Day 3 embryos – to transfer or not to transfer? Day 3 embryo transfer dilemmas

Foteini Chouliara, MD, MSc
Obstetrician & Gynaecologist , Assisting Nature - Human Reproduction & Genetics
From this video you will find out:
  • What happens during an embryo transfer?
  • How many cells does a 3-day embryo consist of?
  • What should an embryo look like on day 5?
  • How are the embryos graded?
  • What percentage of 3-day embryos get to blastocyst?
  • When a day 3 embryo transfer can be indicated?

When can day 3 embryo transfer be indicated?

In this webinar, Dr Foteini Chouliara, Gynaecologist & Fertility Specialist at Assisting Nature discussed day 3 embryos, and when such sources can be considered for transfer.

Dr Chouliara started by explaining how the embryo develops in nature, as soon as ovulation occurs, the oocyte is released by the ovary and is picked up by the fallopian tube. It’s passively moved towards the uterus, if sperm is there and the conditions are optimal, fertilization occurs. At first, the embryo consists of a single cell called the zygote, and then through a series of divisions, it develops into a two-cell organism, a four-cell organism, an eight-cell organism and so on. As the embryo is passively moved along the fallopian tubes, it will spend a few first days of its life inside the fallopian tube. By day 4-5, the developing embryo enters the uterine cavity, and by day 5, if everything goes well, it will form a blastocyst stage on day 5 to day 6. The embryo will hatch out of its protective layer, called the zona pellucida and start the implantation process.

In the IVF setting, the equivalent event to ovulation is the triggering shot and the oocyte pickup. In IVF, instead of the fallopian tube picking up the oocyte, it’s the fertility specialist that is picking up the oocytes, and then is safely passing over the oocytes to the embryologist, who then fertilizes them and places the fertilized eggs into designed culture media and state-of-the-art incubators to allow the embryos to develop and culture further.

Embryo transfer process

Embryo transfer refers to the process of transferring the cultured embryo into an appropriately prepared and receptive uterus. This can take place at any time after fertilization, it can take place on day 2 up to day 6 of embryonic development, but usually, it takes place either on day 3 or day 5. These days are not picked out randomly, they are special days because the embryo reaches special stages of its development, and it’s also the time when it’s possible to grade the blastocysts.

Day 3 embryo

Day 3, or a cleavage stage embryo, consists of 4 to 8 cells. It’s still enclosed in its protective layer, the zona pellucida, and it uses the energy and the chemical components provided by the egg to reach this stage. A blastocyst consists of a lot more cells, which are organized into the inner cell mass and the trophectoderm. The inner cell mass is a group of cells that are going to give rise to the embryo, and the trophectoderm is the outer layer of cells that is going to give rise to the placenta. Almost all embryos can reach day 3, but only a third or half of the embryos can develop into a blastocyst. This is because blastocyst formation requires genomic activation for further growth and differentiation.

Embryo grading

Embryo grading takes place on day 3 and day 5, those embryos that have got the best implantation potential are selected. Day 3 embryo is graded by the number of cells, symmetry, and degree of fragmentation. A very good day 3 embryo is graded 8A. Whereas, in a blastocyst, the embryologists look at the blastocoele degree of expansion. Another important thing is the degree of expansion of the cells, inner cell mass compaction and trophectoderm cell number. A very good quality blastocyst would be 4AA or 5AA, however, it’s significant to remember that the embryos continue to develop, and change. The grading has got its limitations because, at the end of the day, it is based on morphological features, so it can look beautiful, but in reality, it could be chromosomally abnormal.

Nowadays, there is a variety of culture media, and they can emulate in vivo conditions a lot better, and they can accommodate the changing embryonic metabolic and nutritional needs. Culture media can help the embryos to reach the day five stage. At the same time, thanks to the evolution of incubators with separate chambers that could offer more stable conditions as well as time-lapse incubators, which can additionally offer continuous embryo surveillance, there are better laboratory conditions. They improve the embryo traits and can also offer more information to improve embryo grading and the prediction of those embryos that have got better implantation potential, and in the end, improve live birth rates, which is the ultimate goal.

