ABSTRACT
Purpose
Does an association exist between the ICM/TE hatching levels of biopsied blastocysts before vitrification and implantation?
Methods
Total 541 single blastocyst transfers in PGT‐A cycles were collected. The degree of TE/ICM extrusion from the zona pellucida before vitrification was categorized as (1) ICM hatching level ≤ 50% and > 50%, (2) TE hatching level ≤ 25% and > 25%.
Results
TE hatching levels were not associated with clinical pregnancy probability (p = 0.804). Multivariate regression analysis revealed a positive association between ICM hatching level ≤ 50% with implantation probability. Especially in the ICM Grade B group, blastocysts with ICM hatching level > 50% had a significantly lower clinical pregnancy rate (47.4%, 36/76) and ongoing pregnancy rate (39.5%, 30/76) than did blastocysts with ICM hatching levels of ≤ 50% (66.4%, 219/330 and 60.6%, 200/330, respectively) (p < 0.01). Additionally, the proportion of apoptotic ICM cells was significantly higher in the ICM hatching level of > 50% group (90.0%, 9/10) than that in the ICM hatching level of ≤ 50% group (23.1%, 9/39) (p < 0.001).
Conclusion
Blastocysts with an ICM hatching level of > 50% had the lower implantation potential, which may be related to the increased incidence of ICM cell apoptosis.
Keywords: apoptosis, hatching levels, inner cell mass, PGT‐A, vitrification
This graphical abstract illustrates that the ICM hatching level before vitrification is a significant predictor of implantation success in PGT‐A cycles. Specifically, blastocysts with an ICM hatching level of ≤ 50% are associated with higher clinical pregnancy rates, whereas those exceeding 50% show a markedly lower implantation potential. This decreased success rate in the > 50% group is likely attributed to a significantly higher incidence of ICM cell apoptosis.
1. Introduction
Blastocyst ploidy detected using preimplantation genetic testing for aneuploidy (PGT‐A) has been demonstrated to be strongly correlated with implantation [1, 2]. Blastocyst biopsy often involves techniques such as laser‐assisted hatching (LAH), trophectoderm (TE) biopsy, and vitrification [3]. To facilitate TE biopsy, LAH is commonly performed on Day 3 or Day 4 or at the blastocyst stage, creating an opening in the zona pellucida (ZP) and inducing TE cell herniation [4, 5]. It is recommended to perform LAH at the cleavage stage on Day 3 or Day 4 to avoid potential complications such as blastocyst collapse, increased biopsy difficulty, and prolonged biopsy duration [6]. However, LAH on Day 3 or Day 4 may cause blastocysts with varying hatching levels of TE/inner cell mass (ICM) between 0% and 100% before biopsy or vitrification [7].
Although nonphysiological hatching simplifies TE biopsy procedures, potential stress on embryos from these biopsy or vitrification procedures remains a concern [7]. Nevertheless, TE biopsy has been established as a safe method with minimal impact on blastocyst development [4, 8] and implantation potential [9]. Following biopsy, vitrification serves as a common method for blastocyst preservation until frozen embryo transfer. Studies have demonstrated the effectiveness of vitrification in preserving blastocysts with high postwarming survival rates [10, 11, 12]. However, inadequate cryopreservation methods, particularly when combined with blastomere biopsy, can negatively affect embryo viability [13]. In addition, inappropriate vitrification techniques or equilibration times can lead to the accumulation of high cryoprotectant concentrations, which are cytotoxic to embryos [14, 15, 16], potentially affecting survival and clinical outcomes in frozen embryo transfer cycles [17].
Theoretically, optimal vitrification protocols may vary based on blastocyst diameter or expansion levels [18], and therefore blastocysts at different hatching levels after TE biopsy may be affected by the freezing method. Whether direct exposure to the vitrification solution has specific effects on hatching blastocysts following TE biopsy remains unclear; however, studies have demonstrated that vitrified hatching or fully hatched vitrified blastocysts exhibit an elevated risk of reexpansion failure within 1.5 h of warming [9], potentially leading to poorer clinical outcomes [19, 20]. Nevertheless, the potential adverse effects of vitrification on clinical outcomes at different TE/ICM hatching levels after LAH and TE biopsy remain unclear. Therefore, the primary objective of this study was to determine whether previtrification ICM/TE hatching levels influence implantation probability and ongoing pregnancy rates (OPRs) after single blastocyst transfer (SBT) [21] in PGT‐A cycles. The secondary objective was to determine the incidence of apoptosis in blastocysts with varying hatching levels to evaluate potential vitrification‐related damage.
