Embryo stages, progression and pregnancy outcomes

November 10, 2010Carole 593 Comments »

Did I mention I love getting blog topic suggestions from my readers? I get in a rut too. So I was happy to find this request in my inbox. “Carole, please could you discuss the various stages and the required number of cells the fertilized egg goes through up until it is vitrified on day 6? Also please could you explain the terms “cleaved”, “compacted”, “non-expanded” blastocysts and possibly give some percentages as to the chance of pregnancy when, e.g. a compacted 6 day blast is transferred.”

This post also gives me the opportunity to thank Dr. Liz Sanders from the Mississippi Fertility Institute and Dr. Robert Shabanowitz from Geisinger Medical Center for their generous permission to use their embryo images in this blog.

First things first. The first embryonic stage is the fertilized egg or zygote stage. Eggs usually show signs of fertilization between 16-18 hours after insemination. What embryologists look for are two well-defined transient structures called pronuclei in the center of the fertilized egg.  In the picture below, the PN look like two chocolate chip cookies inside the egg. These “cookies” contain the male and female DNA and for normal fertilization, there should be exactly two pronuclei or as embryologists like to shorthand, “2PN”.

Normally fertilized egg with a paternal and maternal pronucleus (2PN) visible. Photo courtesy of Dr. Liz Sanders, Mississippi Fertility Center, Jackson, MS.

What is significant about the 2PN stage? This stage is brief, lasting only a few hours and occurring approximately 16-18 hours after insemination. Seeing more than 2PN (say 3, 4, 6PN or more) are all abnormal numbers of pronuclei which can not be corrected and result in an abnormal embryo which may fail to develop further. 3PN zygotes can cleave and continue to develop but will not produce a healthy pregnancy. Embryologists need to identify these abnormally fertilized eggs and remove them from the viable embryo pool.

Some clinics use a zygote scoring system or Z-score based on the appearance of the PNs to try to identify the fertilized eggs that will cleave and progress from this early stage. If the 2PN stage zygote looks like the egg swallowed two chocolate chip cookies, then the tiny spots within each cookie that look like chocolate chips are the nucleoli. Nucleoli are small, typically round granular bodies composed of protein and RNA that are usually associated with a specific chromosomal site, These nucleoli are involved in ribosomal RNA synthesis and the formation of ribosomes.  Characteristics including the number of nucleoli within each pronucleus, the similarity in size of these nucleoli and whether they are lined up along the edges where the “cookies” touch are all factored into the Z-score.  The usefulness of the Z-score is in dispute and is not used by many clinical labs.

Cleavage stages. When the fertilized egg divides for the first time and forms two cells, it has entered the cleavage stage of development. The term “cleaved” simply means that the cell has divided from one cell to two. Divided =cleaved. The cells in the the two-cell embryo continue to divide, creating a four-cell embryo. Each cell in the four cell embryo divides or cleaves again, forming an eight cell embryo. You can watch a great video of development from the fertilized egg to the blastocyst stage on the NIH stem cell website. A note about “days of culture” related to embryo stages: When I refer to day 3 of culture, day zero of culture is egg retrieval day. Signs of fertilization are visible on day 1 of culture. Cleavage to two-cell stage is typically expected on day 2 of culture and cleavage of the embryo to an eight-cell is expected on day 3 of culture. 

What is significant at cleavage stage? Embryologists have long looked for characteristics at this stage which will identify which embryos will go the distance. Characteristics that are favored by embryologists include same sized cells with little or no fragmentation. The four cleavage stage embryos in the picture at the right are a good example of nice cleavage stage embryos on day 3, when the embryo is expected to have cleaved into at least 8 cells. There is some variability in the cell number that we see on day 3. We expect the best prognosis from embryos that have reached 7-12 cells. If the embryo is only two cells on day 3, this is not a good sign and likely indicates the embryo has ceased to grow. Normal embryos have a fairly strict rate of progression which starts at the time of fertilization. If the time of fertilization is delayed (for example, if rescue ICSI is used), the start time of the embryo’s progression program is delayed and the embryo may reach the eight cell on day 4, not day 3 of culture since fertilization occurred a day later than expected.  But except for delays in fertilization, progression should follow an expected predictable rate. Embryos don’t usually speed up to catch up if they are lagging.

Morula stages of development. The morula stage is characterized by a transformation from a loosely associated group of cells to tightly connected cells that are acting more like a tissue. The process by which cells change from loose association to tight association is called compaction. A compacted morula is a group of cells (usually around 30) which have squeezed together inside the zona. This stage is usually seen on day 4 of culture. The photo to the right shows two typical morula stage embryos that have compacted. The name morula comes from mulberry (Latin: morum), perhaps because the morula looks somewhat like a mulberry.

What is significant at this stage? Sometimes embryos get stuck at cleavage stage and never compact. This is a bad sign and the embryo is no longer viable unless it makes this transition. Embryologists like to see that most of the cells are incorporated into the morula. Cells or large fragments that are left outside of the compacted morula are non-viable. In the picture on the right, you can see a little fragmentation between the morula and zona pellucida (the shell) but not too much. These morula look pretty good. Notice that in each picture from fertilized egg to zona, the zona is still about the same size, but the dividing cells within it are getting smaller and smaller with each division.

