Expert Consensus on the Vitrification of Human Oocytes and Embryos

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Expert Consensus on the Vitrification of Human Oocytes and Embryos

The cryopreservation of human oocytes and embryos is a critical component of assisted reproductive technology (ART). Vitrification, a rapid freezing technique, has become the preferred method due to its ability to preserve the structural and functional integrity of these cells. This consensus document aims to provide comprehensive and systematic guidelines for the vitrification process, ensuring high-quality services in reproductive centers. The guidelines are based on the latest research, clinical practice, and expert opinions, covering aspects such as personnel training, reagent selection, technical procedures, quality management, and storage limits.

Section 1: Training and Qualification of Personnel

The success of vitrification largely depends on the expertise of the embryologists performing the procedure. Therefore, the training of new staff is a critical step. Beginners are trained by highly experienced embryologists who assess their competence based on quality management indicators and their understanding of the fundamental principles of vitrification. These principles include the control of cryoprotectant concentration, operating temperature, and warming/cooling rates.

The training process involves several stages. First, trainees use eight-cell-stage mouse embryos, which are less sensitive to vitrification and thawing, to practice. The standard for training is set such that the survival rate of these mouse embryos must not be lower than that of cleavage-stage human embryos (Day 3) at the same center. After thawing, the mouse embryos are cultured for 24–48 hours, and the blastocyst formation rate should be at least 80%.

Next, trainees work with discarded human oocytes or embryos to familiarize themselves with the size, shape, and handling of these cells during vitrification and thawing. Finally, beginners are allowed to handle a small portion (initially 20%) of oocytes or embryos from patients under supervision. This gradual approach ensures that trainees gain confidence and competence before handling valuable patient samples.

Section 2: Selection and Usage of Reagents and Consumables

The choice of reagents and consumables is crucial for successful vitrification. The use of commercially available vitrification and thawing reagents with proper registration information is strongly recommended. These products should undergo comprehensive quality control processes, including mouse embryo assays, bacterial endotoxin tests, sterility tests, and pH and osmolality tests. ART laboratories are advised to keep two different brands of vitrification and thawing reagents on hand to address emergencies such as failed batches or supply shortages.

Cryopreservation carriers are categorized into open and closed types based on whether the sample is in direct contact with liquid nitrogen. Closed carriers or separate dewars are recommended for gametes and embryos from patients who have tested positive for infections or those from infected carriers. This precaution minimizes the risk of cross-contamination.

Section 3: Vitrification and Thawing Processes for Oocytes, Cleavage-Stage Embryos, and Blastocysts

Vitrification of Oocytes

The vitrification of oocytes involves several preparatory steps. The vitrification reagents should be brought to room temperature (24°C–26°C). Oocytes are typically vitrified 38–40 hours after human chorionic gonadotropin injection and are denuded immediately before cryopreservation. Immature oocytes may undergo in vitro maturation culture for 24–48 hours before vitrification. However, giant oocytes and those with smooth endoplasmic reticulum disks are not recommended for cryopreservation. Each carrier should hold no more than five oocytes.

The vitrification process for oocytes includes the following steps:

  1. Prepare adjacent drops of Basic Solution (BS) and Equilibration Solution (ES) on the lid of a petri dish, with each drop volume being at least 150 µL. Transfer the oocytes to the BS drop, bridge the drops, and leave the oocytes in the center for 3 minutes.
  2. Prepare a fresh ES drop and bridge it with the previous drops. Move the oocytes to the center of the connected drop and leave them for 3 minutes.
  3. Prepare another ES drop (volume ≥40 µL) on a separate petri dish and cover it with oil (≥3 mL). Transfer the oocytes to this drop and leave them for 6–9 minutes until the perivitelline space returns to its original size.
  4. Prepare several drops of Vitrification Solution (VS) (volume ≥40 µL) on another petri dish. Transfer the oocytes to at least 3–5 different positions in the VS to remove residual ES.
  5. Load the oocytes onto the carrier according to the manufacturer’s instructions. Steps 4 and 5 should be completed within 45–60 seconds.

Vitrification of Cleavage-Stage Embryos and Blastocysts

The vitrification of cleavage-stage embryos and blastocysts can be performed at room temperature (24°C–26°C) or at 37°C. The reagents, oil, and petri dishes should be warmed, and a dish containing BS and ES should be prepared. Each carrier should hold no more than two cleavage-stage embryos or blastocysts, and it is recommended to load a single high-quality blastocyst per carrier. Embryos eligible for vitrification include usable cleavage-stage embryos from Day 2–3 and usable blastocysts from Day 5–7.