2. Materials and Methods
2.1. Ethical Approval
The study was carried out in accordance with the Declaration of Helsinki and the Institutional Review Board of Chung Shan Medical University approved this study protocol. This study employed retrospective analysis of clinical data of PGT‐A cycles, and prospectively examined donated aneuploidy blastocysts for apoptosis from Lee Women's Hospital. The clinical study cohort comprised 499 women who underwent PGT‐A (541 cycles) between January 2020 and May 2022. In addition, to demonstrate the extent of apoptosis after vitrification, another prospective study was filed using donated aneuploidy embryos. A total of 49 prospective donated blastocysts were collected between January 2023 and May 2024 for immunocytochemical staining. All the study methods and analyses of data collected from the Lee Women's Hospital database were approved by the Institutional Review Board (IRB) of Chung Shan Medical University, Taichung, Taiwan (CS1‐23027 and CS1‐21181). For the prospective study, written informed consent was obtained from all participating couples. For the retrospective study, the aforementioned Institutional Review Board (IRB) waived the informed consent requirement for retrospective data analysis.
2.2. Patient Selection, Embryo Culture and TE Biopsy and Vitrification
Ovarian stimulation, oocyte retrieval, in vitro fertilization (IVF) procedures, assessment of embryo morphology, and PGT‐A were conducted following previously established protocols [22, 23]. Conventional IVF or intracytoplasmic sperm injection was employed for fertilization. Subsequently, all embryos were cultured in a time‐lapse system by using sequential cleavage and blastocyst media supplemented with 15% SPS (Quinn's Advantage Cleavage Medium; Sage BioPharma) until Day 5 or Day 6. LAH was performed on Day 4 to create a hole of approximately 10 μm in the ZP. Prior to TE biopsy, blastocyst quality was assessed according to the criteria established by Gardner and Schoolcraft [24]. Only qualified blastocysts (Grades 4, 5, 6, AA, AB, BA, BB, or BC) with a diameter of ≥ 150 μm were subjected to TE biopsy. Following a 2‐h period incubation after biopsy, the biopsied blastocysts were then vitrified using Cryotech (Cryotech, Japan) according to the protocol described by Chen et al. [22, 23]. On the basis of next‐generation sequencing results, aneuploidy blastocysts were excluded from single embryo transfer cycles.
2.3. Assessments of Morphology of Biopsied Blastocysts Before Vitrification
To assess the potential influence of TE biopsy and vitrification protocols on clinical outcomes, blastocyst characteristics including ICM/TE grades, hatching levels, and position relative to the LAH hole were evaluated as potential confounding factors. Image J open‐source software was used to measure the TE/ICM hatching area [25]. The morphology and grading system of biopsied blastocysts before vitrification are provided in Figure S1. The ICM (TE) hatching level was defined as the ratio of the ICM (TE) hatching outer area to the total ICM (TE) area. Cycles were divided into two groups on the basis of ICM hatching area ratio: (1) ICM hatching level ≤ 50% and (2) ICM hatching level > 50%. Similarly, TE hatching levels were divided into two groups on the basis of the TE hatching area ratio: (1) TE hatching level ≤ 25% and (2) TE hatching level > 25%. ICM location relative to the LAH hole was defined as either “ICM near the hole (The distance between the ICM and the hole is less than 50% of the blastocoel diameter)” or “ICM far from the hole (The distance between the ICM and the hole is more than 50% of the blastocoel diameter).”
2.4. Warming and Frozen Embryo Transfer
On the basis of next‐generation sequencing results, aneuploidy blastocysts were excluded from embryo transfer cycles. One usable blastocyst was selected for each patient in the warming and transfer cycle. Embryo transfer protocols and procedures were performed as previously described [22, 23]. Serum β‐human chorionic gonadotropin (hCG) was measured 12 days after transfer. If β‐hCG was positive, clinical pregnancy was defined according to the presence of a gestational sac on transvaginal ultrasound at 6 weeks of gestation. A miscarriage before 20 weeks was defined as a spontaneous abortion. The ongoing pregnancy was defined as a fetus with heart activity after 20 weeks of gestation.
2.5. Evaluation of Apoptosis and Proportion of Apoptotic Cells
The incidence of apoptosis in donated vitrified blastocysts after warming was evaluated using terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate (dUTP) nick end labeling (TUNEL) staining [26]. The DeadEnd fluorescent TUNEL system (Promega Corporation, Madison, WI, USA) was employed to detect DNA fragmentation in apoptotic cell nucleoplasms (TUNEL+ appears green). Hoechst 33342 DNA stain was employed to visualize nuclear morphology. Following a 2‐h incubation period, the warming blastocysts were fixed in formaldehyde (4% in phosphate‐buffered saline [PBS]) for 10 min and then washed in PBS for 15 min. The blastocysts were then permeabilized in 0.1% Triton‐ × 100 for 2 min, and the embryos were incubated in fluorescent‐conjugated dUTP for 1 h at 37°C in darkness. Finally, after careful washing with PBS, the blastocysts were stained with Hoechst 33342 (5 μg/mL, 3 min), and fluorescence was observed using a Labopho‐2 microscope (Nikon, Japan). TUNEL+ nuclei localized within embryonic cells with a normal nucleus and intact plasma membrane were classified as internalized apoptotic cells. The proportion of blastocysts containing apoptotic cells was calculated. To assess the distribution of apoptosis in ICM or TE cells, the 49 donated blastocysts were further categorized into the following subgroups: (1) proportion of blastocysts containing apoptotic ICM cells in groups of ICM hatching level of > 50% or ≤ 50%, (2) proportion of blastocysts containing apoptotic TE cells in groups of TE hatching level of > 25% or ≤ 25%, and (3) additionally, ICM cell was classified as an “outside cell” or “inside cell” on the basis of its position relative to the LAH hole in the ZP. Furthermore, a total of 14 hatching blastocysts had both outside and inside ICM cells. These hatching blastocysts were further analyzed for the apoptosis occurrence of ICM in the inside and outside of the ZP.
