What is the structure of an embryo?

The word embryo is the term for the first eight weeks of human embryonic development. At nine weeks, the word fetus is used for the duration of the pregnancy. When a sperm fertilizes an egg (also called an oocyte), this forms a zygote, which is the first embryonic cell. Initial growth happens quickly, with this single-celled zygote dividing into a two-cell, then four-cell, then eight-cell embryo by Day 3. On Day 4, it is a compacted ball of cells called a morula. If growth continues, it becomes a blastocyst on Day 5.

A blastocyst has three components:

• ‍Blastocele: a fluid-filled blastocyst cavity (or blastocoele)
• ‍Inner cell mass (ICM)
• Trophectoderm (or trophoblast): an outer cell layer

The ICM will ultimately become the embryo, while 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 genetic testing, not the ones from the inner cell mass.

A glycoprotein (carbohydrate linked to a protein) shell called the zona pellucida surrounds the oocyte, then the embryo, during the initial stages of embryonic development. 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 uterine lining (also called the endometrium).ⁱ

How are blastocysts developed in the lab?

During IVF, embryo development begins in a clinic’s embryology lab. The embryos grow and mature in a petri dish marked with a unique identifier, such as a barcode, and with safety measures in place to maintain embryo identification. Embryologists oversee this process. Their skills and expertise are important to the success of an IVF cycleⁱⁱ — especially for technical processes such as embryo biopsy and intracytoplasmic sperm injection (ICSI), in which a healthy sperm is injected directly into each egg to promote fertilization.ⁱⁱⁱ

The petri dish is filled with culture medium, a fluid which provides optimal nutrients for developing embryos.^iv Decades of research have contributed to the development of embryo culture media and improvements in their quality. Numerous studies have investigated the importance of effective culture media,^v such as their impact on blastocyst yield and, thus, IVF success.^vi,vii

The petri dish is then housed in an incubator for approximately two to five days as the embryo develops. Incubator conditions are intended to mimic physiological conditions in the fallopian tubes. It is critical that temperature, oxygen, carbon dioxide, pH, and humidity levels in the incubators are kept at optimal levels, because embryos are extremely sensitive.^viii,ix Additionally, the air quality in an embryology lab can impact IVF success rates, and most embryology labs have very specialized air-filtering systems.

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 step is done daily, while other labs only provide a report on certain days of development. Most incubators now have individual chambers so that embryos are less disturbed if neighboring embryos are removed for monitoring.

In contrast, some incubators use “time-lapse monitoring,” which consists of an incubator with a transparent bottom and camera underneath to monitor embryos, so that they do not have to be physically removed from the incubator for evaluation.^x The evidence is mixed as to whether time-lapse monitoring improves the success of IVF.^xi

Stages of embryo development in IVF: What happens after egg retrieval

Developmental changes in the embryo occur quickly; however, they do not all occur at the same rate. The following sections outline the overall framework of events that occur during the first several days of IVF.

Day 0 to 1: Fertilization and 2 pro-nuclei (2PN) zygote formation

The day of the egg retrieval, also called oocyte pick-up or egg aspiration, is considered Day 0 of the IVF cycle. Often, the results of retrieval are a mix of mature, immature, and post-mature eggs. Clinics might report these results to patients using terms for the eggs’ stage of development:

• ‍Metaphase II: These mature eggs are in a stage of genetic development called metaphase II of meiosis II, often abbreviated as MII or M2.
• ‍Metaphase I: These eggs are in a genetically immature stage of meiotic maturation, abbreviated as MI or M1. In some clinics, these eggs can be matured in the embryology lab in a process called in vitro maturation (IVM).
• ‍Germinal vesicle: These eggs are very immature, abbreviated as GV.

