Expert consensus on the vitrification of human oocytes and embryos

Introduction

The cryopreservation of human oocytes and embryos is a key factor in assisted reproductive technology (ART). Based on the most recent guidelines and consensus^[1–8] and in combination with clinical practice, the Chinese Association of Reproductive Medicine has called on experts to summarize the technical process and details pertaining to the vitrification of human oocytes and embryos. The intention is to provide a set of comprehensive and systematic guidelines for vitrification and improve the quality of services offered at reproductive centers.

Section 1: Providing Training to Obtain Qualified Personnel

The new staff will be trained by highly experienced embryologists who act as instructors and determine if these beginners meet the criteria based on quality management indicators and their expertise and experience in the field. The newly trained staff will proceed to the next stage of training upon fulfilling such criteria. (i) Principles: The new staff understand the basic principles of vitrification as well as the fundamental concepts and control methods for cryoprotectant concentration, operating temperature, warming/cooling rate, etc. (ii) Animal samples: Eight-cell-stage mouse embryos may be used for training. Mouse embryos exhibit low sensitivity to vitrification and thawing processes. Hence, the training standard is set in such a way that the survival rate of the embryos is not less than that of the cleavage-stage human embryos (Day 3) at the same center. The eight-cell-stage mouse embryos will be cultured for 24–48 h after thawing, and the blastocyst formation rate should be ≥80%. (iii) Discarded human oocytes or embryos: The staff become familiar with the size and shape of oocytes and embryos and the vitrification and thawing operation. (iv) Clinical practice: A small portion of oocytes/embryos from patients will be assigned for handling by beginners, and the initial proportion is ≤20%.

Section 2: Selection and Usage of Reagents and Consumables

The use of commercial vitrification and thawing reagents with registration information is recommended and not self-made products. Those with comprehensive quality control processes, including mouse embryo assays, bacterial endotoxin tests, sterility tests, and pH and osmolality tests, are preferred. ART laboratories can routinely be equipped with two different brands of vitrification and thawing reagents to deal with emergencies such as failed batches or unstable supplies. Cryopreservation carriers could be categorized into open and closed types based on whether the sample in the carrier device 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 and those from infected carriers.

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

Vitrification of oocytes

Preparation

Return the vitrification reagent to room temperature (24°C–26°C). The oocytes should be vitrified 38–40 h after the human chorionic gonadotropin injection and be denuded immediately before cryopreservation.^[1] Immature oocytes are suggested to be subjected to in vitro maturation culture for 24–48 h, followed by vitrification. Giant oocytes and those with smooth endoplasmic reticulum disks are not recommended for cryopreservation. The number of oocytes in each carrier should not exceed five.

Examples of Operation

(i) Adjacent drops of Basic Solution (BS) and Equilibration Solution (ES) are to be prepared on the lid of the petri dish at a drop volume of ≥150 μL. After the oocytes are transferred to the BS drop, the two drops are to be bridged and the oocytes are to be transferred to their center and left for 3 min [Supplementary Figure 1]. (ii) Prepare a fresh ES drop and bridge it with the aforementioned drops, then move the oocytes to the center of the connected drop and leave it for 3 min. (iii) Prepare another ES drop (volume ≥40 μL) on another petri dish and cover it with sufficient oil (≥3 mL). The oocytes are left for 6–9 min after being transferred to the drop. This step is to be completed when the perivitelline space returns to its original size [Supplementary Figure 2]. (iv) Prepare several drops of Vitrification Solution (VS) at the volume ≥40 μL on another petri dish. Transfer the oocytes to at least 3–5 different positions in the VS to remove residual ES. (v) Load oocytes on the carrier in accordance with the operating method of the cryopreservation carrier being used. Steps 4 and 5 are done in 45–60 s.

Vitrification of cleavage-stage embryos and blastocysts

Preparation

The vitrification can be performed either at room temperature (24°C–26°C) or at 37°C. Warm the vitrification reagent, oil, and petri dish, and prepare a dish containing the BS and ES. The number of cleavage-stage embryos or blastocysts loaded in each carrier should be ≤2, and it is suggested that a single high-quality blastocyst be loaded on each carrier. Embryos eligible for vitrification include usable cleavage-stage embryos from Day 2–3 as well as usable blastocysts from Day 5–7.^[2]

Examples of Operation

(i) Transfer the embryos to the BS and leave them for 1 minute (optional). (ii) Transfer the embryos to the surface center of ES. The embryos will fall freely, and they are to be left for the corresponding period based on the operation temperature [Supplementary Figure 3]. This step is to be completed when the embryo returns to its original size [Supplementary Figure 4]. (iii) Prepare several drops of VS on another petri dish. Transfer the embryos in the VS to at least 3–5 different positions to remove the residual ES. (iv) Load embryos on the carrier.