2.6. Statistical Analysis
The outcome variables included clinical pregnancy probability and parameters such as the age of the women (years), endometrial preparation protocols, hormonal levels, endometrial thickness, oocyte source, embryo day, TE/ICM grade, pre‐vitrification TE/ICM hatching levels and ICM position relative to the LAH pore. Generalized estimating equations (GEEs) with logistic regression were employed to assess the effect of confounding factors on binary clinical pregnancy data (yes/no). The Mann–Whitney U, chi‐squared, and Fisher's exact tests were employed to assess nonparametric data. Finally, all statistical analyses were performed using SPSS Statistics version 23.0 (IBM SPSS, Armonk, NY, USA), and p < 0.05 was considered statistically significant.
3. Results
3.1. Clinical Pregnancy Rates Are Correlated With ICM Hatching Level and ICM Grade
A total of 541 PGT‐A cycles with SBT from 499 patients were analyzed in this study. The patient demographics including age, BMI, duration of infertility, endometrial preparation protocols, hormonal levels, endometrial thickness, oocyte source, embryo day, TE/ICM grade, pre‐vitrification TE/ICM hatching levels and ICM position relative to the LAH pore, and chromosomal status are presented in Table 1. The mean age of the analyzed women was 36.6 ± 4.4 (24–45) years. The overall clinical pregnancy rate (CPR), miscarriage rate, and OPR following SBT were 65.6% (355/541), 9.6% (34/355), and 59.1% (320/541), respectively.
TABLE 1.
Description of the study population by treatment outcome.
| Parameters | Values |
|---|---|
| Cycles | 541 |
| Women age (year) | 36.6 ± 4.4 (24–45) |
| BMI (kg/m2) | 22.2 ± 3.6 (16.4–45.2) |
| Duration (years) | 2.8 ± 2.8 (0–21.4) |
| Endometrial preparation protocols | |
| Hormone replacement therapy | 76.5% (414/541) |
| Nature cycle | 23.5% (127/541) |
| E2 level on ET day (pg/mL) | 267 ± 277 (20–4628) |
| P4 level on ET day (ng/mL) | 42.3 ± 39.2 (2.2–327.4) |
| Endometrial thickness | 10.9 ± 2.2 (7–20) |
| Oocyte source | |
| Autologous | 90.8% (491/541) |
| Egg donation | 9.2% (50/541) |
| Embryo day | |
| Day 5 | 73.4% (391/541) |
| Day 6 | 26.6% (144/541) |
| TE grade | |
| A | 16.5% (89/541) |
| B | 75.0% (406/541) |
| C | 8.7% (46/541) |
| ICM grade | |
| A | 25.0% (135/541) |
| B | 75.0% (406/541) |
| TE hatching levels before vitrification | |
| ≤ 25% hatching | 60.4% (327/541) |
| > 25% hatching | 39.6% (214/541) |
| ICM hatching levels before vitrification | |
| ≤ 50% hatching | 82.3% (445/541) |
| > 50% hatching | 17.7% (96/541) |
| ICM location before vitrification | |
| ICM near the hole of hatching site (≤ 50%) | 57.9% (313/541) |
| ICM far away the hole (> 50%) | 42.1% (228/541) |
| Embryo ploidy | |
| Euploid | 82.6% (447/541) |
| Mosaic | 17.3% (94/541) |
| Mosaic types | |
| Segmental | 87.2% (82/94) |
| Whole‐chromosomal | 12.8% (12/94) |
| Chromosome abnormal number | |
| 1 | 64.9% (61/94) |
| 2 | 19.1% (18/94) |
| > 2 | 16.0% (15/94) |
| Clinical outcomes | |
| Clinical pregnancy rate (CPR) | 65.6% (355/541) |
| Monozygotic twins | 3.9% (14/355) |
| Ectopic rate | 0.3% (1/355) |
| Miscarriage rate | 9.6% (34/355) |
| Ongoing pregnancy rate (OPR) | 59.1% (320/541) |
Abbreviations: BMI, body mass index; E2, estradiol; ET, embryo transfer; ICM, inner cell mass; P4, progesterone; TE, trophectoderm.
