Understanding the Different Stages of Embryo Development

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Embryo development is a delicate, multi-stage process. From the moment of fertilization, growth follows a pattern, one that can be influenced by many factors. With in vitro fertilization (IVF), growth closely mimics what happens in the human body during a natural pregnancy, when an embryo grows from a single cell to the blastocyst stage. However, rather than occurring in a fallopian tube or the uterus, the initial phases take place in a petri dish in an embryology lab, and typically in a fertility clinic while being monitored by a trained embryologist.

What happens during these first few days – the rate of growth and the changes in structure – directly impacts whether an embryo will result in a healthy live birth. Having a better understanding of how embryos grow during the IVF process can help patients understand what a desirable outcome looks like and whether their embryos have achieved certain milestones.

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What is the structure of an embryo?

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The word embryo is the term given to the first nine weeks of human growth. After that – and for the duration of pregnancy – the word fetus is used. When the sperm fertilizes the egg (oocyte), this forms a zygote, the first embryo cell. Initial growth happens quickly, with this single-celled zygote dividing into a 2-cell, then 4-cell, then 8-cell embryo by Day 3. On Day 4, it is a compacted ball of cells called a morula, which becomes a blastocyst on Day 5 assuming that growth continues.

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A blastocyst is a fluid-filled structure with three components: a fluid-filled cavity, an inner cell mass (ICM), and a trophectoderm. The ICM will ultimately become the embryo, while the outer wall layer, the trophectoderm, will become the placenta. At this point, optional genetic testing is available via an embryo biopsy. It is typically the trophectoderm's cells that are collected for this testing -- not the ones from the inner cell.

A glycoprotein (carbohydrate linked to a protein) shell called the zona pellucida surrounds the embryo. The embryo will hatch out of the zona pellucida at the blastocyst stage. In an IVF cycle, the zona can be pierced by a laser or with a special acid solution during a process called assisted hatching to help with this step.  Hatching enables implantation by allowing the trophectoderm to come into contact with the lining of the uterus (endometrium).ⁱ

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How are blastocysts developed in the lab?

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Embryo development in IVF occurs in a clinic’s embryology lab. The embryos grow and mature in a petri dish that is marked with a unique identifier, such as a barcode, and with safety measures in place to ensure embryo identification is maintained. Embryologists oversee this process, and their skill and expertise are important to the success of a cycle.ⁱⁱ This is especially the case for technical processes such as embryo biopsy and intracytoplasmic sperm injection (ICSI)ⁱⁱⁱ in which a healthy sperm is injected directly into each egg to help promote fertilization

The petri dish is filled with culture media, a fluid which provides optimal nutrients^iv for developing embryos, and then housed in an incubator for 2-to-5 days as the embryo(s) develop. Numerous studies have investigated the importance of effective culture media,^v such as its impact on blastocyst yield thus, IVF success rates in IVF.^vi,vii‍

Incubator conditions are intended to mimic conditions in the uterine tubes and so it is critical that the embryologists consistently keep temperature, oxygen, carbon dioxide, pH, and humidity in the incubators at optimal levels because embryos are extremely sensitive.^viii,ix Even the air quality in an embryology lab can impact success rates, and most embryology labs have very specialized air filtering systems.  

Most incubators now have individual chambers so that embryos are less disturbed if neighboring embryos are removed for monitoring and transfer. In most labs, in order to check on the growth of the embryo, the petri dish must be removed from the incubator and studied under a microscope. Sometimes this is done daily, while other labs only provide a report on Day 5.  

In contrast, some incubators use “time-lapse monitoring,” which involves incubators with transparent bottoms and cameras underneath to monitor embryos on a screen, so that they do not have to be removed for microscopic evaluation.^x The evidence is mixed as to whether time-lapse monitoring improves success of IVF.^xi

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What is the IVF process after egg retrieval? Stages of Embryo Development

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Though changes happen quickly, they do not all happen at the same rate. The following sections outline the overall framework of what happens during the first several days of IVF.

What happens from Day 0 to Day 1?  

Fertilization and 2 pro-nuclei (2PN) Zygote formation

Egg (oocyte) retrieval, also called oocyte pick-up or egg aspiration, is considered Day 0. In most cases, there is a mix of mature eggs (eggs in a stage of genetic development called metaphase of meiosis II, MII), immature, and post-mature eggs. In some labs, immature eggs can be matured in the embryology lab in a process called in vitro maturation (IVM).

The mature egg(s) will either be co-incubated in a petri dish with sperm for insemination (IVF) or inseminated by direct injection of sperm into the egg (ICSI). During traditional IVF, the sperm swim and penetrate the egg in the petri dish; therefore, adequate sperm parameters (i.e., count, motility) are recommended. ICSI involves the embryologist injecting a single sperm into each mature egg. ICSI was originally designed to address poor sperm motility, morphology, and/or count, but many labs now use ICSI as the standard for all cases.^xii

The next day (Day 1), typically 16 to 20 hours later, embryologists check for correct fertilization by looking for two pronuclei (2PN, DNA from the sperm and egg) under the microscope.^xiii Eggs that failed to fertilize during IVF or ICSI will be discarded. In addition, eggs that fertilized incorrectly, such as those with no pronucleus (0PN) or only one pronucleus (1PN), will be discarded by many labs, despite some evidence that these zygotes may successfully result in live births.^xiv

If fertilization is successful, a zygote (fertilized egg) will have been formed - the zygote is the first cell of the newly formed embryo. Although grading is possible, most labs do not grade zygote quality.^xv

What happens on Day 2?