Day 5 embryo transfer

Having all these things in mind, considering when to transfer the embryos having in mind the advantages of highly designed culture media, having very good incubators, and having highly skilled embryologists, it seems best to transfer day 5 embryos. Most studies and reviews have shown that a day 5 embryo transfer is superior to any earlier embryo transfer. It allows for a better selection of viable embryos, which might have looked similar on day 3. It also allows transferring of fewer embryos back into the womb and perform single embryo transfers, therefore, minimizing the risk of higher-order pregnancies and all the obstetrical and neonatal complications that go along with it.

Additionally, by transferring on day 5, there is a better synchronization of the embryos with endometrium and the implantation window, even the uterine contractility is reduced, which allows for the embryos to stay exactly where they’ve been placed. By transferring embryos with higher implantation potential, the implantation rates are increased, which in turn means increased pregnancy rates as well as decreased early pregnancy losses. Another reason to transfer day 5 embryos is transparency.

When we are allowing our embryos to cut to an extended culture when we allow them to grow further and reach day 5, we make sure that we can offer our couple the embryos of the best potential possible. That’s transparency, and at the same time, if the embryos do not reach the blastocyst stage, we avoid any futile and unnecessary embryo transfers, so save our couple from necessary heartache and pain.

It’s also very useful for diagnostic purposes, especially in couples with repeated implantation failures. It is possible to see if the embryos are competent enough to reach the blastocyst stage. If they are not, it helps to explore other therapeutic options sooner and find the reason for the failures. Are there any drawbacks to transferring day 5 embryos? The first one is the fact that there is a higher cancellation rate, and although it helps to avoid unnecessary embryo transfers, it could also have a financial impact on the couple as well as psychological stress that the couple is experiencing. To perform a day 5 embryo, an experienced and skilled embryologist is crucial, as well as extended culture in a state-of-the-art lab.

Day 3 embryo transfer

A day 3 embryo transfer might be a good idea if there are poor laboratory conditions, in such cases, it’s preferable to transfer early even though the embryo is not in the appropriate environment as the day 3 embryo should be still in the fallopian tube, not in the uterus, so it is exposed into a more adverse environment. However, it’s a lot better than leaving the embryo in suboptimal lab conditions and leaving a very good day 3 embryos to waste. Day 3 embryo transfer might also be a good idea for couples with fewer embryos available for transfer. For example, couples who are going through natural cycles, poor responders or advanced maternal age. Leaving such embryos to culture further, there is no additional selection advantage. In these cases, it’s also important to remember that it is usually necessary to transfer 2-3 embryos rather than 1.


Deciding when to transfer is not always a straightforward answer, many factors need to be taken into account like the embryo quality, embryo grading, the embryo quantity, how many embryos are available for transfer, the couple’s particular circumstances like the patient’s age, the couple’s history, the risk of multiple pregnancies and the current lab conditions available. Considering all those factors, an individualized choice needs to be made amongst the couple, the treating physician, and the embryologist.

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- Questions and Answers

I had just had FET of 2 day 3 embryos, which failed. They were 10 cells each grade 1 and 2. They were frozen I was 41, and I am 42 now. Do you think the failure was due to the embryos being abnormal? I have been advised to freeze the embryos on day 3 as I only had a few on retrieval. I have 7 day 3 embryos left. What would you recommend, transfer 3 in one go instead of t2? Do you also recommend assisted hatching?

Initially, if there were so many embryos available, I think I would have gone for day 5 Embryo transfer to start with. That way, we could have a better selection, so instead of choosing out of 7 embryos and doing repeated embryo transfers, I think it would have been preferable to leave them to day 5. You would have fewer embryos to transfer but as we said in the presentation with better implantation potential, so possibly, achieving our goal sooner rather than later. Since they’ve been frozen at day 3, there are actually 2 choices, first one is to transfer on day 3, and yes, I do agree that maybe go for 3 instead of 2 to increase your chances. You could also thaw them and leave them to grow to day 5 and transfer blastocyst rather than day 3 embryos, so that could be another idea. About assisted hatching, you could also use this.