Univariate GEE regression indicated that embryo day, ICM grade, TE and ICM hatching levels were significantly associated with clinical pregnancy probability (p < 0.05, Table 2). After adjustment for all analyzed variables, multivariate regression analysis revealed that only ICM grade and ICM hatching levels were significantly associated with the implantation probability. Blastocysts with an ICM hatching level ≤ 50% exhibited a higher likelihood of achieving clinical implantation than did blastocysts with an ICM hatching level > 50% (odds ratio [OR]: 1.800, 95% confidence interval [CI]: 1.022–3.171, p = 0.042). Similarly, ICM grade A was positively associated with clinical pregnancy probability (OR: 1.615, 95% CI: 1.019–2.561, p = 0.041). No significant correlations were observed among embryo transfer day, TE hatching levels, and CPR (p > 0.05) after other variables were controlled for in the multivariate regression analysis. Notably, TE grade did not exhibit significant associations with clinical pregnancy probability.
TABLE 2.
Results of univariate and multivariate regression for clinical pregnancy.
| Variants | Univariate regression | Multivariate regression | ||||
|---|---|---|---|---|---|---|
| OR | 95% CI | p | OR | 95% CI | p | |
| Women age (years) | 0.990 | 0.952–1.029 | 0.596 | — | — | — |
| BMI (kg/m2) | 1.035 | 0.981–1.092 | 0.213 | — | — | — |
| Duration of infertility (year) | 0.954 | 0.899–1.013 | 0.127 | — | — | — |
| Hormone replacement therapy vs. natural cycle | 0.942 | 0.622–1.426 | 0.778 | — | — | — |
| E2 level on ET day (pg/mL) | 1.000 | 0.999–1.001 | 0.922 | — | — | — |
| P4 level on ET day (ng/mL) | 1.001 | 0.997–1.006 | 0.513 | — | — | — |
| Endometrial thickness | 1.069 | 0.978–1.168 | 0.142 | — | — | — |
| Autologous cycle vs. egg donation | 1.498 | 0.845–2.657 | 0.167 | — | — | — |
| Embryo Day 5 vs. Day 6 | 1.785 | 1.200–2.656 | 0.004 | 1.389 | 0.888–2.172 | 0.150 |
| ICM Grade A vs. Grade B | 1.681 | 1.073–2.632 | 0.023 | 1.615 | 1.019–2.561 | 0.041 |
| TE Grade A vs. Grade C | 1.711 | 0.810–3.615 | 0.159 | — | — | — |
| TE Grade B vs. Grade C | 1.502 | 0.810–2.784 | 0.197 | — | — | — |
| Euploid vs. Mosaic | 0.910 | 0.569–1.455 | 0.694 | — | — | — |
| Mosaic type‐whole chromosomal vs. euploid | 0.770 | 0.250–2.375 | 0.649 | — | — | — |
| Mosaic type‐segmental vs. euploid | 1.161 | 0.699–1.927 | 0.565 | — | — | — |
| Abnormal chromosome number > 2 vs. 0 | 0.628 | 0.224–1.765 | 0.378 | — | — | — |
| Abnormal chromosome number 2 vs. 0 | 0.797 | 0.301–2.110 | 0.647 | — | — | — |
| Abnormal chromosome number 1 vs. 0 | 1.429 | 0.782–2.610 | 0.245 | — | — | — |
| TE hatching levels ≤ 25% vs. > 25% | 1.554 | 1.081–2.233 | 0.017 | 1.062 | 0.663–1.699 | 0.804 |
| ICM hatching levels ≤ 50% vs. > 50% | 2.100 | 1.346–3.277 | 0.001 | 1.800 | 1.022–3.171 | 0.042 |
| ICM hatching site near the hole vs. far away the hole | 0.847 | 0.589–1.218 | 0.370 | — | — | — |
Abbreviations: BMI, body mass index; E2, estradiol; ET, embryo transfer; ICM, inner cell mass; OR, odds ratio; P4, progesterone; TE, trophectoderm; Whole Chr., whole chromosome aneuploidy.