The blastocyst stage. Reaching the blastocyst stage of development is considered a very favorable sign for implantation and pregnancy. In a typical IVF cycle, some embryos fail to go on at each stage. It is unusual for 100% of a patient’s fertilized eggs to get to blastocyst stage but it can happen. Embryologists talk about expanded blastocysts, non-expanded blastocysts and hatching blastocysts- all stages in the continuum of blastocyst development. By the blastocyst stage, the embryo has reached 50-150 cells and is starting to strain at the confines of the zona pellucida. This straining is not simply due to cell division but also active pumping of fluid by embryo cells into the inner space of the blastocyst, forming a cavity or blastocoel. The filling of this space with fluid expands the blastocyst and we call this embryo an expanded blastocyst. Before creation of this fluid space, the embryo is called non-expanded. You can see a group of blastocysts that have expanded in the photo to the right. The expansion of the blastocyst helps thin the zona and eventually helps to rupture the zona and let the blastocysts escape or hatch from the zona pellucida. In the expanded blastocyst, the embryologist can see the inner cell mass (ICM) within the blastocyst. I have labeled the ball of cells that make up the ICM in the photo. The ICM contains the cells that will give rise to the actual cells of the fetus. The other cells that surround and protect the ICM and line the inner side of the zona pellucida are the trophectoderm cells. The cells of the trophectoderm give rise to the fetal part of the placenta. The mother also provides cells to the placenta. 

What is significant at this stage? The embryo must have a inner cell mass. The absence of an ICM means game over for the embryo since these cells have died off within the blastocyst. These blastocysts are not transferred. The other troublesome sign is when the blastocysts seems to have a low number of cells, suggesting that the transformation program began before cell division was completed, leaving the embryo with an inadequate cell base for development. The blastocyst stage typically occurs on day 5 of culture and we would see hatching early on day five, especially if the zona was hatched earlier for embryo biopsy. Sometimes the blastocyst will not become expanded until day 6. Differences in culture medium or other features between programs may explain why some programs see full expansion and hatching on day 5 and others see this more on day 6. In our program, we expected to see most if not all the embryos in a patients group of embryos reach this stage on day 5.

There’s another interesting feature of blastocysts and that is their ability to expand- and contract. Expanded blastocysts may “collapse” in culture and look unexpanded. With time, the blastocyst will re-pump the fluid and “re-expand”.  A “compacted” blastocyst is likely a transient condition in which the fully pumped up blastocyst has “deflated’. As long as the blastocyst is capable of expansion, this temporary deflation is not a problem. In fact, prior to vitrification, many programs routinely deflate their blastocysts to optimize the vitrification procedure. After freezing and thawing, a sign of recovery is re-inflation or re-expansion of the blastocyst, showing that the embryo is alive and pumping- literally.

In vitro artifact or source of identical twins? Interestingly, one study using time lapse photography of collapsing and re-expanding blastocysts found a connection between the frequency of collapse and the size of collapsing blastocysts and an increasing frequency of monozygotic or identical twins from IVF. Researcher Dianna Payne described her theory that the frequent collapse was a sign of local areas of cell death. The frequent collapses allowed embryonic cells to move and relocate to a second site within the blastocyst, setting up two ICMs that could lead to identical twins. Excessive cycles of  collapse and reexpansion could kill the blastocyst if it becomes unable to expand. In another study, the ability of a blastocyst to reexpand after thawing was used as a predictor of better pregnancy outcomes.

Hatching Blastocysts. The photo to the right shows an empty zona and four fully expanded blastocysts in various stages of hatching. You can see a bubble of cells sticking out (hatching) out of the left side of the top left embryo. Directly below this embryo, you can see an embryo that is completely free of its shell and its empty shell or zona pellucida has floated to the top of the photo. If you look closely, you will notice that the edges of this hatched embryo is irregular and not shiny like those of the blastocysts that are still enclosed by the zona. The two smaller blastocysts to the right of the hatched blastocysts are still expanding, note their relatively smaller size.

In the picture below, you can see another photograph of a blastocyst in the middle of hatching, half in and half out of its shell. You can see an area in the middle of the embryo that appears more open. This is the blastocoel. Notice how thin and small the zona looks relative to the first photo of the fertilized egg. Some of the newer culture mediums are better designed to allow the natural thinning of the zona in preparation for hatching, making assisted hatching procedures to artificially open a hole in the zona largely unnecessary except for cases in which embryo biopsy is required. Embryo biopsy (removal of one or more cells) from the embryo for genetic analysis requires that a hole is made in the zona at either the eight cell or blastocyst stage embryo.

What does all this embryo progression, embryo scoring and achieving blastocyst stage mean for a person’s chance of pregnancy? Determining which embryo will implant and make a baby is the holy grail of embryology. Evaluation or scoring based on appearance of  the fertilized egg, cleavage stage or blastocyst stage embryos have all been proposed by embryologists to determine which embryos have pregnancy potential and which don’t.  Some clinics have done retrospective studies of embryo progression- a functional test. The embryo progression of sibling embryos was compared from patients who got pregnant to patients who did not get pregnant after day 3 transfer.  Did these sibling embryos stall out or progress to blastocyst stage? Generally speaking, patients whose excess embryos went to blastocyst stage were more likely to get pregnant than those patients whose remaining embryos did not progress to blastocyst stage. So progression is a good functional test of viability and selection of embryos at day 5 of culture is a good tool to identify the embryos that can make it at least that far. Genetic testing for embryonic abnormalities that prevent pregnancy may be the key to identifying the embryos that make babies but those tests are still under development. Testing of embryo metabolism or metabolomics is another promising arena for developing new predictive tools to determine embryo viability.

