Embryo grading process – Let’s assess embryos together

Ing. Adéla Minks Šašková, PhD, Director of Unica Prague, Czechia: I am the Director of Unica Prague. My background is in chemical embryology. I began my career as a scientist working on oocyte maturation before transitioning into IVF. After several years in embryology and leading multiple laboratories, I moved into my current leadership role. Unica Prague is part of a medical group that includes 4 clinics: 1 in Spain and 3 in the Czech Republic.

We do not start with the embryo itself but with the oocytes on the day of follicle puncture. On day zero, we assess whether the oocytes are mature and suitable for fertilisation, specifically looking for MII-stage oocytes.

The following day, we assess fertilisation by checking for pronuclei, with normal fertilisation defined by the presence of two pronuclei. After this, embryo development can be monitored in two ways. If we use time-lapse incubation, we can observe development continuously. If we use benchtop incubators, embryos are assessed on day three, at approximately 72 hours, focusing on cell number.

On day five, when embryos reach the blastocyst stage, we assess them using the Gardner grading system. This includes evaluation of blastocyst expansion, the trophectoderm, and the inner cell mass, which will form the fetus.

Mafalda Ratom, Director of the Human Reproduction Laboratory at IERA Lisboa, Quirónsalud, Portugal: I am the Director of the Human Reproduction Laboratory at IERA Lisboa. I am a senior-level certified clinical embryologist and began my career through research in oocyte maturation, at a time when no formal training pathway for embryology existed. I trained through hands-on laboratory work in both private and public clinical settings in Lisbon before joining the IERA Lisboa project 6 years ago. I am also an invited university professor and co-founder of the Master’s programme in Clinical Embryology in Portugal.

As part of a Spanish clinical group, we traditionally use the ASEBIR classification system developed by the Spanish Association of Embryologists. This system is based on extensive datasets and provides a structured, day-by-day evaluation of embryos, starting at the oocyte stage.

The ASEBIR system is very similar to the Gardner system but uses letters rather than numbers to describe blastocyst expansion. The correspondence between the two systems is very direct, with the primary difference being familiarity.

We also assess fertilisation at standard time points and use time-lapse incubation. For me, the transition to time-lapse technology was transformative. Earlier in my career, embryos were removed from incubators once daily for assessment. With time-lapse, we gained continuous access to developmental information without disturbing culture conditions.

Although we monitor embryos continuously, we communicate with patients at key time points: fertilisation, day three, and the blastocyst stage. These are the most informative stages for patient counselling and decision-making.

Ing. Adéla Minks Šašková, PhD – Director of Unica Prague, Czechia: One major advantage of time-lapse technology is the ability to review embryo development retrospectively, particularly in cycles that result in pregnancy and live birth. It also enables additional scoring methods to identify embryos with the highest implantation potential.

There are many algorithms and AI-based tools available, although some operate as “black boxes” where the precise scoring criteria are not fully transparent. These tools can be helpful, particularly in identifying developmental events that might be missed outside a time-lapse system.

I believe that in the future, best practice will involve each laboratory developing its own algorithms based on its specific conditions. These tools will complement traditional grading systems and help prioritise embryos for transfer.

Mafalda Rato, Director of the Human Reproduction Laboratory at IERA Lisboa, Quirónsalud, Portugal: The ASEBIR system involves continuous embryo evaluation, not only at the blastocyst stage. We assess morphology, cell number and size, fragmentation, and division timing. All developmental information is considered when ranking embryos by implantation potential.

Time-lapse technology allows us to compare embryos in parallel, identifying those that divide too quickly or too slowly relative to others. While we do not rely on the built-in algorithms of the time-lapse system, we use our own in-house data to guide decision-making.

Each laboratory must adjust its scoring system based on its culture media and internal conditions. New consensus criteria are emerging that align time-point observations with time-lapse technology, reflecting the ongoing evolution of embryo assessment. Artificial intelligence will likely become an important addition, particularly for detecting subtle developmental patterns beyond human perception.

