Blastocyst biopsy and vitrification are required to efficiently conduct preimplantation genetic testing. An approach entailing the sequential opening of the zona pellucida and retrieval of 7-8 trophectoderm cells in day 5-7 post-insemination limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions.
Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (>40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally ≤30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated.
The main goal of modern human embryology is to maximize the number live births per stimulated cycle and reduce costs, time and efforts to achieve a pregnancy. To accomplish this goal, validated approaches for embryo selection should be employed to identify reproductively competent embryos within a cohort obtained during an IVF cycle. According to the latest evidences, blastocyst culture1 combined with comprehensive chromosomal testing and vitrified-warmed euploid embryo transfer (ET) is the most efficient framework to increase IVF efficiency2. Clearly, aneuploidy testing requires an embryonic specimen, which at present is mostly represented from few cells retrieved from the trophectoderm (TE), i.e., the section of the blastocyst that gives origin to embryo annexes (e.g., the placenta) during pregnancy. Beyond karyotype analysis, also single gene mutations might be assessed from a TE biopsy as part of a clinical strategy known as preimplantation genetic testing (PGT; -A for aneuploidies, -SR for structural chromosomal rearrangements, -M for monogenic diseases). Other oocyte/embryo biopsy methods have been theorized and adopted clinically across the last decades, namely polar bodies biopsy and blastomere biopsy. However, their use is reduced nowadays since their procedural drawbacks (e.g., higher workload and risk for reproductive impact) and diagnostic limitations (e.g., single cell analysis issues) implicitly hinder a sufficient balance between costs, risks and benefits (for a review see3).
In this paper, one of the main protocols for TE biopsy is thoroughly described together with the subsequent vitrification, warming and transfer procedures required. The workflow here outlined is ideal for a busy PGT unit.
As already described previously by our group4,5, the procedure involves the sequential opening of the zona pellucida of fully-expanded blastocysts and removal of few TE cells (on average 7-8). Compared to the day 3 laser-assisted hatching-based blastocyst biopsy method6, this procedure might ease the daily schedule of an IVF unit where delicate procedures, such as blastocyst biopsy and vitrification, must be timely performed. As soon as the blastocyst reaches its full expansion, the biopsy can be carried out by selecting the TE cells to remove, thereby preventing the risk of herniation of the inner cell mass (ICM), which would otherwise render the procedure challenging. In literature, a third protocol of blastocyst biopsy has been also described, which involves laser-assisted hatching being performed once the embryo has already reached the blastocyst stage, few hours before the procedure5,7. However, this approach is more time-consuming and mainly suits IVF units that are implementing TE biopsy in the hands of limitedly experienced operators and in view of a moderate-low daily workload.
Intracytoplasmatic sperm injection (ICSI)8 should be a consolidated technique if aiming at conducting genetic analyses in IVF. Similarly, a proper culture system to safely harvest embryos to the blastocyst stage is crucial for the implementation of TE biopsy strategy. An adequate number of incubators, as well as the use of low oxygen tension are key prerequisites to this end, not to compromise the blastocyst rate9. At the same time, an efficient cryopreservation program is needed to safely manage a PGT cycle. In the last decade, the implementation of vitrification has boosted embryo cryo-survival rates even up to >99%10,11. This provided sufficient time to perform genetic testing and postpone embryo transfer to the following menstrual cycle, on a non-stimulated and probably more receptive endometrium12.
Both TE biopsy and vitrification are demanding tasks requiring stringent skills and their effectiveness might vary across unexperienced operators. A specific training period is therefore advocated before allowing each operator to perform these procedures clinically; moreover, the maintenance of the operators’ skills should be assessed periodically by monitoring key performance indicators (KPI) for cryopreservation and biopsy procedures. Each IVF clinic should set internal KPIs to this end, which must approximate the ones published by international consortia and/or the outcomes published by reference laboratories.
TE biopsy, vitrification-warming and witnessing procedures are validated techniques at our unit, that have been standardized across all the operators involved as reported in three previous publications11,13,14.
The protocol for human blastocyst biopsy, here described, follows the guidelines of G.EN.E.R.A. Human Research Ethic Committee.