The bottom line is that even with all embryo characteristics that have been proposed as predictors of implantation and pregnancy, there is not yet one test which accurately predicts which embryos will develop into babies. I am hopeful that a combination of existing evaluation methods and future analytical tests will one day identify those embryos that will produce a healthy pregnancy and child.

© 2010, Carole. All rights reserved.

593 Responses to this entry

  • Susie Says:

    Hi Carole, I just had a 3 day transfer of 3 frozen donor embryos. A transfer two years ago from the same set produced my son and a twin which sadly was lost in the first trimester. With my son and his twin we had two grade 1 8-cell embryos that were compacting. This time we have a grade 1 8-cell embryo, a grade 1 10-cell embryo and a grade 4 3-cell embryo (this last one not expected to grow further) but no compacting yet seen on the 8 or 10 cells. My question is whether these are essentially the same “quality” from your perspective as my first successful transfer? In other words is the absence of visible compaction on day 3 a bad thing with otherwise grade 1 embryos at 8 and 10 cells? And yes I compared pictures and they look the same to me, but I don’t know what compacting looks like exactly. Many thanks!!

  • Carole Says:

    HI Susie,
    I wouldn’t worry about lack of compaction on an eight cell embryo on day 3, this is typical. We usually see compaction on day 4, where all the cells sort of squish together like a mulberry and become attached to one another. These sound just fine. Good Luck!! Carole

  • Susie Says:

    Thanks so much for your super speedy reply! If I may, would you put the 10 cell in this same category?

  • Carole Says:

    Yes, the 10 cell probably would still fall in that category but I would hope to see compaction starting pretty soon after 10 cell. Good Luck! Carole

  • Susie Says:

    Carole, thanks so much for your reassurance! I am in fact pregnant, with a beta that went from 87 to 188 on days 11/13 post transfer. So it looks like one of the two took! Thanks again!

  • Carole Says:

    Fabulous news! Congratulations! 🙂

  • sm Says:

    Carole, I just received PGS results stating that we have 12 euploid (frozen) blasts, and the clinic states that, because they only freeze high quality (BB or higher) blasts, they select the embryo/s to thaw and transfer based on which one matured first. I asked to see the grading, anyway, just because I’m interested, but I’m curious what you think…would it make sense to ask them to select an embryo that reached blast stage a little later (all 12 reached blast in 5-6 days) if it was graded higher? One interesting fact is that the very first embryo that got to blast was actually anaploid, as were 4 others. The clinic has great success rates.

  • Carole Says:

    Hi sm,
    I think that any euploid blastocyst will likely have a good chance to implant. Since you have so many chromosomally normal embryos to choose from, trust your clinic to pick the one that, in their experience, will do best in their system. It is in their best interest to get you pregnant ASAP. Wishing you MUCH GOOD LUCK going forward! Carole

  • Rachel Says:

    Hi Carole,

    Great site! Very informative!
    I wonder if I could have your insight into my situation with slow developing embryos?

    IVF#1 2 follicles, 1 egg. On the morning of transfer (day 3) it was only a 2 cell, it went to a 4 cell in the afternoon before transfer. It was otherwise perfect in terms of cell size and fragmentation and got the highest grade. Negative result.

    IVF#2 12 follicles, 11 eggs, 5 mature, 4 fertilised. On the morning of day 2 there was one 2 cell and one that was just about to split, the other two where still just 1 cell. By late afternoon of transfer day (day 2) the leading embryo was still 2 cells and the one that was splitting went back to a 1 cell and another one had made it to 2 cells. Again both of these embryos where otherwise perfect and got the highest grade. Transferred both, negative result.

    Both cycles have been the long protocol, down regulation with buserelin injection and stims with gonal f. Both times I have ended up on my clinics max dosage of gonal f (450iu) as I respond poorly and my follicles stop growing/slow down (although I started at a higher dose the second time). We are both in our mid to late 20’s with no problems except for me having a low-ish AMH. I previously have responded well to clomid (2-4 follicles each time at 100mg) so I’m inclined to believe its the protocol/drugs that is the problem…..

    Any advice/insight would be very much appreciated 🙂

  • Carole Says:

    Dear Rachel,
    I am sorry you are having such a difficult time. In each case, the fact that the embryos did not reach 8 cell stage on day 3 is consistent with the negative outcome. It may be due to the stim propotocol but unless you have IVF data from the CLomid stims (these are usually combined with intercourse or IUI) it is comparing apples and oranges. By this I mean, that two follicles on ultrasound after clomid does not mean that the eggs would have necessarily progressed any better in the lab. Had you gotten pregnant on the CLomid cycles, it would provide evidence that at least one (maybe two) mature high quality eggs were available for fertilization. Having said that, I would probably get a second opinion from another RE who has very good pregnancy statistics. There are many variations on stim cycles and some REs are better versed on all the options available for difficult to stim patients. Good Luck!! Carole

  • Rachel Says:

    Thanks Carole, I did think that about the clomid but thought it was worth a mention anyway! Very few people I have found seem to have this as a recurring problem and I have begun to wonder if its an explanation for the ‘unexplained’ infertility. I have read lots about slower embryos having less chance of success but nothing about why they are slower or what is causing it/what can be changed. My clinic seem unable to explain it so I’m just trying to research as much as possible before our 3rd cycle.