Ing. Adéla Minks Šašková, PhD, Director of Unica Prague, Czechia: I do not think time-lapse should be the main criterion when choosing a clinic. Good embryology practice existed before time-lapse technology, and experienced embryologists can achieve excellent outcomes without it.

Time-lapse can be helpful in specific situations, particularly in patients with a long history of unsuccessful treatment, as it allows closer evaluation of embryo development. It is a valuable tool, but not essential in all cases.

Mafalda Rato, Director of the Human Reproduction Laboratory at IERA Lisboa, Quirónsalud, Portugal: I agree. Time-lapse should not be a primary decision factor. Many clinics without time-lapse have benefited indirectly from the knowledge this technology has generated.

However, in certain cases—such as low ovarian reserve, repeated implantation failure, or when PGT-A is planned—access to continuous embryo images can be particularly valuable. Time-lapse incubators also provide very stable culture conditions, which may reduce stress on embryos in sensitive cases.

Ing. Adéla Minks Šašková, PhD, Director of Unica Prague, Czechia: This depends on whether time-lapse or group culture is used. With time-lapse and sufficient experience, it is possible to predict blastocyst potential based on division timing, cell number, and morphology.

With group culture, predictions are more difficult because embryos are not continuously tracked. However, even in a single culture without time-lapse, cell number, symmetry, and fragmentation on day three can provide useful predictive information.

Mafalda Rato, Director of the Human Reproduction Laboratory at IERA Lisboa, Quirónsalud, Portugal: We inform patients on day 3 using a grading system from A to D. A and B embryos have the highest likelihood of reaching the blastocyst stage, C embryos are intermediate, and D embryos are unlikely to progress.

Predictions also depend on maternal age, male factor infertility, and sperm quality. While extended culture is often appropriate, there are cases where a day three transfer may better serve the patient. Embryo arrest in culture does not always mean the same outcome would occur in the uterus, and decisions should be individualised and discussed with patients.

Ing. Adéla Minks Šašková, PhD, Director of Unica Prague, Czechia: PGT-A can be very useful for older patients, but it is not recommended for everyone. It is a screening tool that assesses ploidy, but it does not identify all genetic or implantation-related issues.

In cases of repeated implantation failure, other factors such as uterine environment or immunological conditions should also be considered. This decision should involve the IVF specialist and be based on the patient’s full history.

Mafalda Rato, Director of the Human Reproduction Laboratory at IERA Lisboa, Quirónsalud, Portugal: We also do not recommend PGT-A for all patients. In Portugal, its use is regulated and generally limited to patients over 37 or those with repeated implantation failure or pregnancy loss.

In cases of repeated failed euploid transfers, it is essential to assess uterine factors, endometritis, adenomyosis, microbiome alterations, and other clinical aspects. Implantation is complex, and the embryo is only one part of the equation.

Ing. Adéla Minks Šašková, PhD, Director of Unica Prague, Czechia: This is a key question discussed at almost every embryology and IVF meeting. Since the introduction of time-lapse systems, this has been one of the main topics in our field.

Many tools attempt to predict correlations between ploidy and morphology or morphokinetics. However, I have not yet seen a tool that convincingly works in clinical practice. These tools require extremely large datasets, ideally linked not only to ploidy but also to live birth outcomes.

Most current programs are based on studies involving thousands or tens of thousands of embryos, sometimes up to 50,000, but in my opinion, this is still not strong enough to reliably predict ploidy. To truly validate such tools, we would need data from hundreds of thousands or even millions of embryos with known outcomes.

Mafalda Rato, Director of the Human Reproduction Laboratory at IERA Lisboa, Quirónsalud, Portugal: Regarding artificial intelligence systems, I agree that we are not there yet. Some programs attempt to identify aneuploid embryos and have produced viability studies and preliminary data, but they are still limited.

What I find more clinically relevant is observing specific abnormal fertilisation patterns, such as triploid embryos or embryos with one pronucleus (1PN). A true 1PN embryo, where fertilisation occurred abnormally, will usually arrest. With time-lapse, we can often see differences in morphokinetics without using artificial intelligence.