NOTE: Refer to the Table of Materials for materials required. Further material required entails laboratory footwear and outfit, surgical facemask, hair cover, surgical gloves, a permanent non-toxic marker, forceps and disinfectant. The use of surgical gown, disposable surgical gloves, facemask, hair cover is mandatory to prevent risk of contamination. All the working areas, as well as the equipment involved in the process, must be cleaned thoroughly with laboratory disinfectant (e.g., Oosafe) before starting any procedure. All consumables and media used should be sterile and individually packaged or aliquoted. It is suggested to use a dedicated workstation for biopsy and tubing and limit the access of the area only to the operators involved in the procedure (embryologist and witness).
1. Preparation on the Day Before the Biopsy Procedure
2. Preparation on the Day of the Biopsy Procedure
3. Blastocyst Selection and Grading
4. Trophectoderm Biopsy
5. Tubing
NOTE: The whole procedure must be carried out in the presence of a witness and inside the laminar flow hood at room temperature. During the procedure, keep the PCR tubes in a cold tube rack on ice (Supplementary Figure 1).
6. Blastocyst Vitrification
7. Artificial shrinkage of Non-biopsied Blastocysts
8. Transferable Blastocyst Warming
9. EmbryoTransfer
Figure 6 represents a scheme of all the outcomes of a biopsy procedure that can be adopted to standardize the protocol and monitor the performance of each operator. The main procedural outcome is the timing to complete the biopsy/biopsies; the main technical outcome is the quality of the plot produced after genetic testing that might result in either a conclusive or inconclusive diagnosis, the latter of which requires a re-biopsy of the undiagnosed blastocyst; the main biological outcome is the rate of cryo-survival and re-expansion versus degeneration after warming; lastly, the main clinical outcome is the live birth rate after vitrified-warmed blastocyst transfer. In three previous studies we reported the KPIs defined at the center(s) for the technical, biological and clinical outcomes11,13,16. Hereafter, we instead report how the KPI for the timing of biopsy was defined. Moreover, the putative influence of the timing between biopsy and vitrification on the post-warming behavior of euploid blastocysts was also investigated.
In a 2 year period, a total of 1,544 trophectoderm biopsy procedures were conducted by 7 operators (Table 1). All biopsied blastocysts were then moved back to the incubator into a post-biopsy culture dish until vitrification. All the relevant data were collected in a relational database. All the timings of biopsy and between biopsy and vitrification were retrospectively obtained from the software of the IVF electronic witnessing system. The data were then exported and analyzed for statistics.
The cryo-survival rate of euploid blastocysts after trophectoderm biopsy and vitrification-warming was N = 571/572, 99.8%. The re-expansion rate at 1.5 h after warming was N = 556/571, 97.4%. Among the 15 not re-expanded blastocysts, one resulted in a live birth after being transferred in utero. The live birth rate after vitrified-warmed euploid single blastocyst transfer was N=227/572, 39.7%.
Definition of the ideal timing of biopsy
Table 1 summarizes the relevant data of the biopsy procedures conducted. Overall, 1.89 ± 1.03 (range 1-4) blastocysts were biopsied per procedure in 8.24 ± 4.23 min (range 3-22). The mean timing of biopsy varied because of both the number of blastocysts biopsied per procedure, from a minimum of 5.78 ± 2.94 min (range 3-16) when only one embryo was laid in the dish to a maximum of 12.93 ± 4.43 min (range 6-22) when the embryos sequentially biopsied were 4. Another relevant parameter was the operator involved in the procedure: the most expert (N = 443 procedures) was the fastest (7.41 ± 3.6 min, range 3-22), while the least experienced (N = 42) was the slowest (14.19 ± 4.24 min, range 6-22). Indeed, a generalized linear model entailing both the “number of blastocysts biopsied per procedure” and the “operator” variables perfectly explains the “timing of biopsy” with a R2 = 0.48 and a power = 1. This analysis was useful to define that ideally ~6 min is enough for a blastocyst biopsy procedure when only an embryo is laid in the dish, while ~9 min, ~12 min and ~13 min for 2, 3 and 4 blastocysts, respectively. Clearly, the whole procedure entails also moving the embryos from the culture to the biopsy dish and from the latter to the post-biopsy culture dish after the procedure, as well as changing the biopsy pipette between sequential biopsies.
