Day 3 vs. Day 5 Culture

July 9, 2010Carole 21 Comments »

One of my readers asked me to comment on day 3 versus day 5 embryo transfers. This is one of those topics that embryologists have heated debates about because both sides can come up with evidence supporting their clinical approach. My disclaimer is that I have always favored day 5 culture of embryos to the blastocyst stage and was an early adopter of the first sequential culture mediums that made extended culture possible. Why am I a ‘believer” in day 5 culture?

1. Culturing the embryo to the blastocyst stage of development synchronizes it with the uterus. In natural conception, on day three after fertilization, the embryo is happily floating in the Fallopian tube, not the uterus. The embryo reaches the blastocysts stage just as it enters the uterus in nature so you would expect a blastocyst stage cultured embryo to be better synchronized to the uterine environment.  The composition of sequential mediums were developed to mimic the changing natural embryonic environment, particularly in regard to metabolic changes that  happen during those first five days of embryonic development.

So why did clinical IVF start out with doing day 3 transfers? Because embryos stopped growing after three days in the original IVF medium so they had to go back to the uterus or they would die! For a short while, zygote intrafallopian transfer (ZIFT) was advocated as a protocol to synchronize the embryo with mom at the time of transfer. In ZIFT, you underwent surgery  twice, two days in a row: first, to get the eggs which where fertilized in the lab, then to transfer the fertilized eggs back into the Fallopian tubes on the second day. It may have been better synchronized physiologically but most patients were not happy about two surgeries.

2. The ability to reach blastocyst stage can be used as a criteria for embryo selection. You may remember an earlier post “The embryo beauty pageant and why your eggs don’t care if you yoga”, discussing the limited utility of appearance-based predictors of embryo viability and pregnancy. It turns out that the ability to go the distance- at least to day 5- is a better indicator of embryo viability than what an embryo looks like on day 3.  Great looking embryos on day 3 may not be the ones that are able to progress to blastocyst stage. Those of us who like blastocyst culture believe that those embryos that fail to progress to day 5 in culture would have also failed to progress if transferred to the uterus on day 3. Programs who prefer day 3 culture argue that embryos are “better off” with less culture. Given the asynchrony problem, I don’t agree. In our hands, we could put back one or two progressive blastocysts and have as good or better chance of pregnancy than transferring more on day 3.

3. Transferring fewer blastocyst stage embryos can be used as a strategy to lower the rate of multiple gestation pregnancies. Why do we transfer more on day 3 than day 5? Because we are factoring in our uncertainty about which embryos are likely to implant. We expect some of them to fail. Of course, the down side is that if we transfer more, we are also likely to increase our chances of getting more than one to implant which is counter to good obstetrical medicine because multiple pregnancies tend to be more difficult and more likely to end badly than singleton pregnancies. By selecting progressive blastocyst stage embryos, one or two can be transferred with good pregnancy rates, resulting in singleton and twin pregnancies and avoiding triplet pregnancies. Of course, embryos can split and a twin transfer can result in three babies but that is relatively rare.

4. Culturing to day 5 allows clinics to offer pre-implantation genetic diagnosis in the original fresh cycle. PGD is increasingly being offered through IVF clinics. Embryos are cultured for three days, biopsied on day 3 to collect a sample, the sample is shipped off to the genetics lab and embryo genetic results are back to the lab by the morning of day 5, allowing an afternoon embryo transfer. Without the ability to culture to day 5, you have to freeze a traumatized just biopsied embryo on day 3 and wait for another month to thaw and transfer the unaffected embryos. Freezing embryos does not improve them and may hurt them. PGD is really not compatible with day 3 culture because it requires two additional days to get the genetic analysis report back. Some programs offer day 5 culture just for PGD cases, but since PGD is a small percent of most cycles, the staff is less familiar with using day 5 culture if it is applied to just “special cases”.

So given all those reasons to adopt day 5 culture, why don’t all clinics practice day 5 culture? Here are some of the reasons clinics don’t do day 5 culture.