2-to-4-Cell Cleavage Stage

At this stage of development, the zygote will ideally divide (called cleavage at this stage) into two cells and then cleave again into four cells. It is optimal if the embryo’s cells will be approximately equal in size, with no irregular structures visible, and there will be low fragmentation (i.e., few cell fragments have broken off).^xvi Many labs in the United States will not disturb a Day 2-stage embryo, but transfers on this day occur in some countries.

What happens on Day 3?

8-Cell Cleavage Stage

Many labs will provide a Day 3 update on the development of a patient’s embryos. When they view the developing embryos under the microscope, embryologists ideally want to see an embryo with seven to nine cells (eight is ideal) on Day 3.^xvii Embryos with fewer or more cells at this stage are considered less likely to be viable. Those embryos with fewer than seven cells may be developing slowly, or they may have arrested, meaning they have stopped developing. Embryos that have arrested will likely be discarded. Embryos with more than nine cells at this stage are defined as “accelerated,” and this may also be associated with poor viability.^xviii

Day 3 is an important milestone for an embryo, because this is the point at which embryonic genome activation (EGA) occurs.^xix Up until EGA, the maternal genome, which comes exclusively from the egg, directs the embryo’s development. Many embryos will arrest (stop developing) after Day 3 because they fail to develop once the embryonic genome is in control. This is most often because the embryonic genome is not genetically competent. Thus, it is normal to see a significant drop off in the number of embryos after Day 3 as some will fail to make this transition and arrest.^xx

Some embryologists grade Day 3 embryos based on cell number, fragmentation, and cell symmetry.^xxi There is debate about the value of embryo grading on Day 3. For example, a 2019 study showed that ongoing pregnancy rates were not significantly different in grade I, II and III embryos (66 percent, 66 percent, and 64 percent, respectively).^xxii In contrast, a study of 423 Day 3 fresh embryo transfers showed significantly different implantation rates for grade I, II and II embryos (42 percent, 40 percent, 29 percent, respectively).^xxiii Overall, some published studies found poor predictive value of Day 3 grading, while others show it is a useful selection tool.

Embryo culture methods, such as culture media, vary between labs. In some cases, the fluid in which the embryos are developing is changed on Day 3 (two-step or sequential culture media). Other labs use a one-step culture medium that is not changed. It is currently unclear whether changing the culture medium is beneficial or could disturb the embryo’s development.^xxiv,xxv

In some clinics, the embryo may be transferred into the uterus on Day 3. While there is an increasing trend toward Day 5 blastocyst transfers, Day 3 transfers are still relatively common, particularly in cases with few embryos per cycle and/or a known low blastocyst development rate during prior cycles. In addition, Day 2/3 cleavage-stage embryo transfers may be preferred in countries with specific restrictions on embryo culture. A 2016 meta-analysis, which combined results from five randomized controlled trials, showed that the cumulative pregnancy rate for cleavage-stage (Day 2 or 3) transfers was similar to blastocyst stage transfers (Day 5 to 6).^xxvi However, the quality of the evidence was rated as low, and additional studies were suggested to confirm the results.  

What happens on Day 4?

Morula Stage

If growth to this stage continues correctly, the cells compact into a morula, which is solid ball of approximately 16 to 32 cells.

Embryo transfers on Day 4 are not common and are typically performed for logistical reasons. For example, a clinic may complete an embryo transfer on Day 4 if they will be closed on Day 3 or Day 5, or if the patient has a conflict. This should not cause concern, as evidence shows that Day 4 transfers generally have similar success rates to Day 5 transfers. One study indicated the clinical pregnancy rates of Day 4 and 5 transfers were 49.5 percent and 51.9 percent, respectively.^xxvii

What happens on Day 5?

Blastocyst Stage

By Day 5, most morulae have reached the blastocyst stage with a size of 70-to-100 cells. The inner cell mass is now distinct from the fluid-filled cavity in its center, and the trophectoderm securely surrounds them both.^xxviii The stages of blastocyst ("blast”) growth are typically categorized as: early blast, blast, expanding blast, hatching blast, and hatched blast.^xxix

Approximately 30 to 50 percent of correctly fertilized eggs (2PN zygotes) are expected to become Day 5 blastocysts.^xxx Of these, a lower percentage are deemed “good quality” blastocysts based on the blastocyst grading system. In 2017, the European Society of Human Reproduction and Embryology set aspirational benchmarks, suggesting embryology labs should strive for blastocyst rates of 60 percent in the future.^xxxi For now, the recommendation is that labs aim for a 40 percent blastocyst rate as a best practice goal.