Figure 7 plots the mean timing of biopsy for each operator along the study trimesters (from the 1st to the 8th). The dotted red line identifies the mean overall value of 8.24 min. Such a graph is useful to monitor the mean performance of each practitioner. For instance, the most expert operators (1 and 2) showed a constant decrease of this timing, which suggests a trend typical of a learning curve. All the operators from 3 to 6 were instead sufficiently constant in their performance around the mean overall value across the trimesters. Anytime they showed a peak in the mean value from a given trimester (e.g., operator 3 in the 7th trimester, operator 4 in 6th trimester, operator 5 in the 3rd trimester), they were warned in order to revise their performance. Operator 7 (i.e., the least experienced) showed timings typical of an embryologist that has just finished his/her training. Possibly, he/she will meet the standards internal to the lab as the expertise would increase.
Importantly, the time of biopsy was similar across re-expanded and not re-expanded euploid blastocysts at 1.5 h from warming (9.52 ± 4.23 min, range 3-22 versus 10.5 ± 5.68 min, range 4-22; t-test = 0.37). Likely, implanted (N = 229) and not implanted (N = 343) vitrified-warmed euploid blastocysts also showed comparable biopsy timings (9.77 ± 4.15 min, range 3-22 versus 9.41 ± 4.36 min, range 3-22; t-test = 0.39). Possibly then, a timing ≤22 min to biopsy up to 4 blastocysts does not affect embryo behavior after warming. Therefore, we defined this value as maximum threshold.
Similarly, no difference was shown in terms of live birth rate across the different biopsy operators, as already reported previously13 (Supplementary Table 1).
Another important parameter to monitor each operator’s performance is the rate of inconclusive results after diagnosis, which should be as close as possible to the general performance of each laboratory. Ideally this rate should not exceed 2.5% and might decrease with time due to an increasing expertise in biopsy and tubing procedures16. The target number of TE cells to retrieve are 7-8 according to two previous studies13,16. To this end, it is suggested to take a picture of the biopsied fragment for quality control purpose (see some examples in Figure 3). Such picture might be checked in case of inconclusive diagnoses to evaluate whether the cause was imputable to the dimension/quality of the fragment (i.e., low quality of the molecular analysis), to the tubing (i.e., DNA amplification failure) or to some issues in the processing of the sample in the genetic laboratory.
Definition of the ideal timing between biopsy and vitrification
In the study period, 572 euploid blastocysts were warmed to undergo an embryo transfer after a diagnosis of euploidy. Figure 8A shows each warmed blastocyst as a black circle distributed across the increasing timing between biopsy and vitrification and clustered in two groups according to the outcome under investigation: re-expanded or not re-expanded within 1.5 h from warming. All the blastocysts (N = 117/117) vitrified within 30 min, 97.6% (N = 245/251) of the blastocysts vitrified between 31-90 min, and 95.1% (N = 194/204) of the blastocysts vitrified beyond 90 min re-expanded, respectively (no re-expansion rates: 0%, 2.4% and 4.9%). Therefore, we set 30 min and 90 min as the early and late thresholds of time between biopsy and vitrification.
Figure 8B shows the re-expansion rate in the three groups (≤30 min, 31-90 min, >90 min) further sub-clustered according to the blastocyst quality and day of preimplantation development. Especially for poor quality and/or day 7 blastocysts, the timing between biopsy and vitrification seems crucial to achieve re-expansion after warming. Specifically, the odds-ratio of re-expansion after warming corrected for both blastocyst quality and day of biopsy in blastocysts vitrified within 30 min from biopsy versus blastocysts vitrified beyond 90 min was 3.05 (95% CI 1.01-9.4, p=0.05). Instead, the period in between these two thresholds (31-90 min) represented a grey area that might or might not have an impact.
Only 1 out of 15 not re-expanded blastocysts resulted in a live birth after transfer. Therefore, we lastly investigated the live birth rate achieved after warmed euploid single blastocyst transfer clustered in the three groups according to the timing between biopsy and vitrification. The highest live birth rate was achieved by transferring euploid blastocysts vitrified ≤30 min from the trophectoderm biopsy (N = 56/117, 47.9%). However, this result did not reach statistical significance when compared to the same outcome obtained either with blastocysts vitrified between 31 and 90 min (N = 92/251, 36.7%; Fisher’s exact test = 0.06), or with blastocysts vitrified >90 min from the biopsy (N=81/204, 39.7%; Fisher’s exact test = 0.16). Therefore, either a negative effect on blastocyst reproductive competence is negligible or the sample size in this dataset (N = 572) was insufficient to reach statistical significance.