3.2. Selecting Blastocysts With ICM Hatching Levels ≤ 50% and ICM Grade A May Yield Favorable Clinical Outcomes
After biopsy and incubation for 2 h, blastocysts were categorized into four groups according to ICM grade and ICM hatching levels before vitrification. Biopsied blastocysts with ICM grade A and ICM hatching levels ≤ 50% exhibited the highest CPR (75.6%, 87/115, Table 3) and OPR (69.6%, 80/115). Conversely, blastocysts with ICM Grade B and an ICM hatching level of > 50% exhibited the lowest CPR (47.4%, 36/76) and OPR (39.5%, 30/76). Notably, in the ICM grade A group, no significant differences in CPR or OPR were observed between blastocysts with ICM hatching levels ≤ 50% (CPR: 75.6%, 87/115; OPR: 69.6%, 80/115) and those with ICM hatching levels > 50% (CPR: 65.0%, 13/20; OPR: 55.0%, 11/20). However, in the ICM Grade B group, both the CPR and OPR were significantly higher for blastocysts with ICM hatching levels of ≤ 50% (CPR: 66.4%, 219/330; OPR: 60.6%, 200/330) than for blastocysts with ICM hatching levels > 50% (CPR: 47.4%, 36/76; OPR: 39.5%, 30/76) (p < 0.01). Although the miscarriage rates were higher in the blastocysts with ICM hatching levels > 50% (15.4% and 16.7%, respectively) than in those with ICM hatching levels ≤ 50% (8.0% and 8.7%, respectively), this difference was not statistically significant.
TABLE 3.
Comparison of clinical pregnancy rates among groups categorized on the basis of ICM grades and ICM hatching levels.
| ICM grade | A | B | ||
|---|---|---|---|---|
| ICM hatching level | > 50% | ≤ 50% | > 50% | ≤ 50% |
| Cycles | 20 | 115 | 76 | 330 |
| Women age (year) | 36.2 ± 3.3 | 35.6 ± 4.4 a , b | 37.4 ± 4.1 a | 36.8 ± 4.4 b |
| BMI (kg/m2) | 22.0 ± 3.0 | 22.4 ± 4.1 | 21.9 ± 2.9 | 22.2 ± 3.6 |
| Duration of infertility (year) | 3.1 ± 2.7 | 2.3 ± 2.2 | 3.3 ± 3.3 | 2.8 ± 2.9 |
| Hormone replacement therapy (%) | 85.0 (17/20) | 78.2 (90/115) | 85.6 c (65/76) | 73.3 c (242/330) |
| E2 level on ET day (pg/mL) | 210.6 ± 77.1 | 275.8 ± 427.1 | 243.5 ± 140.2 | 275.7 ± 240.7 |
| P4 level on ET day (ng/mL) | 35.1 ± 20.0 | 49.7 ± 48.0 d | 35.7 ± 28.5 d | 41.8 ± 38.6 |
| Endometrial thickness | 10.7 ± 1.6 | 10.8 ± 1.9 | 10.6 ± 2.2 | 11.0 ± 2.2 |
| Clinical pregnancy rate (%) | 65.0 (13/20) | 75.6 e (87/115) | 47.4 e , f (36/76) | 66.4 f (219/330) |
| Miscarriage rate (%) | 15.4 (2/13) | 8.0 (7/87) | 16.7 (6/36) | 8.7 (19/219) |
| Ongoing pregnancy rate (%) | 55.0 (11/20) | 69.6 g (80/115) | 39.5 g , h (30/76) | 63.3 h (209/330) |
Abbreviations: BMI, body mass index; E2, estradiol; ET, embryo transfer; P4, progesterone.
p = 0.005.
p = 0.004.
p = 0.024.
p = 0.010.
p < 0.001.
p = 0.002.
p < 0.001.
p < 0.001.
TE hatching levels were associated with ICM hatching levels. All blastocysts with an ICM hatching level > 50% also exhibited higher TE hatching levels (> 25%). Furthermore, even within the TE hatching level > 25% group, blastocysts in the ICM hatching level > 50% group exhibited a significantly lower CPR (51.0%, 49/96; Table S1) and OPR (42.7%, 41/96) than did blastocysts in the ICM hatching level ≤ 50% group (CPR: 66.1%, 78/118; OPR: 59.3%, 70/118) (p < 0.05).
3.3. Increased Incidence of ICM/TE Apoptosis After Vitrification of Blastocysts at High ICM/TE Hatching Levels
The TUNEL assay was conducted on donated blastocysts (n = 49) to evaluate apoptosis and to calculate the proportion of blastocysts containing apoptotic cells (Figure 1). Apoptosis occurred in both ICM and TE cells following vitrification in blastocysts with varying hatching levels (Figure 1). The proportion of blastocysts containing ICM apoptotic cells was significantly higher in the ICM hatching level > 50% group (90.0%, 9/10) than in the ICM hatching level ≤ 50% group (23.1%, 9/39; p < 0.001). However, no significant differences in the proportion of blastocysts containing apoptotic TE cells were observed between groups of the TE hatching level > 25% group (100%, 16/16) and the TE hatching level ≤ 25% group (78.8%, 26/33; p = 0.080). Furthermore, a total of 14 hatching blastocysts had both outside and inside ICM cells. These hatching blastocysts were further analyzed for the apoptosis occurrence of ICM in the inside and outside of the ZP. These blastocysts had a significantly higher occurrence of ICM apoptosis occurring outside of the ZP (71.4%, 10/14) than that within the ZP (14.3%, 2/14; p = 0.043).
FIGURE 1.