Blastocyst grading is intended to categorize an embryo's quality as "good, fair, or poor", which can be used to prioritize which embryo(s) to transfer first and help predict likelihood of success.^xxxii,xxxiii However, grading is subjective and has high inter- and intra-embryologist variability.^xxxiv Overall, blastocysts are graded based on their stage of development (degree of expansion), as well as the quality of their structure (morphology).

A number is assigned according to the stage of blastocyst expansion (i.e., early, expanded, hatching), and two letters are assigned for grading.^xxxv,xxxvi These are assessed by an embryologist under a microscope. For example, a “5AA” blastocyst refers to hatching (5) blast with a tightly packed ICM (A) and a good quality TE forming a cohesive covering layer (A).  

Better blastocyst grades may be correlated with higher IVF success rates.^{xxxvii,xxxviii} For example, a 2021 study determined implantation was more likely with good-quality blastocysts (79.8 percent) rather than poor-quality ones (48.1 percent).^xxxix However, the evidence is conflicting, and other studies show that blastocyst grading is not a consistent, dependable way to predict clinical pregnancy or live birth.^xl,xli  

What happens on Day 6 and Day 7?

Blastocyst Stage

During in vivo (in the body) fertilization, as in couples who are conceiving at home, Day 6 of embryo development is when the blast has moved into the uterus and begins hatching out of the zona pellucida in preparation for implantation on Day 7.^xlii

With IVF, most embryos have already been frozen or transferred by Day 6, however some are slower to develop. As such, embryologists may continue to monitor them up to Day 7 to see if they reach the blastocyst stage and can be frozen.  

Although Day 6 blasts tend to have lower clinical pregnancy and live birth rates than Day 5 blasts,^xliii this does not mean that they will not develop into healthy babies. For example, a 2018 study observed live birth rates of 30 percent and 17 percent in Day 5 and Day 6 frozen embryo transfer, respectively.^xliv  

It is less common to culture “slowly growing” embryos to Day 7, and overall, they have lower success rates than Day 5 or 6 blasts. However, one small study observed a live birth rate of 44 percent in Day 7 euploid blasts (compared to 77 percent in Day 5 euploid blasts). Although the number of Day 7 blast transfers was small, it provides an argument for monitoring embryos beyond the typical 5 or 6 days.

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Why and when do embryos arrest (stop developing)?

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In each stage of the IVF cycle, attrition of numbers is expected – typically, not all retrieved eggs will be mature, not all mature eggs will fertilize, and not all fertilized eggs will develop into blastocysts.

Many embryos will arrest (meaning they stop dividing) during the stages of development prior to reaching the blast stage. The arrested embryos are discarded. As such, the total number of embryos is expected to decrease at each stage, i.e., from Day 0 to Day 3 to Day 5. The largest drop-off is usually seen between Day 3 to 5, as described above.  

It is normal to lose 50-70 percent of the original embryos.^xlv In other words, they arrest before reaching the blastocyst stage. The most common reason that pre-implantation embryos stop developing is due to chromosomal abnormalities, such as aneuploidy (too few or too many chromosomes). Other reasons include metabolic factors or structural problems in the embryo.^xlvi

Prior to a decade ago, most embryos were transferred at the Day 2 or Day 3 stage, but advancements in culture techniques have allowed embryos to develop in vitro to the blastocyst stage. This has paved the way for an increase in success rates per transfer, but it also means that more embryos will arrest in culture prior to being transferred.

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What happens next? 

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In IVF, Day 2 through Day 5 embryos can be transferred back to the uterus, with Day 3 and 5 being the most common. An embryo can be transferred fresh, within the same cycle as the egg retrieval, or it can be cryopreserved (frozen) for later transfer. Often 1 or 2 embryos will be transferred fresh and the remaining embryos will be cryopreserved.    

Prior to embryo transfer, some patients opt for preimplantation genetic testing (PGT) to improve their odds of success. There are different types of PGT: PGT-A tests for chromosomal aneuploidies (abnormalities), PGT-M screens for specific single gene (monogenic) disorders, and PGT-SR tests for chromosomal structural rearrangements. A biopsy of 5-to-10 trophectoderm cells is taken and sent for genetic screening. PGT allows for selection of genetically competent (“normal”) embryos to transfer to the uterus.  

The number of embryos transferred is situational.  In most cases, it is one or two. Due to the risks of a multiple pregnancy (twins, triplets or more), there is a push toward single embryo transfer (SET). SET is particularly advised in younger women, either when donor eggs are used or in the case of good quality blasts and/or PGT-euploid embryos. Any remaining embryos which are not transferred are frozen (cryopreserved) and can be used in a later transfer.

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Conclusion

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The different stages of embryo growth during the IVF process can be difficult to parse but are important to understand. The rate of growth and changes in structure help guide decisions on embryo transfer and can help predict the likelihood of successful implantation and live birth. Understanding this process will help patients monitor the progress of their IVF plan and determine the right questions to ask of their clinic.

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