Figure 1: Parameters for blastocyst grading. Expansion: (A) fully hatched, (B) in hatching, (C) fully expanded, and (D) not expanded. The ideal stage is C, while a blastocyst D should be given more time to achieve full expansion, unless this stage is reached in day 7; Inner cell mass (ICM) morphological quality: 1 (noticeable with several strictly packed cells), 2 (discernable with several but roughly packed cells) and 3 (difficult to distinguish with very few low-quality cells); trophectoderm (TE) morphological quality: 1 (well-organized epithelium with several cells), 2 (loose epithelium with few cells) and 3 (few and/or large low-quality cells). Please click here to view a larger version of this figure.
Figure 2: Summary of the sequential zona pellucida (ZP) opening and trophectoderm (TE) cells retrieval approach for blastocyst biopsy. (a) Orient the blastocyst with the inner cell mass (ICM) close to the holding pipette and far from the spot where the selected TE cells will be retrieved. Secure the blastocyst on the holding pipette; (b) open the ZP through 2-3 laser shots; (c) blow some culture media through the hole; (d) the blastocyst will detach from the ZP; (e) enter the ZP and suck 5-10 TE cells in the biopsy pipette; (f) move backwards with the biopsy pipette to stretch the selected fragment and expose the junctions between the cells; (g) fire at the junctions between the cells and continue stretching the fragment until the TE cells are released from the body of the blastocyst; (h) the blastocyst after TE biopsy is collapsed; (i) take a picture of the biopsy fragment for quality control and transfer it to the PCR tube that will be sent to the genetic laboratory. Please click here to view a larger version of this figure.
Figure 3: Examples of biopsy fragments: (a-c) desirable fragments; (d) lysed fragment; (e) small fragment with degenerated cells; (f) small, partially lysed and degenerated fragment. Please click here to view a larger version of this figure.
Figure 4: Artificial shrinkage. (a) Orientate the blastocyst so that the inner cell mass (ICM) is far from the targeted section of the trophectoderm (TE); (b) Fire 2-3 laser shots in a row at the junctions between TE cells and moving outwards; (c) Wait for the blastocyst to collapse before starting vitrification. Please click here to view a larger version of this figure.
Figure 5: Examples of blastocyst degeneration (a), cryo-survival but no re-expansion (b) and cryo-survival and full re-expansion (c) 1.5 h post-warming. Please click here to view a larger version of this figure.
Figure 6: Summary of the different outcomes of trophectoderm biopsy that might be used to monitor the performance of an operator and define the key performance indicators internal to each laboratory. The main procedural outcome is the timing of biopsy. The main technical outcome is the rate of conclusive (euploid or aneuploid) and inconclusive diagnoses (re-biopsy required) obtained; the latter might be caused by DNA amplification or low-quality molecular data, both resulting in not-interpretable chromosome copy number profile plots. The main biological outcome is the rate of cryo-survival and re-expansion or degeneration after biopsy, vitrification and warming. The main clinical outcome is the rate of live births or negative pregnancy outcomes achieved after vitrified-warmed blastocyst transfer. Of note, while the procedural outcome is exclusively dependent on the operator and the number of blastocysts to biopsy per procedure, all other outcomes might be affected from other confounders independent from the biopsy operator (e.g., the steps and operators involved in the molecular analysis, the morphological quality of the blastocyst, the day of biopsy) that should be accounted to properly evaluate his/her performance. Please click here to view a larger version of this figure.
Figure 7: Mean timing of biopsy per operator across the 8 study trimesters. The table summarizes the related number of procedures and the mean number of blastocysts biopsied per procedure by each operator in the 8 study trimesters. The dotted red line within each graph represents the overall mean timing of biopsy (8.24 min). The error bars are the standard deviations. Please click here to view a larger version of this figure.
Figure 8: Re-expansion after warming versus timing between biopsy and vitrification. (A) shows not re-expanded and re-expanded blastocysts 1.5 hr after warming. Each blastocyst is represented by a black circle across the increasing timings. The vertical continuous black lines represent 30 min set as early threshold and 90 min set as late threshold. (B) shows the re-expansion rates in the three groups (timing between biopsy and vitrification: ≤30 min, 31-90 min, >90 min) further clustered according to blastocyst quality and day of biopsy. Please click here to view a larger version of this figure.