1. Fear that the unnatural culture environment will cause problems with embryo development or genetic imprinting. This argument can be extended to argue against doing IVF at all. What is more “unnatural” than growing embryos in a lab? Ironically, this handwringing is not typcially accompanied by calls to postpone clinical introduction of new protocols or for longitudinal studies of how IVF kids are doing at one year, 5 years, eighteen years, etc.. A recent study presented at the European Society for Human Reproduction and Embryology (ESHRE) showed that in vitro maturation of eggs before they are used in IVF may be responsible for  abnormally large birth weights requiring C-section delivery. This sobering report suggests that some human embryos are also subject to genetic imprinting problems from some culture conditions. Reassuringly, these symptoms of Large Offspring Syndrome were not observed in offspring conceived with traditional IVF with or without ICSI, but seemed limited to cases in which in vitro maturation was used.  Also, there was no evidence that these large infants had other problems, at least initially, but the report called for long term outcome studies.

In my opinion, this demonstrates that culture conditions for in vitro maturation of eggs do not adequately mimic natural conditions for egg maturation and it may be premature to use in vitro egg maturation protocols clinically. Traditional IVF uses ovarian stimulation drugs to goose egg maturation but maturation is happening in the patient’s ovary, not in a dish. I am not aware of  convincing scientific evidence that extending culture to day 5 is inherently bad. Of course, if the wrong medium is used or labs mix and match sequential mediums to cut costs, then all bets are off. I was very happy with Vitrolife medium, which had two decades of animal research (sheep and cow) backing up the composition of the medium before they even considered clinical implementation of a human version. I don’t now and never had any financial  interest in Vitrolife but my experience with its product line is the basis for my favorable experience with sequential culture.

The fear of prolonged culture is also expressed in the idea that the embryo is “better off inside the mom”  and so should be transferred as soon as possible. One prominent program actually transfers at day 2 (two-cell stage) to the uterus  and gets pregnancies so the human embryo can be very accommodating, but that doesn’t mean it is enjoying optimal conditions. It does make us all feel warm and fuzzy to be on the side of nature and motherhood so this scientifically weak argument still gets traction.

2. Day 5 culture is more work for the lab and costs more money in terms of labor and supplies. Instead of having to monitor patient embryos for four days (retrieval plus 3 days of culture), now you have the embryos around for six or even seven days. If you have a small staff, they are not happy with you because they are working seven or more days in a row, without a break. The crew can get cranky, especially if the program doesn’t batch cases. In some programs, staff may get paid more for working over a weekend, increasing labor costs. Day 5 culture means you need to buy more and different medium to continue culture past day 3. Sequential medium is more expensive than simple culture medium.

3. Day 5 culture requires that the lab make some adjustments in their work flow. A lot of labs got excited by day 5 culture when it was first introduced, tried it and decided they didn’t like it. Sometimes, it boiled down to how it was administered in the program. Some programs have all these criteria for whether a particular case gets to go to day 5 or not, so you end up doing different protocols on different patients or even on different embryos within the same case. Programs that hate day 5 usually hate day 5 because they are freezing embryos on day 3, day 5 and sometimes even zygote stage, because cases are all over the place for day of transfer. Sometimes, they even split cases, freezing some embryos on fertilization day and some on day three when they transfer and leftovers on day 5. Keep in mind, that each developmental stage requires a different freezing protocol if they are using slow freeze methodology which adds time and aggravation to the IVF day. This complicated decision tree for sequential culture makes no sense to me, adding complexity without benefit.

A much simpler model is to do day 5 culture for every case and freeze all non-transferred embryos on day 5. Simple. Elegant. Fewer mistakes.  I have had embryologists tell me, we can’t let everyone go to day 5, we’ll have too many patients whose embryos fail to advance and they’ll have no transfer on day 5. In my experience, this happened less than one percent of the time in our program. If it is happening a lot, I would argue that the problem may be with egg maturity or with how the lab is using the sequential medium. Day 5 culture does require attention to detail, particularly when changing the embryos to the new medium on day 3 to ensure that all traces of the previous medium are removed. But, even in cases with few eggs, we have had good success with day 5 culture since these few eggs were usually mature.

Conclusions. In our program, we found that pregnancy rates increased across the board when we adopted routine blastocyst culture and we were able to almost eliminate triplet pregnancies in favor of twins or singletons which was also a good thing. However, there is more than one way to grow an embryo and human embryos are surprisingly adaptive, so you can find good programs that prefer day 3 culture. And I am sure they would be happy to tell you why!