The mature egg(s) will either be co-incubated in a petri dish with sperm for conventional insemination or inseminated by direct injection of sperm into the egg (called intracytoplasmic sperm injection, or ICSI). During conventional insemination, the sperm swim and penetrate the egg; therefore, adequate sperm count and motility are recommended for patients using this method. With ICSI, the embryologist injects a single sperm into each mature egg. ICSI was originally designed to address poor sperm count, motility, and/or morphology, but many labs now use ICSI as the standard practice for all patients.^xii

The next day (Day 1), typically 16 to 20 hours later, embryologists check for correct fertilization by looking under the microscope for two pronuclei (2PN), indicating DNA from the sperm and egg.^xiii If fertilization is successful, the fertilized egg is then called a zygote. The zygote is the first cell of the newly formed embryo.

Eggs that failed to fertilize are typically discarded. In addition, eggs that fertilized incorrectly (e.g., with no pronucleus (0PN) or only one pronucleus (1PN)) are discarded by many labs, despite some evidence that these zygotes may potentially result in successful live births.^xiv,xv It is also possible for a fertilized egg to have three or more pronuclei (3PN, 4PN, etc.), and most labs typically discard these embryos as well.^xvi Although grading zygotes is possible, most labs do not grade zygote quality.^xvii

Day 2: 2- and 4-cell cleavage stage

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

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 optimal) on Day 3.^xix 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 have been arrested, meaning they have stopped developing. Arrested embryos are likely to be discarded at the end of the cycle. Embryos with more than nine cells at this stage are defined as “accelerated,” which may also be associated with poor viability.^xx

Day 3 is an important milestone for an embryo, because it is the point at which embryonic genome activation (EGA) occurs.^xxi Up until EGA, the maternal genome, which comes exclusively from the egg, directs the embryo’s development. After EGA, the embryo’s own genome takes over. Many embryos will arrest (stop developing) after Day 3 because they fail to develop once the embryonic genome is in control — 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.^xxii

Some embryologists grade Day 3 embryos according to their number of cells, fragmentation, cell size, and cell symmetry.^xxiii Based on these criteria, Day 3 embryos may be ranked as good, fair, or poor or rated on a scale of 1 to 5 or I to III or another grading system. 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).^xxiv In contrast, a study of 423 Day 3 fresh embryo transfers showed significantly different implantation rates for grade I, II and III embryos (42 percent, 40 percent, 29 percent, respectively).^xxv Overall, some published studies found poor predictive value of Day 3 grading, while others show it is a useful selection tool.

Also, on Day 3, in some labs, embryologists change the fluid (or culture medium) in which the embryos are developing — a practice called two-step or sequential culture media. Other labs use a one-step culture medium that is unchanged and supplies the embryo with all nutrients needed for development until Day 7. It is currently unclear whether changing the culture medium is beneficial or could disturb the embryo’s development.^xxvi,xxvii

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 patients with few embryos and/or a known low blastocyst development rate during prior cycles. In addition, Day 2 or Day 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 or 6).^xxviii However, the quality of the evidence was rated as low, and additional studies were suggested to confirm the results.

Day 4: Morula stage

On Day 4, a properly developing embryo should consist of approximately 16 to 32 cells. At this stage of development, the embryo is called a morula, which will then form a compacted mass of cells in preparation for blastocyst development.

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 timing 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 Day 5 transfers were 49.5 percent and 51.9 percent, respectively.^xxix

Day 5: Blastocyst stage

By Day 5, most morulae have reached the blastocyst stage with a size typically over 100 cells. A group of cells called the inner cell mass is now distinct from the trophectoderm cells, which make up the outer layer of cells. There is also a visible fluid-filled cavity in the center of the embryo known as the blastocoele, or blastocyst cavity.^xxx The stages of blastocyst (or "blast”) growth are typically categorized as: early blast, blast, expanding blast, hatching blast, and hatched blast.^xxxi

Approximately 30 to 50 percent of correctly fertilized eggs (2PN zygotes) are expected to become Day 5 blastocysts.^xxxii In 2017, the European Society of Human Reproduction and Embryology (ESHRE) set aspirational benchmarks, suggesting embryology labs should strive for blastocyst rates of 60 percent in the future.^xxxiii For now, the recommendation is that labs aim for a 40 percent blastocyst rate as a best practice goal.