Apoptosis in hatched blastocysts after vitrification (sample size = 49). (A–F) Blastocysts with different ICM/TE hatching levels stained with Hoechst 33342 (left, blue color) and TUNEL (right, green color), The yellow arrow points to the ICM area, and the white arrow points to the TE area, (A) A blastocyst with ICM hatching level ≤ 50%, (B) A blastocyst with ICM hatching level > 50%, (C) A blastocyst with TE hatching level ≤ 25%, (D) A blastocyst with TE hatching level > 25%, (B,C and D) A hatching blastocyst containing apoptotic ICM cell distributed outside of the ZP, (E) Proportion of blastocysts containing apoptotic cells. According to the distribution of apoptosis in ICM or TE blastomeres, 49 donated blastocysts were further divided into the following subgroups: (1) proportion of blastocysts containing apoptotic ICM cells (grouped by ICM hatching level: > 50% or ≤ 50%, n = 49), (2) proportion of blastocysts containing apoptotic TE cells (grouped by TE hatching level: > 25% or ≤ 25%, n = 49), and (3) proportion of hatched blastocysts containing apoptotic ICM cells distributed inside or outside of the ZP (n = 14).
4. Discussion
To the best of our knowledge, this study was the first study to examine the relationship between pre‐vitrification ICM/TE hatching levels and implantation potential of biopsied vitrified blastocysts after PGT‐A cycles. The morphology and related ICM location of the biopsied blastocysts were also evaluated. Multivariable regression results revealed an association between CPRs and ICM hatching levels or ICM grades. Furthermore, blastocysts with an ICM hatching level of > 50% exhibited a significantly increased incidence of post‐vitrification ICM apoptosis.
ICM grade was identified as another vital factor influencing the CPR following PGT‐A SBT. Blastocysts with ICM grade A consistently exhibited a higher CPR than did blastocysts with ICM Grade B irrespective of the ICM hatching level (> 50% or ≤ 50%; Table 3). These findings support the prioritization of ICM grade A blastocysts for frozen single embryo transfer, consistent with previous studies suggesting that ICM grade is the strongest predictor of pregnancy success and live birth [27]. Although a minor degree of blastocyst damage after vitrification may not significantly reduce implantation potential [28], a reduction in the number of ICM cells following cryopreservation was linked to reduced survival rates [29] and reduced implantation potential [30, 31], potentially leading to implantation failure or pregnancy loss [29]. We suggest that a higher ICM grade may serve as a protective factor, mitigating potential damage caused by biopsy and vitrification procedures and ultimately contributing to an improved CPR and OPR. Furthermore, among ICM grade A blastocysts, those with ≤ 50% ICM hatching levels demonstrated higher clinical pregnancy and ongoing pregnancy rates compared to those with > 50% ICM hatching levels (CPR: 75.6% vs. 65.0%; OPR: 69.6% vs. 55.0%), though these differences did not reach statistical significance. The limited sample size within this subgroup likely constrained the power of the analysis for ICM Grade A blastocysts. Nevertheless, the numerical disparities suggest a preliminary trend favoring reduced ICM hatching levels. These observations require validation in larger, multicenter cohorts to definitively establish the clinical impact of hatching degree on high‐quality blastocysts.
In our study, we observed significant decreases in the CPR and OPR in blastocysts with an ICM hatching level > 50% compared with those in blastocysts with an ICM hatching level ≤ 50%. Conversely, the miscarriage rates in blastocysts with an ICM hatching level > 50% appeared higher than that in blastocysts with an ICM hatching level ≤ 50%. Therefore, we suggest that the effect of ICM hatching levels on the CPR may be more pivotal than that of TE hatching levels after biopsy and vitrification. The findings of this study suggest that several factors may contribute to the lower implantation rate observed in the blastocyst group with an ICM hatching level > 50%, including increased cryoprotectant exposure and mechanical effects. The transfer of fully hatched euploid blastocysts after vitrification has been reported to yield significantly lower success rates compared with the transfer of blastocysts in other stages of development [32], suggesting that increased cryoprotectant exposure due to TE biopsy‐induced hatching may compromise tolerance to vitrification and warming procedures [32, 33] suggested that the ZP confers a degree of protection for ICM and TE cells. The absence of the ZP may contribute to lower clinical outcomes, particularly in TE biopsy cycles. Furthermore, it has been reported that fully hatched euploid embryos are less likely to survive during the vitrification process [33]. The lack of the ZP may alter dehydration and cryoprotectant penetration processes [28, 34, 35], potentially leading to increased intracellular accumulation of cryoprotectants [21]. Although high concentrations of cryoprotectants are necessary for vitrification, they are also cytotoxic to embryos [14, 15, 16]. This cytotoxicity may be associated with altered protein expression [36], changes in intracellular pH [37], and increased intracellular calcium levels [24]. Additionally, cryopreservation solutions can generate formaldehyde, which may further contribute to cellular damage [38]. However, cryopreservation may have a more detrimental effect on ICM survival compared with TE cells [29]. Animal studies have demonstrated variations in permeability of the ICM and TE cells to water and cryoprotectants [39] and TE cells exhibit higher lipid content than does ICM, which has been associated with improved cryopreservation survival [40, 41, 42]. Consequently, we propose that vitrification may have a more significant detrimental effect on hatched ICM than on TE cells, resulting in reduced embryo implantation ability. Thus, blastocysts with an ICM hatching level of > 50% have a greater proportion of ICM cells exposed to cryoprotectants outside the ZP, potentially leading to increased cryoprotectant accumulation and compromised clinical outcomes [18]. In summary, our findings suggest that blastocysts enclosed in the ZP may have a survival advantage over hatched blastocysts, particularly those with an ICM hatching level > 50% or those that are fully hatched, during freezing and warming processes.