Triploid embryos, for example, may result from polyspermy or failure of the oocyte to expel extra chromosomes. These embryos often appear very fast-growing and morphologically beautiful, but they will not implant. Time-lapse markers can help identify these patterns. For a thesis project, this could be a valuable area of investigation.

Ing. Adéla Minks Šašková, PhD, Director of Unica Prague, Czechia: The culture process itself is the same. The final decision is usually made on day five. If there are multiple embryos, the decision may be whether to proceed with transfer or PGT-A testing.

In some cases, especially when there is only 1 or 2 embryos with potential, we may prefer a fresh transfer rather than testing, as PGT-A is an invasive procedure. Even when performed carefully, it still involves a biopsy. Therefore, the choice depends on the patient’s history and is always discussed with the patient.

Mafalda Rato, Director of the Human Reproduction Laboratory at IERA Lisboa, Quirónsalud, Portugal: Day-3 transfers can be either fresh or frozen, but we no longer biopsy embryos on day 3. This technique was abandoned in Portugal several years ago because it was technically challenging, more harmful to the embryo than blastocyst biopsy, and produced confusing results, especially regarding mosaicism.

We may freeze day-three embryos if hormonal conditions, such as elevated estradiol or progesterone levels, make fresh transfer unsuitable. In such cases, we synchronise the uterus for a later day-three transfer.

We also use day-three freezing strategically when accumulating embryos for PGT-A. Because of age-related restrictions in Portugal, patients with low ovarian reserve may need several cycles to accumulate enough embryos. We freeze embryos at day three across cycles, then culture them together to day five in the final cycle and biopsy them at once to increase the chance of obtaining a euploid blastocyst.

Ing. Adéla Minks Šašková, PhD, Director of Unica Prague, Czechia: In our laboratory, embryos graded C on day five are usually kept in culture until day six to assess whether they increase cell number and show further developmental potential. Day-six embryos generally have lower implantation potential, but extended culture helps us differentiate between embryos that are still developing and those that are not.

Embryos with poor trophectoderm development have a lower chance of implantation due to insufficient cell numbers. In donor cycles, if we do not see sufficient potential, we generally do not freeze these embryos. Therefore, if embryos have been frozen, it suggests that some developmental potential was observed.

It is also important to remember that embryo grade is not everything. I have seen healthy children born from embryos graded 3BC, even when expectations were low.

Mafalda Rato, Director of the Human Reproduction Laboratory at IERA Lisboa, Quirónsalud, Portugal:  If ERA testing has already been performed, complementary tests such as EndomeTRIO may be considered. This test evaluates signs of chronic inflammation or dysbiosis and is often used alongside ERA.

Immunology is a very complex and specialised field that must be evaluated on a case-by-case basis by an immunology specialist. It involves specific genetic and immunological interactions between the patient and partner.

Hysteroscopy itself is a relatively simple procedure that allows visual inspection of the uterine cavity to identify polyps, endometriosis, inflammation, or structural abnormalities that may interfere with implantation. Some guidelines also consider the use of specific embryo transfer media, such as those containing hyaluronic acid, as a plausible add-on.

Implantation failure evaluation is complex and multifactorial, and these are some of the key areas that may be explored.

Ing. Adéla Minks Šašková, PhD, Director of Unica Prague, Czechia:  Yes, this is absolutely possible. We have patients for whom we transfer 1 embryo and freeze or test the remaining embryos. This depends on good communication between the patient, physician, and embryologist and can be tailored to the individual situation.

Mafalda Rato, Director of the Human Reproduction Laboratory at IERA Lisboa, Quirónsalud, Portugal: This is a possibility, although it must be discussed carefully with the medical team. In women over 41–43 years of age, where the probability of euploid embryos is low, we generally advise PGT-A and would consider whether this approach is clinically efficient.

Treatment decisions must balance medical suitability, emotional burden, and financial considerations. If patients are well informed and there is no added risk, it can be a shared decision.