Is there anything I could do (I did a hysteroscopy already) to increase my rates of success?

I think it’s a very good idea, especially if we’ve had a failure because some uterine abnormalities are very subtle. They can be a little bit difficult to be picked up just by ultrasound alone, so it’s a good thing that you did a hysteroscopy. Since every embryo is so valuable, all the other aspects need to be well regulated, like the thyroid, vitamin levels, and any other health problems sorted and regulated before your embryo transfer.

I am a patient with a low ovarian reserve and poor response, I had 3 follicles, and only 1 mature reached day 3. Do you recommend transferring or waiting until day 5 with the possibility of losing it? What would the implantation rate be?

These are difficult decisions because you’re faced with the fear and the insecurity of not having an embryo to transfer. If this is something that you cannot handle, then do a day 3 embryo transfer, mostly for psychological reasons, but if you discuss it with your physician and you know the pros and cons, maybe it would be worthwhile to try it and leave it to reach day 5 and that way you can test the competency and the potential of your embryo. To do so, as I mentioned earlier, you need to have excellent lab conditions so that if the embryo doesn’t reach day 5, it’s purely due to the lack of its competence and not due to any external factors.

I had 6 embryos that were not good enough quality on day 6, there were none to transfer. Would you have transferred on day 3?

Yet again, if the lab conditions are good, and this is always checked by the performance indices that every lab has, then I think it was a good idea that you didn’t transfer on day 3 because these embryos would never implant. It’s a risky business leaving them to day 5, it can be a bit disappointing, but at least you can see what’s happening in front of your own eyes, and you can see why this embryo transfer wouldn’t have worked because the embryos, unfortunately, didn’t manage to reach the blastocyst stage.

How do you know if the clinic has excellent lab conditions?

There are different performance indices that every lab needs to have and needs to meet. You can ask your clinic for the fertilization rates and the blastulation rates, and you can have some insight into how things are working, but generally, this shouldn’t be a question that you ask your clinic, it’s a question the clinic itself is asking itself. It’s maintaining a high quality of care and high lab standards. Every clinic should look into these indices itself and improve on itself, and every physician knows how strong a lab is, and I think this plays a very important role in deciding on whether to advise the couple to go for a day 3 or a day 5 embryo transfer.

What could cause a poor blastulation rate?

It’s the embryo itself, and as we’ve said before, it’s not easy for the embryo to become a blastocyst. The activation of the genome has to take place, and it needs to work right. That’s the combination of both the maternal and the paternal genes, and if this works, then yes, we have blastulation, and the embryo can differentiate into what we call the blastocyst. However, having a blastocyst doesn’t mean that we’re going to have a live birth, but it’s another step into the embryonic development that the embryo can take along the way.

What types of abnormalities are difficult to detect through ultrasound but could be detected through hysteroscopy?

It could be subtle abnormalities like a polyp or maybe a small submucosal fibroid or even a septum. Small septums are usually missed on gynaecological ultrasounds, even on 3D ultrasounds, so hysteroscopy remains the gold standard in the evaluation of the endometrial cavity. If it was cheaper and a little bit less invasive, all couples should have a hysteroscopy before any embryo transfer.

Would you take all of the embryos to the blastocyst stage irrespective of the grades? Also, what is an ideal blastulation rate?

If we decide to go for a day 5 embryo transfer, yes, leave all the embryos to test them and see if they will become a blastocyst. A good blastulation rate, is up to the embryologists, but it should be, I think, above 30- 40%.

