have available and also the clinic policy.

Our aim in moving ahead from Day 3 to 4 is to better the process of embryo selection for transfer. Most IVF clinics in India prefer to do Day 3 embryo transfer. If the decision to grow them to the next stage is taken, the embryos are shifted from one dish to the next and returned to the incubator. This is done to ensure that the embryos meet the nutrition required for the next stage.

Cleavage-stage embryo is transferred in the uterus where it grows and floats in the uterus for two days until it becomes a blastocyst and then implants. Any embryo has to progress up to the blastocyst stage before it can implant and culminate into pregnancy. On an average, only half of all Day 3 embryos will continue to grow to the blastocyst stage and the other half will stop growing after Day 3. Embryos stop growing because there is something wrong with their chromosomes. If an embryo stops growing in the lab it would most likely not have made a baby if it had been transferred.

On Day 3 we know (1) how many cells the embryos have, (2) if the embryos are in a good condition internally and externally up to this point, and (3) we have three options—embryo transfer, vitrification, or culture to Day 5.

It is good to clarify your doubts with your embryologist.

Embryologist: “You have four B and two C grade embryos. We would advise you to keep the embryos till Day 5 and then do a transfer.”

Patient: “Why can’t we transfer the two B grade embryos today?”

Embryologist: “We can do that. But keeping the four embryos of good grade (A and B are considered good) till the blastocyst stage would give us better selection.”

Day 4 – Compacted Morula or Morula

This day and stage is very dynamic. Nothing is clearly signaled by the embryos. Hence, they are not checked on this day and embryo transfer is not planned for this day as well.

No decisions should be made this day.

In order to take the embryo up to the blastocyst stage, labs have to be extremely specialized. Be sure to ask the right questions about the capacity and credentials of the lab right at the beginning. It would be good to know the number of full time embryologists employed at the facility.

Decisions about the day of transfer are crucial and you need to be in well-qualified hands.

Clinician (to the embryologist): “Can you please take a look at this report?”

The handwritten embryology report handed over to the embryologist said that the patient consulting the clinician at the moment had gotten two embryos transferred on Day 4 (in the Morula stage) at an infertility clinic she had consulted earlier.

Embryologist: “The outcome of that cycle was negative because the transfer was done on Day 4; you don’t know whether the embryo was growing or arrested at that stage. The embryo should not be transferred on Day 4.”

Day 5 / Day 6  – Blastocyst stage

On this day, embryos should be reaching the blastocyst stage of development. They not only continue to divide and increase in cell numbers, but the cells also differentiate into specific cell types. By this time embryos start to outgrow the space inside the “shell” that surrounds it and when the blastocyst bursts through this membrane it is prepared for implantation into the uterine lining.

There are two cell types in the Day-5-embryo or the blastocyst.

One cell type forms the Inner Cell Mass (ICM) that will eventually grow into the fetus.
The other cell type, known as Trophectoderm Epithelium (TE) is a sheet of cells that will go on to make the placenta. Together these cell types make a fluid-filled sphere with the TE cells on the outside and the ICM inside. Think of a balloon. If you blow up a balloon and put a ping-pong ball inside, that is what a blastocyst looks like. The latex of the balloon is the TE and the ping-pong ball is the ICM. Both of these cell types are necessary to establish a healthy pregnancy. You cannot have a baby without a placenta and you cannot have a pregnancy without a fetus. When we grade embryos at the blastocyst stage, we assign a letter grade to each of the cell types. This is the most advanced stage until which the embryos can be kept in the lab. The embryos which have the potential progress and the weak get arrested. The best embryos are either transferred or frozen on Day 5, depending on the individual case. In general, pregnancy rates are better when blastocyst-stage embryos are transferred rather than Day-2- or Day-3-embryos. On Day 5 we know the number of embryos that have continued to grow and the quality of these embryos.

Going up-to this stage has its pros and cons. They are:


  1. Pregnancy rates are higher.
  2. Even in a normal cycle, when the embryo reaches the blastocyst stage, it becomes attached to the uterus. Hence, carrying out the transfer on Day 5 is a case of the embryo being present in the right place at the right time, thereby maximizing the chances of pregnancy.


The embryo may not progress up to the blastocyst stage, and there might not be a transfer.


Most clinicians and embryologists will advise their patients that even if the embryo does not progress to the blastocyst stage, waiting till then is not a bad thing, as doing this would enable the lab to investigate the reason for non-conception by the patient with greater clarity. It will also help in providing more information to improve the subsequent IVF cycles. Moreover, not all embryos grow at the same rate. Some will grow faster and be ready for transfer or freezing by Day 5 and some will need another day’s growth to get to an advanced stage. This is normal. In such a case, the embryologist will inform the patient that it is better to observe the embryos for an additional day. Scientific evidence suggests that an embryo on Day 6 is as good as Day 5.

Leave a Reply

Your email address will not be published. Required fields are marked *