To further investigate the effects of vitrification on hatching blastocysts and ICM cells, we employed the TUNEL assay to assess apoptosis incidence. Although the biopsied blastocysts maintained intact morphology following vitrification, TUNEL staining revealed apoptosis in both ICM and TE cells across most donated blastocysts. Notably, blastocysts with an ICM hatching level of > 50% or a TE hatching level of > 25% exhibited a higher incidence of apoptosis in the ICM or TE cells (Figure 1). Furthermore, cells located outside the ZP exhibited a higher apoptosis incidence than did those remaining inside the ZP after vitrification. Blastocyst apoptosis is a vital indicator of compromised embryonic developmental potential [43, 44]. A mouse study indicated that TE biopsy itself may not induce apoptosis in blastocysts because DNA integrity remained comparable between intact and biopsied blastocysts [45]. However, following TE biopsy and vitrification, biopsied vitrified blastocysts exhibited significantly lower DNA integrity compared with intact vitrified blastocysts [45], suggesting a potential association between vitrification and blastocyst apoptosis after TE biopsy [45]. Consistent with these findings, other studies have documented increases in DNA fragmentation immediately after thawing [43, 44] and DNA damage in blastocysts subjected to all cryopreservation techniques [46]. Furthermore, blastocysts with a greater degree of expansion were more susceptible to cryopreservation‐induced DNA damage [46]. The apoptotic cascade can be triggered in bovine embryos following vitrification [47], and apoptosis appears to be positively correlated with cell number. Blastocysts with a lower total cell number exhibited a higher dead cell index [48]. Thus, apoptosis may cause a decline in the number of ICM cells after warming and may also be related to reduced survival rates [29]. In the present study, the observed increase in ICM apoptosis in the group with an ICM hatching level of > 50% was accompanied by a lower CPR and a higher miscarriage rate. Thus, we propose that a greater number of hatched cells, particularly those outside the protective ZP (especially ICM cells), were highly susceptible to the detrimental effects of vitrification, which would explain the negative impact on the clinical outcomes observed in this study.
This study proposed that in addition to the impact of vitrification, mechanical factors associated with ICM hatching levels may also contribute to implantation failure. Blastocysts with extensive hatching may be relatively susceptible to damage during embryo transfer procedures because of compromised structural integrity. Our laboratory experience suggests that highly hatched embryos require specialized manipulation skills to prevent accidental damage (e.g., sticking to the pipette used during transfer). Minimizing mechanical stress during manipulation has been proven essential for preserving embryo viability. We found that precise regulation of vitrification volumes and the embryo transfer process effectively mitigates risks associated with excessive blastocoel collapse and surface adhesion. To further reduce hydrodynamic shear stress, we employed wide‐bore pipettes (≥ 300 μm) combined with controlled, low‐pressure aspiration. Moreover, pre‐coating and rinsing all handling tools with media enriched with hyaluronan [49], albumin [50], or hydroxypropyl cellulose (HPC) [51] proved essential in preventing embryos from sticking to inner surfaces—a critical step for ensuring high recovery rates during vitrification and subsequent implantation.
Additionally, LAH typically creates a small opening (~10 μm) in the ZP. If the size of the ICM exceeds the inner diameter of the ZP opening, the ICM may become trapped when passing through this opening [52]. Blastocysts with a higher ICM hatching level are at higher risk of ICM incarceration [3]. Furthermore, during cryopreservation, freezing‐related ZP hardening may occur [53, 54], potentially exacerbating hatching difficulties and further increasing the risk of ICM incarceration during transfer [55]. ICM herniation has been linked to increased risk of ICM splitting before implantation [56, 57]. The smaller ICM fragment resulting from splitting may lag behind because of insufficient cell mass [58]. Although evidence related to ICM splitting or incarceration leading to a decreased implantation rate is currently unavailable, avoiding excessive blastocyst herniation before transfer is generally recommended [3]. Furthermore, the mechanical stress generated by trapping of embryonic cells (expansion or PGT biopsy) increases nuclear DNA shedding and may induce chromosome segregation errors [59]. The present study suggested that LAH‐induced or cryopreservation‐related ZP hardening may lead to ICM fragmentation and cell loss during embryo transfer. Additionally, highly hatched blastocysts may be susceptible to extrusion during manipulation and may cause TE cells to be expelled from the ZP, thereby affecting embryonic integrity.
