Abstract
Background
Recent studies have attempted to improve laboratory efficiency while preserving clinical outcomes by shortening the time to warm cryopreserved embryos, though it is still unclear for oocytes. This study thus aimed to evaluate the effects of a modified warming protocol (MWP) on embryonic development and pregnancy outcomes of vitrified donor oocytes.
Methods
The data of this retrospective cohort study were collected from women who underwent donor cycles (fresh or vitrified oocytes) at Lee Women’s Hospital, Taiwan, from January 2019 to August 2024. The sample included 13,103 donor oocytes, divided into three groups: conventional warming protocol (CWP) group (n = 8506), MWP group (n = 980), and fresh group (n = 3617).
Results
Survival rates after oocyte warming were similar between the CWP and MWP groups (93.7% vs. 93.9%, P > 0.05). Oocyte degeneration rates post-intracytoplasmic sperm injection (ICSI) were similar for vitrified-warmed and fresh oocytes (2.7–3.4% vs. 2.8%). Normal fertilization was lower for vitrified-warmed oocytes (79.5–79.6% vs. 83.0%, P < 0.05), while abnormal fertilization was higher (9.1–10.1% vs. 3.3%). Blastocyst formation and usable blastocyst formation were lower in the CWP group (57.5% and 35.4%) compared to MWP (77.3% and 51.4%) and fresh groups (69.2% and 48.5%). Ongoing pregnancy/live birth was higher in the MWP group than in the CWP group (66.7% vs. 50.4%, P < 0.05). Multivariate analysis showed a positive association between MWP and usable blastocyst formation (adjusted incidence rate ratio = 1.423, 95% CI = 1.268 to 1.597, P < 0.001), as well as ongoing pregnancy/live birth (adjusted odds ratio = 1.899, 95% CI = 1.002 to 3.6, P < 0.05).
Conclusions
This study suggests that the MWP enhances the blastocyst formation potential and pregnancy outcomes of vitrified-warmed oocytes, making it similar to that of fresh oocytes. Thus, the MWP may replace the CWP as the standard protocol for optimizing donor cycle outcomes.
Clinical trial number
Not applicable.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13048-025-01776-2.
Keywords: Donor oocytes, Vitrification, In vitro fertilization, Modified warming protocol
Background
By the early 2000s, oocyte vitrification had become a standard practice in fertility clinics worldwide. The first successful pregnancy and live birth from vitrified oocytes represented a pivotal milestone in assisted reproduction, leading to the widespread adoption of vitrification in assisted reproductive technology. Since then, vitrification has become a cornerstone of fertility preservation, particularly for women receiving cancer therapy and those participating in egg donation programs [1–3].
To mitigate cryoprotectant (CPA) toxicity during freezing, oocytes are initially equilibrated with the selected CPA at room temperature. This controlled equilibration enables the CPA to effectively penetrate the cell membrane, minimizing the risks of osmotic shock and toxicity. Subsequently, the oocytes were immersed in a vitrification solution (VS) with increased concentrations of CPA and rapidly cooled to an ultralow temperature to complete the vitrification process [4]. In the Cryotec thawing cycle, cryopreserved oocytes are rapidly warmed to 37 °C in a thawing solution (TS, 1 min); this approach prevents the formation of ice and maintains the structural integrity of the glass-like state [4]. During warming, the CPA is swiftly diluted and removed to further mitigate the risks of cryoprotectant-induced damage and toxicity. A gradient dilution approach involving the incubation of oocytes in dilution solution (DS, 3 min) and wash solution (WS, 5–6 min) at room temperature is followed to minimize osmotic stress, ensuring the uniform rehydration of the oocytes and mitigating the risk of membrane rupture [1]. Despite being effective, this conventional warming protocol (CWP) is complex and time-intensive [5].