N of procedures | N blastocysts biopsied per procedure | Mean timing of biopsy ± SD, range (min) | |
Operator 1 | 443 | 2.01 ± 1.09, 1-4 | 7.41 ± 3.6, 3-22 |
195 | 1 | 4.75 ± 1.96, 3-16 | |
111 | 2 | 7.83 ± 2.45, 3-18 | |
71 | 3 | 10.27 ± 2.41, 4-16 | |
66 | 4 | 11.48 ± 3.81, 6-22 | |
Operator 2 | 290 | 1.81 ± 0.98, 1-4 | 7.87 ± 4.13, 3-22 |
142 | 1 | 5.69 ± 3.32, 3-16 | |
89 | 2 | 8.48 ± 2.79, 3-18 | |
30 | 3 | 11.37 ± 3.72, 4-20 | |
29 | 4 | 13.1 ± 3.89, 9-22 | |
Operator 3 | 287 | 1.98 ± 1.05, 1-4 | 9.10 ± 4.65, 3-22 |
121 | 1 | 6 ± 2.19, 3-15 | |
89 | 2 | 9.6 ± 3.87, 3-22 | |
38 | 3 | 12.66 ± 4.55, 4-22 | |
39 | 4 | 14.13 ± 4.8, 6-22 | |
Operator 4 | 217 | 1.66 ± 0.87, 1-4 | 7.58 ± 3.45, 3-22 |
118 | 1 | 5.58 ± 1.96, 3-14 | |
66 | 2 | 8.92 ± 2.91, 4-22 | |
21 | 3 | 11.48 ± 2.34, 5-16 | |
12 | 4 | 13 ± 4.26, 6-19 | |
Operator 5 | 144 | 2.03 ± 1.08, 1-4 | 9.43 ± 4.24, 3-22 |
59 | 1 | 6.15 ± 2.5, 3-16 | |
43 | 2 | 10.07 ± 2.73, 6-16 | |
20 | 3 | 12.6 ± 2.89, 9-18 | |
22 | 4 | 14.09 ± 4.43, 6-22 | |
Operator 6 | 121 | 1.67 ± 0.94, 1-4 | 7.79 ± 3.93, 3-22 |
70 | 1 | 6.19 ± 2.95, 3-16 | |
32 | 2 | 8.12 ± 1.72, 3-11 | |
9 | 3 | 12.78 ± 3.31, 9-18 | |
10 | 4 | 13.5 ± 6.19, 6-22 | |
Operator 7 | 42 | 1.62 ± 0.94, 1-4 | 14.19 ± 4.24, 6-22 |
27 | 1 | 11.85 ± 5.53, 6-16 | |
6 | 2 | 16.5 ± 3.73, 11-22 | |
7 | 3 | 19.86 ± 3.34, 13-22 | |
2 | 4 | 19 ± 4.24, 16-22 | |
Total | 1544 | 1.89 ± 1.03, 1-4 | 8.24 ± 4.23, 3-22 |
732 | 1 | 5.78 ± 2.94, 3-16 | |
436 | 2 | 8.85 ± 3.14, 3-22 | |
196 | 3 | 11.72 ± 3.70, 4-22 | |
180 | 4 | 12.93 ± 4.43, 6-22 |
Table 1: Total mean timing of biopsy and mean number of blastocysts biopsied in each procedure according to biopsy operator. The mean timing of biopsy has been also shown according to each sequential number of blastocysts biopsied per procedure. A generalized linear model that includes both the “biopsy operator” and “number of blastocysts biopsied per procedure” variables perfectly correlates with the “timing of biopsy” (R2 = 0.48, power = 1).
Supplementary Figure 1: Main devices and supports required for the procedure. Please click here to download this file.
Supplementary Table 1: Logistic regression analysis does not show any significant association between the biopsy operator and live birth after vitrified-warmed euploid blastocyst transfer. Please click here to download this file.
Only well-experienced skilled embryologists who have completed their training period should perform both TE biopsy and blastocyst vitrification. Furthermore, a witness is required to monitor the procedures and guarantee an efficient traceability during i) the movements of the biopsied blastocyst from the biopsy dish (Supplementary Figure 1) to the post-biopsy dish (Supplementary Figure 1), then to the vitrification plate (Supplementary Figure 1) and lastly to the vitrification support (Supplementary Figure 1); ii) the transfer of biopsied TE cells from the biopsy dish to the PCR tube (Supplementary Figure 1); iii) the warming and transfer steps post-diagnosis. For a detailed description of all witnessing steps refer to a failure modes and effects analysis (FMEA) previously published14.