To the best of our knowledge, this study was the first study to analyze the impact of pre‐vitrification blastocyst hatching morphology and degree (expansion and diameters) on clinical outcomes in PGT‐A and SBT cycles. However, several limitations of this study warrant consideration. Specifically, this study was limited by the use of a single vitrification protocol and a single LAH day. Accordingly, future studies should explore the potential need for tailored vitrification protocols optimized for blastocysts with varying hatching levels. Such optimization would involve striking a balance between maximizing dehydration and viscosity while minimizing cytotoxicity [45]. However, the optimal exposure times for blastocysts to equilibrium solutions remain unclear; thus, further investigation is necessary to determine suitable durations that align with the physiological capabilities of blastocysts [18, 21, 60]. Finally, a larger randomized clinical trial is warranted to confirm the present findings. In addition, a shorter incubation period after biopsy before vitrification may deserve further study for decrease of hatching portion. Data from our previous work suggests that facilitating partial or full blastocyst reexpansion can improve implantation success, as the kinetics of reexpansion often reflects an embryo's underlying metabolic vigor and developmental competence. However, this recovery window presents a trade‐off: prolonged culture may trigger spontaneous hatching, thereby increasing the susceptibility of the exposed inner cell mass (ICM) to mechanical damage. Alternatively, vitrifying collapsed blastocysts shortly after biopsy—ideally within 1 h—has been shown to minimize structural stress and preserve post‐thaw viability [61]. Under conditions where hatching progression is difficult to forecast, immediate vitrification thus serves as a pragmatic approach to safeguards. Defining the exact temporal balance between structural stability and physiological recovery remains a priority for optimizing clinical throughput.
On the basis of the observed negative impact of extensive ICM hatching, LAH performed at the time of blastocyst biopsy may be a viable strategy to minimize excessive ICM exposure and potentially improve outcomes [3]. However, LAH itself can induce blastocyst collapse and thus can extend the biopsy duration [6]. Beyond morphological parameters, the impact of embryonic developmental speed—specifically Day 5 versus Day 6—on clinical success cannot be overlooked. While recent large‐scale cohort studies demonstrate that euploid Day 6 embryos can achieve favorable results [62], Day 5 blastocysts generally maintain a competitive edge in implantation and live birth rates within unselected populations [63]. We accounted for this potential confounding by incorporating ‘embryo day’ as a covariate in our multivariable logistic regression analysis. Notably, the association between hatching stage and clinical outcome remained robust even after adjusting for the day of blastulation. This persistence confirms that the degree of hatching exerts an independent effect on embryo viability, suggesting that both developmental pace and hatching progression provide distinct, additive value in the clinical selection process.
The findings of this study indicate that regarding PGT‐A cycles, a comprehensive evaluation incorporating the ICM grade and blastocyst hatching levels may be beneficial for the selection of embryos with the highest potential for clinical pregnancy. Prioritizing blastocysts with ICM grade A and minimal ICM hatching levels for embryo transfer may optimize clinical outcomes.
Funding
The authors have nothing to report.
Ethics Statement
The study was carried out in accordance with the Declaration of Helsinki, and the Institutional Review Board of Chung Shan Medical University approved this study protocol (approval number CS1‐23027 and CS1‐21181). For the prospective study, written informed consent was obtained from all participating couples. For the retrospective study, the aforementioned institutional review board (IRB) waived the informed consent requirement for retrospective data analysis.
Consent
Consent for publication was obtained from each patient.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Figure S1: Morphology and grading system of biopsied blastocysts before vitrification. (A) ICM positioned near the hatching hole; (B) ICM positioned far from the hatching hole; (C) TE hatching ≤ 25%; (D) TE hatching > 25%; (E) ICM hatching ≤ 50%; (F) ICM hatching > 50%.
Table S1: Comparison of clinical pregnancy rates among groups categorized on the basis of ICM and TE hatching levels.
Acknowledgments
The authors thank all members of the embryology team at Lee Women's Hospital, in particular YI‐TING CHEN, PEI‐WUN LIN and SHU‐HUEI LIN, for help in the collection of the vitrification data and image analysis.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1: Morphology and grading system of biopsied blastocysts before vitrification. (A) ICM positioned near the hatching hole; (B) ICM positioned far from the hatching hole; (C) TE hatching ≤ 25%; (D) TE hatching > 25%; (E) ICM hatching ≤ 50%; (F) ICM hatching > 50%.
Table S1: Comparison of clinical pregnancy rates among groups categorized on the basis of ICM and TE hatching levels.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.