  • Louise Says:

    Hi Carole
    On 7th August 2013 I posted that following an IVF cycle we had transferred a day 5 early blast. I was initially concerned that out of 8 fertilised embryos this was the only one available for transfer and it hadn’t yet developed into a full blast. I just wanted to feedback to you and others who may be reading this and in a similar situation that it worked and I’m currently 24 weeks pregnant. We couldn’t be happier!
    Thanks for your help and advice.

  • Carole Says:

    Congratulations!! Thanks for sharing your great news, I wish you and your family all the best in this New Year.

  • Malie Says:

    Hi Carol
    I just would like to ask regarding the embryo development.
    at my day 4 , there is only 9 cells ? is there still a chance for this to go to next stage on day 5 or 6 ( Blastocyst ) ?
    and be frozen for the next cycle?

    I am 37 years old.


  • Cally Says:

    Dear Carol,
    Thank you so much for the great blog! You are providing us all with so much instructive infos! I am very gratefull for the time you decided to dedicate to this!

    I am 36 years old, my husband is 43, it is our 2nd cycle of IVF. I got 14 eggs retreived and now I have 6 expanded blasts but only 1 is graded B/C and the other are C, then 2 regular blasts 1 B/C and 1B, all were frozen, no fresh transfer ( I think they said it was better to avoid any risk of OHSS)
    I was wondering how the grading influences the outcome, are there any studies about the Relationship between grading and chances of pregnacies/birth.
    I red in the thread that some clinic would only freeze “top quality” bastocycts, that would meen in another lab I would have found myself with maybe 1 or 2 embryos left -only instead of 6 if they had kept only the “good quality” ones? Am I getting my hopes up in wishing I can get 2 pregnancies out of this “batch?” Our dream is to have 2 kids so with that in mind, would it be safer to have another cycle before FET in order to “bank” more bastocycts before I get even older?….I would really appreciate your input even though I know the odds in that matter are highly unpredictable 😉

  • Carole Says:

    Hi Cally,
    I think you are in good shape and should try not to worry too much. I am sure that you feel disappointed that you didn’t have a fresh transfer but there is research showing that with the current freezing techniques (vitrification) you are often better off NOT to have a fresh transfer ESPECIALLY if your estradiol levels got very high, not just to avoid OHSS but also because implantation is less likely to work in an heavily estrogen dominant uterine lining. So this is probably all for the best. And I also would not stress about the grading. Yes, there are lots of papers about grading and implantation rates and little agreement. You can get pregnant with embryos that “scored” poorly and not get pregnant with embryos that scored high. The important point is that you have a lot of embryos that reached blastocyst stage, regardless of grade. You are 36 so that’s only a little beyond what is considered young in IVF so no, I wouldn’t bank a lot of embryos and delay an FET. I would try not to worry too much, distract yourself with other things and plan to have a great FET when your doctor thinks the timing is good. WIshing you MUCH GOOD LUCK!!! Carole

  • Mira Says:

    Dear Carol,
    Thank you so much for the great blog! Lots of great information. Thank you for helping us all!

    I am 39 years old, my husband is 52. We have had 2 IVF attmepts.
    During the first IVF, I’ve got 14 eggs retreived, out of them 10 matured, 10 fertilized. On day 5, 2 blastocist were transfered – grade 3AB and 3BA. No embrios were frozen.The result was negative.
    Durung the second IVF attempt, I’ve got 9 eggs retreived, out of them 5 matured, 4 ferlilized. On day 5, 2 blastocist were transfered – grade 5AB and 4AB. No embrios were frozen. The result was negative again.
    1.Do you think the negative results are due to a bad embrios or other reasons and what they might be?
    2. During the next attempt, do we have to consider a third day transfer?
    Thanks for your help and advice

  • Carole Says:

    Hi Mira,
    Thanks for your kind words about the blog. In answer to your question about why you have had a couple of negative results, one of the likely possibilities is that some of these embryos have an abnormal number of chromosomes (either more or less). This condition is called aneuploidy and is probably the main reason why older women (over 35) have difficulty conceiving. As we age, the final steps in the process to make a mature egg become less well regulated and more mistakes happen. Talk to your doctor about the possibility of pre-implantation genetic screening (PGS) which involves sampling a few cells from each embryo (embryo biopsy) and checking whether the embryo is genetically normal or not- then only transfer the normal ones. If you want to do this, be aware that it adds several thousand dollars to the cost of the IVF cycle. You’ll also want to know that the lab you pick is very experienced with the process. And there is a chance you might find out that all the embryos are abnormal which is upsetting.
    In order to get the benefit of the genetic testing, you have to let the embryos grow out to day 5, to get a good biopsy of several cells from a blastocyst stage embryo and you might have to freeze all the embryos while you wait for results from the genetic testing lab. I don’t see any advantage for you in doing a 3 day transfer. Your embryos are reaching blastocyst stage so that is not the issue. Talk to your doctor and perhaps get a second opinion from another doctor before you do another cycle to make sure you understand all the options and the risks vs benefits for each. Good Luck!!!