Not all embryos that reach the blastocyst stage are deemed “good quality.” 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 the likelihood of success.^xxxiv,xxxv However, grading is subjective and has high inter- and intra-embryologist variability (that is to say, different embryologists at different times might give the same embryo a different grade).^xxxvi

Overall, blastocysts are graded based on their stage of development (degree of expansion), as well as the quality of their structure (called morphology). Grading is completed by an embryologist looking at the embryo under a microscope. The embryologist assigns each embryo a number corresponding to the stage of blastocyst expansion (e.g., 1: early blastocyst, 2: blastocyst, 3: full blastocyst, 4: expanded blastocyst, 5: hatching blastocyst, and 6: hatched blastocyst). Then they assign the embryo a letter grade (A, B, or C) for each of two aspects of its structure, the inner cell mass and the trophectoderm.^{xxxvii,xxxviii} For example, a “5AA” blastocyst refers to a hatching blast (5) with a tightly packed inner cell mass (A) and a good-quality trophectoderm forming a cohesive covering layer (A).

Better blastocyst grades may be correlated with higher IVF success rates.^xxxix,xl 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).^xli 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.^xlii,xliii

Days 6 and 7: Blastocyst stage

For couples who are conceiving at home, Day 6 of embryo development is when the blastocyst has moved into the uterus and begins hatching out of the zona pellucida in preparation for implantation on Day 7.^xliv

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,^xlv it 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 transfers, respectively.^xlvi 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. (Euploid is the term for embryos that contain the full set of 46 chromosomes seen in most human beings.) Although the number of Day 7 blastocyst transfers was small, it provides an argument for monitoring embryos beyond the typical 5 or 6 days.

Why and when do embryos arrest (stop developing)?

In each stage of the IVF cycle, some loss 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 their cells stop dividing) before they reach the blastocyst stage. As such, the total number of live embryos is expected to decrease at each stage, from Day 0 to Day 5. The largest drop-off is usually seen between Day 3 and Day 5. The arrested embryos are typically discarded, as they are no longer living.

It is normal to lose 50 to 70 percent of the original embryos.^xlvii In other words, up to 70 percent of embryos typically 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.^xlviii

A decade ago, most embryos were transferred at the Day 2 or Day 3 stage, but advancements in culture techniques now allow embryos to develop in the lab to the blastocyst stage. These techniques have paved the way for an increase in success rates per embryo transfer, but the yalso mean that more embryos will arrest in culture prior to being transferred.

What happens next? 

In IVF, Day 2 through Day 5 embryos can be transferred back to the uterus, with Day 3 and 5 being the most common days for transfer. An embryo can be transferred fresh, within the same cycle as the egg retrieval, or it can be cryopreserved (frozen) for later transfer. Often patients will choose to transfer one or two fresh embryos on Day 5 after egg retrieval and freeze the remaining embryos created during that IVF cycle.

Prior to embryo transfer, some patients opt for preimplantation genetic testing (PGT). There are different types of PGT: PGT-A tests for chromosomal abnormalities, PGT-M to screen for specific single gene (monogenic) disorders, and PGT-SR to screen for chromosomal structural rearrangements. In all types of PGT, an embryologist takes a biopsy of five to 10 cells from the embryo’s trophectoderm and sends them for genetic screening. This process allows patients to select embryos with “normal” PGT results (in which no chromosomal abnormalities were found) to transfer to the uterus.

The number of embryos a patient transfers at a time depends on their circumstances. In most cases, either one or two embryos are transferred. Due to the risks of a multiple pregnancy (twins, triplets, or more) when transferring two or more embryos to the uterus at one time, there is a push toward single embryo transfer (SET). SET is particularly advised in younger females, either when donor eggs are used or in the case of good quality blasts and/or PGT-euploid embryos. Any remaining embryos that have not been transferred are frozen and can be used later.

Conclusion

The stages of embryonic development during the IVF process are complex. The rate of growth and changes in structure help guide decisions on embryo transfer and 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 at their clinic.