The vitrification process for embryos includes the following steps:

  1. Transfer the embryos to the BS and leave them for 1 minute (optional).
  2. Transfer the embryos to the center of the ES drop. The embryos will fall freely, and they should be left for the corresponding period based on the operating temperature. This step is complete when the embryo returns to its original size.
  3. Prepare several drops of VS on another petri dish. Transfer the embryos to at least 3–5 different positions in the VS to remove residual ES.
  4. Load the embryos onto the carrier.

Thawing of Oocytes, Cleavage-Stage Embryos, and Blastocysts

The thawing process involves the following steps:

  1. Prepare a drop of Thawing Solution (TS) (≥200 µL) on a petri dish, cover it with oil, and warm it to 37°C.
  2. Prepare several drops of Dilution Solution (DS) and BS on a petri dish, cover them with oil, and keep them at room temperature.
  3. Remove the carrier from the liquid nitrogen and immerse it in the TS quickly (within 1 second), leaving the oocytes/embryos for 45–60 seconds. If the sample floats, move it to the center of the drop.
  4. Transfer the sample to the DS and leave it for 3 minutes. Note: A small amount of the primary liquid should be carried over during the transfer.
  5. Transfer the sample to the BS and leave it for 5 minutes.
  6. Transfer the sample to a new BS and leave it for 5 minutes.

Timing of related operations after thawing is critical. Intracytoplasmic sperm injection should be performed 2–3 hours after the thawing of oocytes. Cleavage-stage embryos can be thawed a day before or on the day of embryo transfer. For blastocysts, it is recommended to perform embryo transfer 2 hours after thawing to observe the re-expansion of the blastocyst, which aids in assessing its survival.

Section 4: Artificial Shrinkage of Blastocysts

Artificial shrinkage of blastocysts is a technique used to improve vitrification outcomes. Laser artificial shrinkage is widely used due to its simplicity. The position for laser drilling should be the cellular junction of the trophectoderm, away from the inner cell mass. The laser intensity should be set according to the specific conditions of each device, and 1–2 laser pulses are usually sufficient. Blastocysts can be vitrified after 10–15 minutes of artificial shrinkage. The duration should not be too long to prevent blastocyst re-expansion.

Section 5: Assisted Hatching

Assisted hatching is a technique used to facilitate embryo implantation. Laser zona pellucida (ZP) thinning is recommended for cleavage-stage embryos or morulae, with the thickness of the ZP reduced by 50%–80% in 25%–50% of the ZP circumference. Laser ZP breaching is performed on blastocysts graded as 4–6, with an opening of 25%–50% of the ZP circumference. ZP thinning can also be performed on early-stage and expansion-stage blastocysts (graded as 1–3) due to their thick ZP.

Section 6: Quality Management

Definition of Successful Survival

The criteria for successful survival vary depending on the type of cell:

  • Oocyte: A surviving oocyte should exhibit normal cell membrane morphology and clear cytoplasm. Negative characteristics, such as darkened cytoplasm, massive vacuolization, cytoplasmic leakage, or abnormal perivitelline space, indicate damage or degeneration during vitrification and thawing.
  • Cleavage-stage embryo: Survival is defined as at least half of the blastomeres remaining intact after thawing. Full survival means all blastomeres are intact.
  • Blastocyst: Survival is defined as ≥75% of the cells being intact or the re-expansion of the blastocoele within 2 hours of thawing.

Key Performance Indicators

Systematic monitoring of key performance indicators (KPIs) is essential for evaluating the effectiveness of vitrification and thawing processes and identifying potential risks. The KPIs include survival rates, blastocyst formation rates, and other relevant metrics. Competency values represent the minimum acceptable performance levels, while benchmark values denote aspirational targets. KPI statistics should ideally be compiled monthly, though laboratories can adjust the frequency based on their caseload. Anomalies in KPIs, such as values falling below competency levels or two standard deviations below the previous year’s average, warrant detailed investigation.

Section 7: Time Limit for Cryopreservation

The optimal storage time for cryopreserved oocytes and embryos is determined based on survival rates, pregnancy outcomes, ethical considerations, and the effective utilization of medical resources. The recommended storage time for vitrified oocytes is no more than 1 year, while for embryos, it is no more than 5 years.

Conclusion

This consensus provides a comprehensive set of guidelines for the vitrification of human oocytes and embryos, covering all aspects from personnel training to quality management and storage limits. By adhering to these guidelines, reproductive centers can improve the quality of their ART services and achieve better outcomes for patients.

doi.org/10.1097/CM9.0000000000002895

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