  • Kirsten Says:

    Hi, thank you for all this great information. I am a 43 year old with secondary infertility. In my previous ivf cycle 12 months ago, I had 31 eggs, 24 fertilised, most went to 5 day but only 6 were used in my treatment (2 x fresh, 2 frozen, 2 not survived freeze) I had successful implantation on the fresh but mc. Bfn on the fet. I am wanting to do aCGH this time but clinic is resistant and only offers 3 day biopsy. From what I’ve read 5 day biopsies are most reliable, least invasive and have best results. Do you agree? Are there any reasons a 3 day biopsy might be preferable. I expect a goid quantity of fertilised eggs this cycle so am of the mindset at the moment aCGH is a good course of action for me. Thanks so much for your insights.

  • Nicole Says:

    I am 29 with borderline FSH (10.1) but good AFC (30) and amh (3.97). I just failed my first fresh IVF with a SET of 1 – 3 day grade A(A being the best my clinic gives) 8 cell compacting embryo. My estrogen was 3,300 on the day of trigger. 22 embryos were retrieved, only 8 were mature. ( I only stimmed for 7 days)
    Out of my 8 embryos,
    1 – 8 A compacting (transferred)
    4- 8A
    All 7 remaining embryos froze. We just thawed two – 8A. They were thawed the afternoon before transfer. Both thawed perfectly with all cells still in tact. One was an early morula, the other was a 12A.
    The RE that did my FET said that we have a great chance that both will stick due to my age and the quality. but then she said that we had a 40% chance at pregnancy. Does that seem right for the stage of the embryos? Also, is it possible that since I have only been pregnant once ( ended in a blighted ovum may 2013) that something could be wrong with my embryos? I have since had another HSG and a saline sonogram and everything checked out fine.

  • Carole Says:

    Dear Nicole,
    For your age, even with a few borderline lab results, it seems that the stimulation might be less than ideal because you had fewer mature eggs than would be expected. In the follow-up appointment with your doctor, ask her to explain what she might do differently in a second stim cycle. In our program, when the stim was so short (7 days) the docs would do what they could to coast the patient on lower doses so that they could get a few more days of stim in before the trigger. My guess is that your doctor would plan to adjust the protocol to get more mature eggs next time with a few days more stim. If she plans to use the exact same protocol again, you might benefit from getting a second opinion from another doctor. You can also ask your doctor what she bases the 40% pregnancy rate on? national statistics? Her own patient’s statistics? To get an idea what average rates are for women your age, look up pregnancy results for all the programs in your state at wwww.sart.org. Good Luck!! Carole

  • Nicole Says:

    Thank you so much for your quick reply. I did ask my RE, he said that he wasn’t sure what happened but he wasn’t concerned due to my 8 embryos looking so good. I assumed it was due to my protocol. I started the cycle with an AFC of 30+. On the day of trigger, I had 4 follicles @ 17nm, the rest were between 9 & 15mm. After my fresh cycle failed, I met with him as I just needed answers, I just drained my savings for the one thing in life that would make my life complete and I just needed reassurance. His reply was, there’s nothing wrong kid, you’ll get pregnant. We’ll just put two in next time. So that’s what I did and now I wait. Are you a fan of ESET or do you favor transferring 2?

  • Carole Says:

    You’re very welcome. I do favor transferring one at a time, generally speaking, because if the conditions are right, and the embryo is healthy, you will get pregnant. Twin pregnancies are the largest risk factor attributed to IVF. I wrote a blog about this topic based on the latest findings from the ASRM meeting in October, 2013. You can read it here: http://fertilitylabinsider.com/2013/10/asrm-2013-update-art-safety-the-twin-effect/ The good news is that even if you have twins, obstetricians have more and more practice from IVF twins so it is usually manageable, even if not ideal.
    Wishing you Much Good Luck! Carole

  • Samantha Upchurch Says:

    What a great blog! My question is this: I am 42 1/2 and on my 7th round of IVF for unexplained infertility. On round 3 we had a wonderful healthy baby boy, and now want a sibling. We are really running out of money and time so want this next round to work. Our clinic does Eeva which we have used on our last 2 rounds, got 6-8 blastocysts each time, implanted 2 each time but never got pregnant and never had any to freeze. For financial reasons our next round will be our last. Should we continue with Eeva or would PGS /array CGH be more likely to give a better outcome given my age? (The latter will be more expensive but it’s worth it f it’s better). I can’t find anything on relative outcomes of these 2 techniques for women of my age, perhaps because they are both quite new? Thanks so much

  • Carole Says:

    Dear Samantha,
    I think the field is still trying to figure out the role of time lapse with or without PGS/array CGH. I am not sure whether eeva alone is a substitute for aneuploidy testing. At 42.5 years old, your probability of pregnancy is most likely reduced because many of your eggs would be expected to be chromosomally abnormal (aneuploid) which is what the PGS tests are for. The problem is that you may find that all your eggs in a particular cycle are aneuploid which would explain why you are not getting pregnant but won’t help you achieve the pregnancy you want. You have to decide whether gambling on another round of IVF with or without PGS will be useful to help you decide about next steps. How many more IVFs can you afford to put yourselves through? At the end of the next one, if you aren’t pregnant, that cycle should at least have some utility to give you information about next steps. I am not sure any of this is really helpful advice for you. Some people in your shoes would try egg donation, embryo donation or regular adoption, all of which are statistically better bets for a sibling for your son. But I understand that these options aren’t necessarily the right choices for you, I just hope the next cycle is more than just another cycle— but that it gives you more information, even if it does not succeed in getting you pregnant. Wishing you MUCH Good Luck!! Carole