The American Society for Reproductive Medicine has issued its opinion on “Blastocyst culture and transfer in clinical-assisted reproduction”, which summarizes the conflicting randomized controlled trials that have compared day 3 to day 5 culture. The bottom line is that patients should look at the publicly available pregnancy outcome data for programs listed on the www.CDC.gov website. Check out my earlier post on how to navigate the CDC site. Whether they use day 3 or day 5 culture, you should look for the best pregnancy rates (50% or higher per transfer) and low multiple rates (rare triplets) to identify the best programs. In the end, these two factors and not culture method are probably the most predictive of your chances of coming home with a healthy baby.

© 2010, Carole. All rights reserved.

21 Responses to this entry

  • E Says:

    thank you so much for this informative post! I had no idea it was so controversial! You are providing a wonderful public service to the IF community through this blog.

  • E Says:

    Here’s another question: is there a difference between freezing 3 day embryos vs. 5 day embryos? I’ve heard that 3 day embryos are more likely to survive the thaw…not sure how true that is though…

  • Carole Says:

    If the freezing techniques are performed correctly, embryos can be frozen with good survival at any stage. We found that vitrification worked best for us at every stage and we did better with vitrification (90% survival) compared to slow freezing procedures (60-70%). Other labs report they do better with slow freezing compared to vitrification. Go with the strengths of the lab you are using.

  • grants for women Says:

    This is such a great resource that you are providing and you give it away for free. I enjoy seeing websites that understand the value of providing a prime resource for free. I truly loved reading your post. Thanks!

  • Prostate Gland Problems Says:

    well written blog. Im glad that I could find more info on this. thanks

  • Haidee Says:

    Fantastic blog full of information! Thank you for the comment you left on my blog, returning the favour! 🙂 My clinic tends to go to Day 5 blasty’s too, I had a really disappointing last IVF cycle though as we had 14 embryos on Day 3 but only one that made it to Day 5 as a viable emby. The rest arrested, however we did have about 6 that got to blasty but I was told they didn’t have the part that makes the baby. This confused me greatly and I then found out at a follow up that my clinic has newly decided not to freeze those they think have only a tiny chance of turning into a baby as it ives too much false hope to the couple. I’m coming up to a third attempt and hoping for some better results! Thank you for sharing your wealth of knowledge!

    Mummy in Waiting
    -ICLW

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  • Trace Says:

    Hi Carole,

    I’ve often come across your website and have found it a very useful resource. This post is one that is particularly topical for us! We are having the 3 day vs 5 day discussion with our embryologist at the moment, and unfortunately she has not been able to back up any of what she’s saying with actual research (other than her own experience, which is valid, but not robust enough for my purposes!). Her focus seems to be very much on success rates per ‘cycle’ which for our clinic at least means per embryo transfer. My husband and I are much more concerned with success rates per egg-collection, as that is the most painful, time consuming, and expensive part of the whole deal. A FET – no bother, I could do one every month for the next six months if need be (assuming it was on a natural cycle) with minimal expense and discomfort / disruption to my life compared to a fresh cycle.

    The studies that I have found looking into the cumulative pregnancy rates per egg-collection all seem to suggest that day 3 transfers offer significantly superior pregnancy rates to blastocyst transfers overall.

    http://www.ncbi.nlm.nih.gov/pubmed/22786480

    I understand that most clinics seem to be focused on advertising their per embryo transfer success rates, but from the perspective of the average cash-strapped couple going through IVF, perhaps the focus should be on success per egg-collection – by far the most expensive and difficult part of the whole deal.

    We have 3 embryos (after showing 20+ follicles, collecting 12 eggs, 8 of which were used for ICSI, but only 3 fertilised), and are being pushed towards day 5 culture by our embryologist – however given the cumulative pregnancy statistics, this approach appears to make no sense for us. This is our last cycle of ICSI.

    On our first ICSI cycle we did egg-share, had 12 collected, 6 were ours to use, all 6 fertilised, and all made it to day 5, but only 3 were deemed ok to use/freeze. I was 29yrs at our first cycle and am 32 for this 2nd cycle. I’m not sure why the drop-off in fertilisation rate – the embryologist can’t see any reason for the difference.

    Just food for thought – hopefully it is helpful to have the patient’s perspective on this topic. If you have any links to journal articles contrary to the Cochrane review link above I would definitely be keen to take a look at them. Thanks again for your very useful website!