All methods described in this paper respect the local regulation (Italian Law 40/2004). According to the Law, in fact, the couple can request to be informed about the health status of the embryos they produced during the IVF cycle. In this regard, a detailed informed consent for PGT must be signed from both partners.
In this paper we described how to implement blastocyst biopsy for PGT in a busy laboratory routine. The application of blastocyst biopsy approach has been an important advancement in the last decade in IVF. First reported by de Boer and colleagues in 200417, it has been soon recognized as a more effective and informative procedure compared to cleavage stage and polar body biopsy approaches3. The value of this procedure mainly resides in a reduction of the technical burdens, but also in a lower incidence of chromosomal mosaicism at this stage of development18,19. Moreover, the removal of few TE cells from a blastocyst has been suggested as a safer procedure than the removal of one blastomere from a cleavage stage embryo. Indeed, in a randomized non-selection study the former approach did not result in any impact on embryo implantation potential, while the latter involved a significant ~20% reduction20.
The protocol mostly used worldwide entails laser-assisted zona opening in day 3 post-insemination. No randomized controlled trial has been conducted to date to compare the different blastocyst biopsy approaches. However, it is reasonable that the lower the number of manipulations and exposures of the growing embryos to suboptimal environmental conditions, the lower the potential invasiveness of the protocol. Moreover, the presence of a hole in the zona pellucida from day 3 of development might affect blastocyst expansion and cause the herniation of ICM cells together with TE cells. For these reasons, we set and implemented the protocol described here that entails the sequential laser-assisted zona breaching and TE cells retrieval as soon as the blastocyst reaches full expansion. Also a different protocol exists that entails a day 5 or 6 assisted hatching5,7. Specifically, the drilling of the zona is performed on day 5 or 6 of preimplantation development on the opposite side with respect to the ICM; the blastocyst is then moved back to the incubator waiting for the spontaneous herniation of TE cells. Clearly, periodic monitoring of the blastocyst must be provided in the following hours, to biopsy it as soon as the TE will start herniating, as well as to prevent the embryo from hatching completely. If on the one hand unskilled practitioners can easily implement this alternative biopsy strategy, on the other hand it is not suitable for a busy laboratory performing several procedures per day. The sequential zona opening and blastocyst biopsy protocol described here is instead less time-consuming and allows a shorter hands-on time and a higher flexibility to schedule the daily activity in the laboratory so to coordinate the timeframes dedicated to biopsy and to vitrification procedures.
Poor quality blastocysts might be more complex to biopsy since the TE cells can be sticky, but more laser shots coordinated with the stretching of the fragment to expose the junctions between the cells are sufficient to remove it from the body of the blastocyst. Fully hatched blastocyst can be biopsied like blastocysts enclosed in the zona pellucida, but they might be trickier to vitrify and warm. In case of inconclusive diagnoses after biopsy, the data reported to date are concordant that no harm seems to derive from a re-biopsy and a following vitrification-warming cycle16,21.
Vitrification is currently used in the center to perform blastocyst cryopreservation since it has been consistently reported safer, more efficient and less time-consuming than slow freezing protocol from a review and meta-analysis of the most recent literature10.
Once the procedure was well-established and the operators properly trained, we performed a retrospective analysis to define the ideal procedural timings for both blastocyst biopsy and vitrification procedures (summarized here in the representative results). As final outcomes, we have assessed the re-expansion rate of euploid blastocyst evaluated at 1.5 h after warming and the live birth rate achieved after vitrified-warmed euploid single embryo transfer. No case of blastocyst degeneration was observed after biopsy, and just 0.2% (N = 1/572) of degeneration rate was reported after warming, confirming the reliability of the biopsy and vitrification approaches adopted. According to the analysis, the timing required to perform blastocyst biopsy does not affect either euploid blastocysts’ viability after warming defined as re-expansion rate, or reproductive potential defined as live birth rate. Although various timings can be observed across operators with different expertise, we may consider blastocyst biopsy safe when the procedure is completed in about 8 min, in a range from 3 to 22 min depending on the number of embryos per procedure (however no data are available for biopsy procedures performed in longer intervals). The mean time spent for blastocyst biopsy from each single operator should be periodically (at least every three months) monitored as KPI. Alongside, the rate of inconclusive diagnosis and the live birth rate after vitrified-warmed euploid blastocyst transfer should be also addressed. Lastly, we outlined the ideal timing between biopsy and vitrification. Since we observed the highest re-expansion rate after warming when blastocysts were vitrified within 30 min from the biopsy, we suggest this value as the ideal threshold. Specifically, the longer the time between biopsy and vitrification, the more the biopsied blastocyst will re-expand before being cryopreserved. This might be harmful for cryo-survival, especially when dealing with poor-quality and/or day 7 blastocysts11. Nevertheless, no significant impact on the clinical outcomes was observed even if vitrification was delayed beyond 90 min. Therefore, in sporadic occasions, such timings might be allowed.