Thawing of the oocytes, cleavage-stage embryos, and blastocysts

Preparation

(i) Thawing Solution (TS): Prepare a drop of TS on a petri dish (≥200 μL), cover it with oil, and warm it to 37°C. (ii) Dilution Solution (DS) and BS: Prepare several drops of DS and BS on the petri dish, and cover them with oil, then keep them at room temperature.

Examples of Operation

(i) Take the carrier out of the liquid nitrogen and immerse it in the TS quickly (within 1 s), leaving the oocytes/embryos for 45–60 s. If the sample floats, move it to the center of the drop [Supplementary Figure 5]. (ii) Transfer the sample to the DS and leave it for 3 min. Note: A small amount of the primary liquid is to be carried over during the transfer. (iii) Transfer the sample to the BS and leave it for 5 min. (iv) Transfer the sample to a new BS and leave it for 5 min.

Timing of Related Operations after Thawing

Intracytoplasmic sperm injection is to be performed 2–3 h after the thawing of oocytes.^[3] The 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 h after thawing to observe the re-expansion of the blastocyst, which aids in assessing blastocyst survival.

Section 4: Artificial Shrinkage of Blastocysts

Laser artificial shrinkage is widely used owing to its simplicity; however, the debate about whether artificial shrinkage improves pregnancy outcomes is ongoing. The position for the laser drilling is the cellular junction of the trophectoderm, and it should be away from the inner cell mass [Supplementary Figure 6]. The laser intensity should be set according to the specific conditions of each device and 1–2 laser pulses are appropriate. The blastocysts can be vitrified after 10–15 min of artificial shrinkage. The duration should not be too long to prevent blastocyst re-expansion.

Section 5: Assisted Hatching

It is suggested that laser zona pellucida (ZP) thinning be performed on cleavage-stage embryos or morulae, with the thickness of the ZP being reduced by 50%–80% in 25%–50% of the ZP circumference. Laser ZP breaching is performed on blastocysts graded as 4–6,^[2] with an opening of 25%–50% of the ZP circumference. ZP thinning can be performed in early-stage and expansion-stage blastocysts (graded as 1–3) as their ZP is thick.

Section 6: Quality Management

Definition of successful survival

(i) Oocyte: A successful surviving oocyte exhibits normal cell membrane morphology and clear cytoplasm. Negative characteristics, such as darkened cytoplasm, massive vacuolization, cytoplasmic leakage, or abnormal perivitelline space, would indicate that the cell structure has been damaged or even degenerated during the vitrification and thawing. (ii) Cleavage-stage embryo: Cleavage-stage embryos are considered to have survived when at least half of the blastomeres are intact after thawing, and full survival means that all blastomeres are intact after thawing. (iii) Blastocyst: Survival is defined as ≥75% of the cells being intact or the re-expansion of the blastocoele within 2 h of thawing.

Key performance indicators

The systematic monitoring of key performance indicators (KPIs) aids in objectively evaluating the vitrification/thawing technology and identifying potential risks in a timely manner. The KPIs for vitrification and thawing are given in Supplementary Table 1. The competency values represent the minimum level of performance for each KPI, and the benchmark values denote aspirational targets.^[1] The ideal frequency of KPI statistics is on a monthly basis.^[4] Additionally, each ART laboratory can determine the appropriate period based on the actual caseload. The following rules are applied to determine whether a real problem exists and warrants a detailed survey: (i) when the KPIs are lower than the competency values; (ii) when the KPIs are lower than two standard deviations of the previous year's average.

Path for abnormal data analysis

The analysis is to be performed according to the path given in Supplementary Figure 7 when the KPIs are abnormal.

Section 7: Time Limit for Cryopreservation

Considering the survival rate of cryopreserved embryos/oocytes, pregnancy outcomes, ethics, and the effective utilization of medical resources, the recommended optimal storage time after the vitrification of oocytes is no more than 1 year, and for embryos, it is no more than 5 years.^[5]

Conclusion

In summary, this consensus provides a set of comprehensive and systematic guidelines for vitrification, including personnel qualification training, selection and usage of reagent consumables, routine vitrification and thawing of oocytes and embryos at different stages, quality management and cryopreservation time limits, to improve the quality of ART services.

Conflicts of interest

None.