  • Carole Says:

    Hi Trace,
    I read the Cochrane review you cited and their conclusions: This review provides evidence that there is a small significant difference in live birth rates in favor of blastocyst transfer (Day 5 to 6) compared to cleavage stage transfer (Day 2 to 3). However, cumulative clinical pregnancy rates from cleavage stage (derived from fresh and thaw cycles) resulted in higher clinical pregnancy rates than from blastocyst cycles. The most likely explanation for this is the higher rates of frozen embryos and lower failure to transfer rates per couple obtained from cleavage stage protocols. Future RCTs should report miscarriage, live birth and cumulative live birth rates to enable ART consumers and service providers to make well informed decisions on the best treatment option available. They conclude that because more cleavage stage embryos were frozen, they had a better cumulative pregnancy rate when FETS were considered. I think that that conclusion is a little simplistic. Yes, we always freeze more at day 3, but that doesn’t mean that we freeze more implantation ready embryos- the ones without a future have not yet revealed themselves because we yanked them out of culture on day 3. By day 5, we have a much better idea of which embryos will go the distance, and which are failing. The better results from FET are due to the fact that FET cycles allow us to optimize the endometrial conditions for implantation because we aren’t also trying to push egg production.

    Aside from doing genetic testing–which it doesn’t sound like you are doing– the main reason to go to day 5 is to identify the most “implantation-ready” embryos among a large pool of embryos. If you- at day 3- already have just a few embryos that are still under consideration, most docs will choose to transfer the 1-2 that you have on day 3. Further culture might result in zero age appropriate embryos for transfer which just upsets the patient. And to be fair, there is some disagreement about whether extended culture doesn’t also carry some risks for inappropriate imprinting. So as for any medical decision, you need to analyze the risks and benefits and choose the option that poses the mot potential benefit with least risk. Either day 3 or day 5 transfer would be a reasonable choice for your situation. There is no one “right” answer. Good Luck!!! Carole

  • Nancy Says:

    I just found out today that my egg retrieval yielded 3 eggs. Based on my follicles, the doctor thought we’d get 6-7 eggs. However, I have read on your site the reasons for the lower egg retrieval as compared to the follicle count. I just turned 41 a few days ago during this process. I have had a few tries before this cycle that were cancelled due to one lead follicle and another cancelled due to low follicle count. I also had a failed ivf a year ago – retrieved 6 eggs but zero fertilization (natural ivf). We didn’t do Icsi because my husbands sperm motility and count are very good. I had 2 previous pregnancies/live births within the last 5 years with iui. My kids were born when I was 35 and 37 years old, but of course even a few years make a big difference.

    My question is, due to the low number of eggs retrieved and depending on what they tell me tomorrow as far as how many fertilized, if any (fingers crossed/praying for good fertilization), would you recommend a 3 or 5 day transfer? We are doing icsi this time due to the zero fertilization last time. When I asked my doctor briefly about the timing for embryo transfer right before the procedure today, she quickly responded – 5 days -blastocyst.. Then, I did my research tonight. I hadn’t even raised the question before. I guess one benefit to me is money – if the egg wasn’t going to make it to blastocyst them does that mean chances are it would have failed a 3 day transfer as well? Thus saving me the money spent on the transfer? I’ve decided This is my last try anyways, but I still want to make the best decision…

  • Carole Says:

    Hi Nancy,
    It is fair to say that if the embryo does not make it to day 5, it will not implant. So waiting and seeing how the embryo does is probably a good idea. There is also a real chance that with only three eggs to start, you might have nothing to transfer. While a very disappointing result, you have saved the money of the transfer and the agony of the 2 week wait only to be disappointed later. I wish you all the best for good fertilization and that all three embryos make it to blastocyst stage! Good Luck! Carole

  • Dee Says:

    Hello Carole,

    My question is regarding 3 day vs 5 day transfer. I have read a study I will post it below: it’s short I promise. It basically says that if your embryos are less than 8 cells at day 3 the uterus is a better home because it resulted in pregnancies. Than again I think this study was done in the year 2000. Perhaps the lab conditions were different than compared to now. But what many RE’s say is that if an embryo doesn’t make it to day 5 it wouldn’t have implanted anyways. Please read below and share your Opinion. Because it’s very confusing.

    Thank you!!
    The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer.
    Racowsky C, et al. Fertil Steril. 2000.
    Show full citation
    Abstract
    OBJECTIVE: To select patients for day 3 vs. day 5 embryo transfer.