The method chosen to retrieve a specimen for PGT should not affect embryo viability, should involve reliable and informative results, should be clinically effective and should be easy to implement thereby reducing the costs and the laboratory workload. Blastocyst biopsy fulfills all these prerequisites. Nonetheless, it still is an invasive procedure that must be performed by skilled operators in a well-equipped laboratory. Currently, the avant-garde of a non-invasive PGT (niPGT) approach is under investigation. Perhaps, in the future, the spent culture media after IVF might be analyzed to conduct chromosomal and/or genetic testing. This is an intriguing future perspective since the costs for the IVF clinic would be lower and all the workload entailed by embryo biopsy would be sidestepped. However, the reliability and reproducibility of spent media analysis for genetic testing are still to be assessed22,23,24, therefore more efforts must be invested to define and validate a protocol that could suit all IVF clinics performing PGT worldwide.
The authors have nothing to disclose.
AG and RM collected the data and drafted the manuscript. DC analyzed the data, drafted the representative results, performed the statistics and revised the manuscript. FMU and LR provided critical discussion of the results and of the whole manuscript.
Equipment | |||
Cold tube rack | Biocision | XTPCR96 | |
Electronic pipette controller | Fisher Scientific | 710931 | |
Flexipet adjustable handle set | Cook | G18674 | Stripper holder |
Gilson Pipetman | Gilson | 66003 | p20 |
IVF Electronic Witness System | CooperSurgical Fertility & Genomic Solutions | RI Witness ART Management System | |
Inverted microscope | Nikon | Eclipse TE2000-U | |
Laminar Flow Hood | IVF TECH | Grade A air flow | |
Laser objective | RI | Saturn 5 | |
Microinjectors | Nikon Narishige | NT-88-V3 | |
Mini centrifuge for PCR tubes | Eppendorf | CSLQSPIN | for 0.2ml PCR tubes |
Stereomicroscope | Leica | Leica M80 | |
Thermostat | Panasonic | MCO-5AC-PE | |
Tri-gas incubator | Panasonic | MCO-5M-PE | 02/CO2 |
Consumables | |||
Biopsy pipette | RI | 7-71-30FB35720 | 30µm ID, flat 35°C |
Cryolock | Cryolock | CL-R-CT | |
CSCM complete | Irvine Scientific | 90165 | IVF culture medium supplemented with HSA |
Embryo Transfer Catheter | Cook | G17934 | |
Flexipet pipette | Cook | G26712 | 140µm stripping pipette tip |
Flexipet pipette | Cook | G46020 | 300µm stripping pipette tips |
Holding pipette | RI | 7-71-IH35/20 | 30µm ID, flat 35°C |
Human Serum Albumin | Irvine Scientific | 9988 | |
IVF One well dish | Falcon | 353653 | |
Mineral Oil for embryo culture | Irvine Scientific | 9305 | |
Modified HTF Medium | Irvine Scientific | 90126 | Hepes-Buffered medium |
Nuclon Delta Surface | Thermofisher scientific | 176740 | IVF dish 4-well plate with sliding lid |
Primaria Cell culture dish | Corning | 353802 | 60x15mm |
Reproplate | Kitazato | 83016 | |
Serological pipette | Falcon | 357551 | 10ml |
Sterile disposable Gilson tips | Eppendorf | 0030 075.021 | 200µl |
Tubing Kit | Provided by the genetic lab | PCR tubes (0.2mL), loading solution, biopsy washing solution | |
Vitrification media | Kitazato | VT801 | Equilibration and vitrification solutions |
Warming media | Kitazato | VT802 | Thawing and dilution solutions |