  • Samantha Says:

    Dear Carole,
    Thank you so much for your very quick reply! I do realise that the odds are stacked against us and that aneuploidy is probably the reason why we’re failing, but we’re not yet ready for egg donation although we may consider it in the future. I was just thinking there’s less of a ticking clock on egg donation so wanted to throw everything at having our own genetic child first before I hit 43. I understand that on average at my age about 90% of my eggs are likely to be aneuploid, (ie 1 in 10 will be ok) so I was thinking if I do enough rounds and we have enough info on the eggs then maybe we could find that one egg. We can afford one last round with our own eggs, then if that fails we’ll pause to earn a little more, then go for a donor egg (as I could do that any time in my early 40s wheras I wouldn’t expect my eggs will be useable beyond 43). So for the best chance on this last round would you suggest PGS? Or do you think Eeva would do as good a job of finding the elusive egg (if indeed there is one) at less cost? Also do you think there is any merit in trying to improve my egg quality eg with melatonin as I believe there is a small scale Japanese study suggesting this might be helpful?
    Thank you so much

  • Alisa Winslow Says:

    Hi Carole:

    I am amazed at what you are doing, you clearly have a passion for your profession. Thank you.

    I am 38 and working with CCRM. Today we transferred our last CCS normal embryo. 100% of cells survived and the embryo was a 5BB in January when it was frozen but today had completely hatched, a 6BB. The embryo was a day 6 blast prior to the second freeze. It had to be thawed in January because the first CCS test was inconclusive.

    Transfer was at 11:45 and the thaw process started at 9 AM. We were reassured by the hatching and also the cell survival, but I am very concerned by the very slow expansion. I am also confused about how it would hatch without expanding and would appreciate your thoughts on that.

    When I asked the doctor he would not give me any stats and deferred to the embryologist who had already instructed me to ask the doctor for his thoughts. So… now I am relying on the internet for reassurance and am not finding much.

    I have a 2 year old daughter from our 34 year old embryos but it took us 4 cycles and 11 embryos to get her (they were 2 day transfers though). We switched to CCRM after 4 of our 2 day embryos failed to survive the thaw from my old clinic.

    At CCRM we had 12 retrieved, 11 fertilize, 5 make ti to blast, and 3 CCS normal. 2 of the normals took until day six to make it to the blast stage and one of those was transferred today.

    Two of the embryos were transferred in January and didn’t make it. This one is our last.

    Here is a picture,


    What are your thoughts? And can you help explain how it would hatch without expanding?

    I am off to go be positive… but would love your insight and expertise.

    Thanks in advance!

  • Alisa Winslow Says:

    Hi- I tried to post earlier but it didn’t show up here. Wondering if it was because I mentioned my clinic.

    Curious about my one remaining CCS normal embryo. It was a 5BB when frozen. Had to be thawed for testing after the first test was inconclusive. So today at transfer was a second thaw.

    Embryologist said it appeared we have 100% cell survival, but expansion was slight. Embryo was thawed around nine and transferred at 11:45/ It had also hatched and was rated a 6BB.

    I am confused as to how it hatched without expanding.

    Here is a photo. Appreciate any guidance you can give because I did not receive much after talking to both the embryologist and the doctor.

    I have a two year old daughter from my 34 year old cycles (two full cycles, 4 transfers, 11 embryos transferred). This cycle is my second full cycle for baby number 2. 2 CCS normal embryos failed to implant back in January.


    Great resource. THANK YOU!

  • Alisa Winslow Says:

    OOPS… and now mu earlier post is magically there. Sorry!

  • Carole Says:

    Hi Alisa,

    The picture you sent was a little blurry, but I wouldn’t get too distressed about the “hatched without expanding” comment. Embryos must expand in order to hatch but just because we didn’t get a picture of it, doesn’t mean it didn’t happen. Embryos can expand, and then collapse, only to reexpand and hatch. Or hatch and collapse. We collapse embryos deliberately before freezing sometimes and they re-expand at thaw. I think your embryo did expand but it may not have been documented or observed. Good Luck with your transfer! Carole

  • Alisa Winslow Says:

    Thanks so much Carole. I wonder if it hatched at the time they thawed it for the second go at the biopsy for CCS testing and just didn’t document it. Either way I hope the little guy expended and is settling in now.

    Appreciate your quick response. This is a terrific and informative site.

  • Lara Says:

    Hello Carole.

    Thank you for all of your very informative responses. My husband and I have done our first cycle of IVF. I have unexplained bilateral tubal blockage, after consisting our son naturally.

    I was on 300 gonal-F and 300 Menopur a day because I have an AMH level of .8. They were able to retrieve 15 eggs, 12 were mature and healthy. Husbands sperm appeared good on all levels, but only 1 egg fertilized. We had a day 3 transfer of a grade a, 12 cell embryo and did the assisted hatching to try to help it as much as possible.

    I have three questions please:

    1. What do you believe would be the possible causes of only 1 egg fertilizing?

    2. How often in a lab do you see a 12 cell day 3 embryo make it to the Blastocyst stage?

    3. With assisted hatching and 12 cells at this stage, would you expect the hatching to occur earlier then a 6 or 8 cell on day 3?