I am 43, and I have 4 embryos left, 2 of mine which have fragmentation and 2 donor embryos, all of which have been frozen on day 3. All of my previous transfers have failed (2 with my embryos and 1 with the donor). My clinic pushes 3-day transfers, but at this point, I’m thinking to thaw and try to push my remaining embryos to day 5, but I fear I will lose them all. Should I attempt to push them to day 5, I’ve just completed an ERA test and have a hysteroscopy scheduled to make sure there are no issues.

We’re looking at 2 separate embryo transfers, one with our own first and then, I guess, with a donor. In this case, maybe there isn’t an advantage to growing them to day 5 because it’s not going to help us to select 1 or the other if you only have 2, both of them are going to be transferred. The only thing that we’re going to miss out on is whether these embryos were good enough to become blastocysts, so if everything works out and you become pregnant, we don’t mind, but if there is a failure, then it would be very important to know when we’re asking ourselves why we failed. It’s mostly for diagnostic purposes, rather than using the day 5 culture to select amongst the embryos.

What is your take on day 6-7 blastocysts?

An embryologist can answer this better than me, but if the lab conditions are optimal, the embryo should reach the blastocyst stage by day 5, and by day 6, it should be ready to hatch. However, if the lab conditions are a little different, it might take a little longer for the blastocyst to reach this stage, but I’m afraid by that time, we will have lost the implantation window. I think it’s better to transfer on day 5 or day 6 at the latest, not longer than those 6 days.

We had 9 donor eggs of which 7 fertilized but only 1 made a good blastocyst to embryo transfer on day 5. I wonder what could cause only one embryo to progress to day 5. The donor was 25, and that one blastocyst was of good quality.

When you’re going for donor eggs, you’re expecting better results for sure, I mean egg quality is the most important thing, but sperm quality is equivalently important. I don’t know how it was the sperm parameters because sometimes when the sperm parameters are poor, regardless of how good the donor is, we do get bad blastulation results.
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Picture of Foteini Chouliara, MD, MSc

Foteini Chouliara, MD, MSc

Dr Foteini Chouliara is an Obstetrician and Gynaecologist with a special interest in Reproductive Gynaecology at Assisting Nature since 2018. She was awarded the Bachelor of Medicine and Bachelor of Surgery degree (M.B., Ch. B) by the University of Glasgow School of Medicine in 2005. After completing Foundation Year 1 post-graduate training she started her residency in surgery following by the residency in Obstetrics & Gynaecology at Hippokration Hospital of Aristotle University of Thessaloniki/ Greece. Since 2017 she is working as a qualified Obstetrics & Gynaecology Consultant. She received her Master of Science (M.Sc.) Degree in “Human reproduction” from the Democritus University of Thrace in 2019. She is also a Research Fellow of the Aristotle University of Thessaloniki carrying out research work on the role of particular biomarkers in the pre-surgical assessment of ovarian tumours as part of her Doctorate. Dr Foteini Chouliara is deeply committed to the supervision of the International Patient Department at Assisting Nature. Her current scientific interests include: Biomarkers of ovarian cyst and their value in Human Reproduction, PCO, Prenatal first trimester diagnosis in Reproductive Patient. She has been a part of scientific publications, presentations, papers, such as: Najdecki R, Chouliara F, Timotheou E, Tatsi P, Chartomatsidou T, Asouchidou E, Pakaki F, Bouchlariotou S, Humaidan P, Papanikolaou E. Single follicular degarelix, a new long-acting GnRH-antagonist for LH surge suppression during ovarian stimulation in oocyte donors. A randomized controlled trial. ESHRE Annual Meeting, Vienna 2019 (oral presentation) Prevalence of antenatal depression and associated factors among pregnant women hospitalized in a high-risk pregnancy unit in Greece, Social Psychiatry and Psychiatric Epidemiology, 2016 Jul. Antenatal depression among women hospitalized due to threatened preterm labour in a high-risk pregnancy unit in Greece, Journal of Maternal and Fetal Medicine, 2017 Mar 21.
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