Several studies have contributed to simplifying the warming protocol. For example, Yan et al. demonstrated that the all-37 °C thawing method can maintain the clinical outcomes of vitrified-warmed embryo transfer without adjusting the temperatures during the warming process [6]. Besides, embryos were thought to be less susceptible to osmotic stress. Recent research has focused on shortening the time required for warming cryopreserved embryos, aiming to enhance laboratory efficiency while maintaining clinical outcomes. Lieberman et al. introduced one of the modified warming protocols (MWP) that eliminates the need for DS and WS, simplifying the CWP into an ultrafast, single-step process, with incubation in TS for 1 min. This MWP not only reduces the time required for blastocyst warming but also improves the outcomes of pregnancy [7]. In addition, it achieves survival and re-expansion rates similar to those observed with the CWP [7, 8]. This reduced warming time mitigates fatigue among embryologists, thereby optimizing clinical workflow.
Oocytes are highly sensitive to cryopreservation-induced stress [9–11], which affects the applicability of MWPs to vitrified oocytes. Although these protocols can improve the efficiency of embryonic cryopreservation, their suitability for vitrified oocytes remains unclear. Thus, the present study sought to determine whether the MWP can be safely and effectively applied to vitrified oocytes without compromising their viability or developmental potential.
Results
A total of 9486 mature oocytes from 452 vitrified donor cycles and 3617 mature oocytes from 174 fresh donor cycles were analyzed in this study (Tables 1 and 2). All vitrified oocytes were thawed, fertilized, and cultured to the blastocyst stage. Blastocysts that met the morphological criteria on day 5 or 6 were selected for either cryopreservation or transfer. Clinical outcomes were compared between vitrified-warmed oocytes and fresh oocytes.
Table 1.
Overview of patient baseline and cycle characteristics
| Groups | MWP | CWP | Fresh |
|---|---|---|---|
| Cycles | 44 | 408 | 174 |
| Donor age, years | 26.2 ± 3.9 | 25.5 ± 3.7 | 25.8 ± 3.6 |
| Donor AMH, ng/mL | 6.9 ± 3.3 | 6.1 ± 2.8 | 6.2 ± 3.3 |
| Donor BMI, kg/m2 | 20.5 ± 1.8a | 21.5 ± 2.9a | 21.6 ± 3.3 |
| Stimulation protocols, % (n) | |||
| GnRHa | 0% (0/44)a | 0% (0/408)b | 50.0% (87/174)ab |
| GnRHanta | 0% (0/44)a | 17.2% (70/408)a | 13.8% (24/174) |
| PPOS | 100% (44/44)ac | 82.8% (338/408)bc | 36.2% (63/174)ab |
| Trigger methods, % (n) | |||
| hCG | 2.3% (1/44) | 9.1% (37/408) | 0% (0/174) |
| GnRHa | 18.2% (8/44)ac | 50.0% (204/408)bc | 81.0% (141/174)ab |
| Dual trigger | 79.5% (35/44)ac | 40.9% (167/408)bc | 18.9% (33/174)ab |
| Total FSH dosage, IU | 2206 ± 315ab | 2410 ± 386b | 2400 ± 351a |
| Serum hormone levels | |||
| Basal FSH, mlU/mL | 6.7 ± 1.8 | 6.3 ± 2.6 | 6.9 ± 2.1 |
| Basal LH, mlU/mL | 5.1 ± 2.2 | 6.4 ± 6.5 | 5.9 ± 5.6 |
| Basal E2, pg/mL | 51 ± 22 | 81 ± 86a | 51 ± 31a |
| Trigger day LH, mlU/mL | 3.4 ± 5.4 | 2.3 ± 3.1 | 2.4 ± 3.3 |
| Trigger day E2, pg/mL | 6375 ± 3810 | 5888 ± 4090 | 6375 ± 4098 |
| Trigger day P4, ng/mL | 1.2 ± 0.9 | 1.4 ± 1.3 | 1.4 ± 0.9 |
a, b, cChi-square and Kruskal-Wallis statistics were used to identify significant differences in the row. The abbreviations “AMH”, “BMI”, “GnRHa”, “GnRHanta”, “PPOS”, “hCG”, “FSH”, “LH”, “E2”, “P4”, “MWP”, and “CWP” denoted the anti-Mullerian hormone, body mass index, gonadotropin-releasing hormone agonist, gonadotropin-releasing hormone antagonist, progestin-primed ovarian stimulation, human chorionic gonadotropin, follicle-stimulating hormone, luteinizing hormone, estradiol, progesterone, the modified warming protocol, and the conventional warming protocol, respectively.