    Thanks so much for your time!


  • Carole Says:

    Hi Lara,
    I am sorry you are having such a hard time. Re: your first question: This very low fertilization rate can be due to at least 3 possible issues – in no particular order- lab technical error or incompetence, immature eggs or a problem with either egg or sperm that was not previously detected (missing sperm or egg receptors). You don’t mention whether it was regular IVF or ICSI but such a low fert rate is more common with regular IVF. Your second question- a twelve cell may be dividing a little too rapidly to be normal-embryos with either faster or slower division rates are more likely to have an abnormal chromosome number (aneuploidy) and so are more likely to stop growing before blast stage.).Re: Q3: Not sure what you are asking re day 3. If it takes longer for a blast to get to blast stage, it would probably also hatch later in time than it’s faster sibling. If you are talking about assisted hatching, not sure what you mean- the tech can hatch the zona before blast stage or any time at blast stage. Clinic protocols vary by clinic. Good luck! Carole

  • Linda Says:

    Hi Carole,

    I am over 40 and struggling to make blasts. I just did an FET of my lone 5BB and it was not a success. I am contemplating whether I should do day 3 transfers or even day 2.
    Two questions:
    1) I am wondering why day 4 transfers are not more commonly done as opposed to day 3, since you are closer to the ideal location of where the embryo would be naturally in utero, but not risking not making it to blast.

    2) I doubt any studies have been done on this, but I started wondering about the “one-size-fits-all” sequential culture media that is used to culture to blast and wondered if older women’s embryos might have different “biochemical” requirements than someone in their 20s and 30s (i.e. might women over 40 need more customized sequential media due to the differences in our bodies biochemically (and eggs and resulting embryos) compared to our younger cohorts) or is an embryo just an embryo regardless of age other than aneuploidy issues? I thought it was an interesting hypothesis anyway…

    Thank you for all that you do with this website to enlighten, calm and teach us!

  • Carole Says:

    Hi Linda,
    I am sorry you are having such a difficult time. There are several reasons why labs persist in offering day 3 transfers: 1) they can’t reliably culture to blast stage, 2) the patient only has 1-2 embryos which would typically be transferred so there is no need for longer culture to pick the 1-2 best among many and 3)it is more labor intensive to culture embryos for 2-3 additional days in culture. It is possible that culture medium could be optimized for some populations of patients but that has not been shown scientifically. Frankly, we are still learning about what embryos need. I don’t know if day 2 or day 3 transfer would be helpful in your case. If the problem is due to aneuploidy, the time in culture would probably not make a difference in the final outcome. If there is another cause related to metabolism, maybe shorter incubation in suboptimal culture may be better. If IVF is covered by your insurance plan or you can afford another cycle easily, you might contemplate an additional cycle to feel you have tried everything but otherwise, it might be time to consider all the possibilities available to you to build a family including egg donation, embryo donation or even adoption. I wish you all the best going forward!! Carole

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  • Sarah Says:

    Hello: yesterday we did a 3-day transfer of one 5 cell and one 11 cell embryo. I am 43. Do those sizes (I.e. Not 8 cell) indicate likely failure? We are at an excellent center. They retrieved 19, 16 were mature, we fertilized with icsi (non obstructive azoospermia), 3 fertilized, only the two we transferred made it it to day three. If this cycle fails, do those number indicate anything about future chances in another ivf (obviously age is against me). Is producing 16 mature eggs a positive (if at my age 1/10 eggs is bad does being able to produce around that number of mature eggs make it more possible to find one useable one)? For women in my age range is there data indicating what are the characteristics of the few successful ones? Thanks in advance.

  • Carole Says:

    Hi Sarah,
    While it is true that cell numbers much lower or higher than 8 cells on day 3 bode less well, it is too soon to give up on this cycle. The more mature eggs you produce, the better your odds that one or two might be normal, but as you said the odds are against you, based on age alone. As far as selecting the successful ones, the most common approach is to use pre-implantation genetic testing for aneuploidy and only transfer those embryos with a normal chromosome number. Consider asking your physician about this option. Other alternatives are donor egg or donor embryo. Wishing you much good luck going forward.

  • Heather Says:

    HI Carole,
    Love your blog! I’m hoping you can give me some advice. I had a IVF-ICSI cycle with TESE sperm when I was 28 (my husband has idiopathic obstructive azoospermia). I had 2 4AA blasts transferred, both took but I gave birth to one baby (I had a vanishing twin at 7 weeks). I have four blasts frozen from this cycle (4AA, 4AB, 4AB, and 4BA). Unfortunately they were frozen using the slow freeze technology. My dr says the thaw rates are about 80% and the success rate is 35- 40% for FET. I have a FET coming up and I’m not sure if I should transfer one or two embryos, especially since they were fertilized with TESE sperm (higher chance of chromosomal abnormalities?) and because they were frozen using the slow freeze method. What would you recommend? Thanks.

