Embryo stages, progression and pregnancy outcomes

November 10, 2010Carole 561 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.

561 Responses to this entry

  • Carole Says:

    Hi Louise,
    I don’t really understand why they are waiting until day 6 to biopsy. Some programs wait until the embryos are already hatching for biopsy because they don’t have the skills to open the shell and pull out a few cells. There may be another reason they are waiting. As always, you should always feel that you have the right to ask these questions of your IVF team. If their embryo freezing/thawing skills are good, you will probably be in decent shape to have one or two attempts at pregnancy. Good Luck!!! Carole

  • Ingrid Says:

    Dear Carole,
    I need to have some information.
    I had a Fivet in a centre in Prague where they transfer to me 2 morulas even if they told me that they were two blastocysts. I had beta zero.
    Now I still have a morula and an early blasto ( grade 1) that I decided to transfer in another centre because I don’t trust anymore in my previous centre.
    The new centre was really surprised that they thawed these embryos at this stage! The new center was pretty honest and they told me that they thaw only XB or HB, i can go and try but they seemed not to be very optimistic. They told me that they dethawed my embryos the day before and they observe them.
    What do you think? I’m thinking every day if to go and to take these embryos or to start with a new eggs donation.
    Tks a lot for your help

  • Ingrid Vola Says:

    Dear Carole, I need to have some information. I had a Fivet in a centre in Prague where they transfer to me 2 morulas at day 5 ( it was a DE) even if they told me that they were two blastocysts. I had beta zero. Now I still have a frozen morula and an early blasto ( grade 1) that I decided to transfer in another centre because I didn’t trust anymore in my previous centre. The new centre was really surprised that they frozen these embryos at this stage! The new center was pretty honest and they told me that they only froze XB or HB, i can go and try but they seemed not to be very optimistic. They told me that they will thaw my embryos the day before and they observe them. What do you think? I’m thinking every day if to go and to take these embryos or to start with a new eggs donation. Tks a lot for your help Ingrid – See more at: http://fertilitylabinsider.com/2010/11/embryo-stages-progression/#comments

  • May Says:

    Hi Carole,

    Thanks for the informative article. I had my retrieval yesterday at 9:30am and I received my day 1 fertilization report this morning at 9:30am (exactly 24 hours after). Based on the 13 eggs we retrieved, 12 were mature. We did ICSI – 4 were fertilized as of this morning. Another 4 were fertilized but “not at 100% yet”. The nurse said they will be put back into the incubator with the rest of them to see if it will continue to progress. My question is… how likely will these 4 embryos that are not 100% fertilized 24 hours after retrieval continue to grow normally? The nurse didn’t say that these were abnormal, just that they fertilized but not at 100% at the time she called me. Any insight you can give is appreciated. Thank you!

  • Carole Says:

    Hi May,
    I don’t understand the “not at 100% yet” comment either. Your egg is either fertilized or not fertilized. Since ICSI was used, there should be pretty synchronized fertilization and a fairly tight window to see all fertilized, especially since all but one were mature at time of ICSI. You have every right to ask them to clarify what they mean. Good Luck!! Carole

  • Carole Says:

    Hi Ingrid,
    It might be worth it to let the new lab thaw and observe the embryos with a plan to transfer them if they continue to grow. You can always start with a fresh cycle if this does not work. Good Luck!! Carole

  • Ingrid Says:

    Dear Dr. Carole,
    thank you for your prompt reply.
    I forgot to ask you if you suggest me a PGD in my next DE fresh cycle or not!
    I had a natural pregnancy at the age of 43 but M/C ( 9+4) due to chromosomal abnormalities
    DE Beta zero
    DE ( frozen embryos) ectopic pregnancy
    Natural pregnancy at the age of 45 M/C due to Trisomy 18.
    The caryotipe of my husband is perfect. Anyway, Do you think it’s better to do also the Array -CGH or this is not the case?
    Thank you so much for your help!

  • Carole Says:

    Hi Ingrid,
    At 45, your risk of producing eggs- and thus embryos- with chromosomal abnormalities is very high so yes, PGD is probably a good idea- if you are using your own eggs. Array-CGH is better than older methods. You may, however, find that all your embryos are abnormal which would be upsetting but if it gives you some insights into the possibility of pregnancy with your own eggs, it might be worth it- just so that you can try something else . IF you are using donor eggs from a young donor, PGD is generally not ordered, because the risk of chormosomal abnormalities are much lower. Good Luck!! Carole

  • Ingrid Says:

    Dear Dr Carole,
    Ref my message dated 29th february, ref the early blasto and the morula, I went in the new centre for the Ket.
    So, they told me that they thawed the embryos the night before and that they were viable! They gave to me the 15pc of chances with my early blasto that became grade 2 and the compacted morula! I was sceptic about the CM but the Dr suggested me to transfer also it!
    In the paper they gave to me there is written: day 5 1EB/2 1 CM/3! What do you think? Do you confirm the 15pc of the center?

  • Carole Says:

    HI Ingrid,
    Yes, we have had pregnancies with compacted morulas, as long as they are stage appropriate- that is – they become compacted morulas on or about day 4 to day 4.5. IF they are much slower or faster than that, it suggests a problem with the embryo. The chances of pregnancy arising from a given embryo is dependent on a huge numbers of factors, egg quality, embryo quality both contribute to embryo quality. The embryo needs to be healthy and the uterine environmnet needs to be able to accept the embryo so there are a lot of moving parts. On top of all that, the technical staff must be capable and well trained because there are opportunities to screw up every step of the process. So their 15% is based on their experience with their patients at their clinic. I think 15% is low but it may very well be what you can expect based on their past data with patients similar to you. Also, this is the second of two embryos transferred; the other is an expanded blast so that one alone should give you a decent chance of pregnancy. Good Luck! Carole

  • Ingrid Says:

    Dear Carole,
    tks for your reply.
    The other was an early blasto. Anyway, my beta were zero.
    I will try with a fresh cycle in this new center.
    I know that they only transfer Exbanded blastos or in hatching blastos.
    I hope it will be the good opportunity.
    Tks a lot for your suggestions and your patience

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