    DESIGN: Retrospective analysis of assisted reproduction technology (ART) cycles comparing outcomes of day 3 and day 5 transfers.

    SETTING: ART program of Brigham and Women’s Hospital.

    PATIENT(S): Patients with day 3 or day 5 embryo transfers (n = 221 and 141, respectively).

    INTERVENTION(S): Cycles with eight or more zygotes were stratified by the number of eight-cell embryos available on day 3 (none, one or two, or three or more).

    MAIN OUTCOME MEASURE(S): Number of blastocysts, implantation rates, ongoing pregnancy rates, and number of fetal heart beats.

    RESULT(S): With no eight-cell embryos on day 3, 0% and 33% pregnancies resulted from day 5 vs. day 3 transfers. With one or two eight-cell embryos on day 3, ongoing and high order multiple rates were not different between day 3 and day 5 transfers. With three or more eight-cell embryos, day 5 transfer resulted in a decrease in multiple gestations but no difference in ongoing pregnancy rates compared with day 3 transfer.

    CONCLUSION(S): With no eight-cell embryos on day 3, a day 3 transfer is warranted. With one or two eight-cell embryos, any benefit of day 5 transfer appears to be equivocal. With three or more eight-cell embryos, day 5 transfer is recommended.

  • Carole Says:

    Hi Dee,
    In answer to your question, yes- IVF culture has been vastly improved- since 2000- especially in culture media to take the embryo from day 0 to day 5- so I think any differences in pregnancy rate found in this old study are most likely due to problems with more primitive sequential medium formulations so I wouldn’t make decisions based on this paper. Follow your clinic’s advice regarding the approaches that yield the best results in their hands. Good luck with your next cycle!! Carole

  • Yash IVF Says:

    This is really good information. Thank you for sharing.

  • Harry Says:

    Dear Carole,

    Thank you for the wonderful blog of yours. I only wish I had come across it earlier.. I am still combing through all the entries, but hope you can help me with my question as I may need the answer really soon (we just did oocyte pick-up today)…

    My wife (age 39) and I (age 38) had suffered many cycles of unsuccessful IVF. My wife had few surgeries for endometriosis previously and poor ovarian response to stimulation. We finally decided on using donor oocytes. I did some research prior and decided we want a day-5 blastocyst single embryo transfer as my wife is worried about multiple pregnancies. We traveled to a foreign country and today was the day of oocyte pick-up. The kind donor had given us 13 oocytes. Our plan was for all embryos to be cryopreserved as my wife is not ready for a fresh cycle transfer (we couldn’t get her and the anonymous donor to be synchronized).

    The bombshell came and I was taken aback when the doctor said the lab will freeze the embryos at day 3 (cleavage-stage). I told him I thought we had discussed and the plan was for a day-5 transfer. He said it is possible to thaw / warm the vitrified day-3 embryos and culture them later to blastocyst before transfer, so it will still be a day-5 transfer.

    My questions are:
    1. Is it better to culture the embryos now to day-5, freeze them, and then do single embryo transfer later, or should we follow the doctor’s advice to freeze them now at day-3 and then culture them later?
    2. If we follow the doctor’s method, how many day-3 embryos shall we thaw at one go, since not all may progress to blastocyst stage? The donor is quite young (age 27) but I found from some studies the rate of blastocyst formation may be around 20%. So if we thaw too few, there may be no blastocysts, while if we thaw too many, we may potentially end up with 2 or more blastocysts. I found only few reports of blastocysts surviving 2 freeze-thaw cycles..
    3. Can the lab continue to grow the embryos from day-3 to day-5 if I tell them within the next 1-2 days? I read from your blog that the culture media (simple vs sequential) is different. Do they need to change the culture medium midway?

    I am trying to do as much research as I can and will try to meet the doctor again tomorrow to discuss. But I am very confused by this change in plans. Really hope you can help and advise!

    Thank you very much and bless you for your help!

    Harry

  • Carole Says:

    Hi Harry,
    Sorry I saw this so late but generally, I would let the lab guide you because they know what has worked best for their patients in their hands. I have seen either approach work well so I would defer to the technique that the lab is comfortable with and with which they have had past success. Depending on their culture method, they may need to change there medium- but again, they can best explain their methods. Good Luck!
    Carole

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