  • Carole Says:

    Hi Heather,
    I think that you’ll have more information on the day of the thaw because the embryologist typically evaluate them for their post-thaw quality- which is especially important with the older slow freeze technology. Here is a link to the ASRM practice committee opinion on how many embryos to transfer http://asrm.org/uploadedFiles/ASRM_Content/News_and_Publications/Practice_Guidelines/Guidelines_and_Minimum_Standards/Guidelines_on_number_of_embryos%281%29.pdf Basically, ASRM recommends 1 embryo for women under 35 with a favorable prognosis and 2 for those with a non-favorable diagnosis. The favorable vs non-favorable variable allows for your physician to include other particulars about your case that might argue for 2 embryos. Unless, there is some reason why you think you can’t afford another FET or can only have one FET procedure, I would probably thaw one and transfer it. If this FET doesn’t work, thaw one and transfer one in a second cycle, and so on. You are more likely to avoid having twins in this one at a time approach. Twins is the biggest obstetrical risk from IVF and carries the potential for trouble for both mother and child. In your case, you have already proven that two of two transferred can implant so the risk of twins is quite real. You need to balance the obstetrical risk of twins against the risk of not becoming pregnant from any single transfer attempt. You don’t need to decide now. At time of thaw, you will have more information about embryo quality post-thaw and can discuss the best option for transfer at that time with your doctor. Good Luck!! Carole

  • SoHopeful Says:

    Hello Carole,

    Thank you so much for your devotion of time to so many anxious and hopeful individuals and couples. Here’s our question:

    Our day 3 lab results were
    2 3-day morulas
    2 good 8 cell
    1 good 7 cell
    1 fair 10 cell
    And a few rated poor

    The doctor asked us which we wanted to transfer, and not knowing we asked him to choose. After consulting with the embryologists they chose to transfer the two morulae.

    Now I’m looking online and finding that 3-day morulae may not be auspicious. Did they make a mistake?

    Thanks so much for your insights.

  • Nam Says:

    Hi Carole,
    I stumbled upon this blog and am glad I found it. You are doing a wonderful task by helping us anxious souls!
    History: I have had 2 m/c and 1 ectopic, trying naturally. So we decided to go for IVF. This is my first IVF/ICSI cycle. !3 eggs retrieved, 11 mature and 9 fertilized.
    Today is day 3 after retrieval. Here are my results:
    2 – 9 cells
    4 – 8 cells
    1 – 7 cell
    1 – 5 cell
    1 – compacting

    We have been advised day 5 transfer. I was thinking that the two 9 cells or the compacting embryo that are “overachievers” are the best to transfer, but on more reading I don’t think this is the case.
    What according to your opinion looks good at this stage to transfer? (I understand things change every day so thigns may look different tomorrow).

    Also, give we have had recurrent pregnancy losses, would you recommend PGD to us? My RE has done all thorough genetic testing and doesn’t think we need it. I am very scared of going through another loss.

  • Carole Says:

    Hi Nam,
    With our new understanding from image capture of embryo development, it seems that being exactly on time (an 8 cell on day 3) might be a better indicator of implantation potential than being faster developing than expected (compacting morula). But you’ll know more on day 5 and then the quality of the blastocyst can also be evaluated. Regarding PGS testing for aneuploidy- that is very much in debate right now. In theory, it should be very effective in preventing recurrent miscarriage but in practice, it has not always worked out that way. Whether that is due to a problem with PGS or it’s implementation by the lab is unclear. You are in pretty good shape right now. Take it one day at a time and be hopeful! Wishing you much good luck!! Carole

  • Carole Says:

    Dear So Hopeful,
    I assume that the other stage appropriate embryos were cryopreserved so even if this transfer does not result in pregnancy, you have future chances ahead of you. Having said that, I wouldn’t assume you won’t get pregnant this time. The embryologist most likely chose the embryos that they expect to do best in their lab under their conditions so be hopeful. Wishing you all the best!! Carole

  • Danam Says:

    Is a 10 celled embryo good. I did a late morning 3 day transfer.

  • Carole Says:

    Hi Danam,
    !0 cells on day 3 would be about right- especially later in the day. Wishing you much good luck!!

  • Tamika Says:

    Hi Carole,

    I am so grateful I stumbled across this blog. I’m 42 and had my 1st IVF in March, fresh cycle of 2 4th. The results were negative. Shortly after I learned about embryo donation and was grateful because my husband and I could not afford another round of IVF and we could not afford purchasing eggs. We were offered 3 2pn embryos which all 3 thawed successfully. The donor is a 31 year old female and 29 year old male who completed their family and donated the remaining 3. Before the cycle my RE discovered that a fibroid that she saw previously was entering the uterus, we postponed cycle to remove the fibroid. On day three I went in for a scan because there was fluid in my uterus and my RE suggested PIO would obsurb the fluid and if none was found on day 3 we could move forward. She found no fluid and said we could go either way, transfer day 3 or wait to day 5. I was already so excited I choose to move forward. Today is day 6 for my embies and the one graded as an A- stopped developing at the morula stage. I am ready so much now on how 5th is the preferred practice and wondering if I made an error…deep sigh. I’m out of money so this is our only hope.

  • Tamika Says:

    Oh forgot to mention the 2 transferred were grade A

  • Tamika Says:

    Geese sorry also forgot to mention the 2 A’s were 8 cells, no fragmentation aND the A- was a 7 cell a little fragmentation.

  • Carole Says:

    Dear Tamika,
    I think you are in good shape with the embryos you transferred. Their appearance and the score is just part of the picture and no one can accurately predict what will happen. Just because one left behind did not progress does not mean that the ones you transferred won’t be fine. Try not to worry too much and distract yourself with something else while you wait. Wish you Much Good Luck for your test! Carole

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