Table 2.
Survival, fertilization, and embryo development outcomes using donor oocytes
| Groups | MWP | CWP | Fresh | |
|---|---|---|---|---|
| Oocytes | 980 | 8506 | 3617 | |
| Survival rates, % (n) |
93.9% (920/980) |
93.7% (7967/8506) |
–– | |
| Fertilization methods | ICSI | ICSI | ICSI | Insemination |
| Degeneration rates, % (n) |
2.7% (25/920)a |
3.4% (268/7967)b |
2.8% (60/2106)c |
0% (0/1511)abc |
| Unfertilization rates, % (n) |
7.6% (70/920)ab |
8.0% (637/7967)cd |
10.9% (229/2106)ace |
15.8% (239/1511)bde |
| Abnormal fertilization rates, % (n) |
10.1% (93/920)ab |
9.1% (725/7967)cd |
3.3% (69/2106)ace |
15.0% (227/1511)bde |
| 1PN rates, % (n) |
7.3% (67/920)ab |
7.3% (579/7967)cd |
2.2% (47/2106)ace |
0.9% (13/1511)bde |
| ≥ 3PN rates, % (n) |
2.8% (26/920)abc |
1.8% (146/7967)ade |
1.0% (22/2106)bdf |
14.2% (214/1511)cef |
| Normal fertilization rates, % (n) |
79.6% (732/920)ab |
79.5% (6337/7967)cd |
83.0% (1748/2106)ace |
69.2% (1045/1511)bde |
| Blastocyst rates, % (n) |
77.3% (566/732)abc |
57.5% (3645/6337)ade |
69.2% (1210/1748)bdf |
64.4% (673/1045)cef |
| Usable blastocyst rates, % (n) |
51.4% (376/732)ab |
35.4% (2246/6337)acd |
48.5% (847/1748)ce |
44.5% (465/1045)bde |
| Day 5 |
27.3% (200/732)a |
17.2% (1091/6337)abc |
31.0% (542/1748)b |
29.5% (308/1045)c |
| Day 6 |
24.0% (176/732)abc |
18.2% (1155/6337)ad |
17.4% (305/1748)b |
14.9% (156/1045)cd |
a, b, c, d, e, f Chi-square statistic was used to identify significant differences in the row. The abbreviations “MWP”, “CWP”, “ICSI”, “1PN”, and “3PN” denoted the modified warming protocol, the conventional warming protocol, intracytoplasmic sperm injection, 2-pronucleus embryos, and 3-pronucleus embryos, respectively.
Cycle characteristics of oocyte donors
Figure 1 depicts the morphology of the vitrified-warmed oocytes. The MWP group exhibited a more intact cell membrane structure, less swelling, and fewer cytoplasmic vacuoles than did the CWP group. Table 1 presents a summary of oocyte donors’ basal and cycle characteristics. No significant between-group difference was observed in donor age or anti-Müllerian hormone (AMH) level. However, the MWP group had a lower body mass index (BMI, 20.5 ± 1.8 kg/m2) than did the other groups (21.5 ± 2.9 kg/m2 and 21.6 ± 3.3 kg/m2). Ovarian stimulation protocols varied across the 3 groups: the progestin-primed ovarian stimulation (PPOS) protocol was exclusively used in the MWP group (100%), whereas the gonadotropin-releasing hormone agonist (GnRHa) protocol was used in the fresh group (50%). Between-group differences were also observed in the trigger method: dual triggers were most common in the MWP group (79.5%), whereas GnRHa triggers were most common in the fresh group (81%).
Fig. 1.
Morphology of vitrified-warmed oocytes after warming. The figure depicts oocytes with (A) a normal structure, (B) small vacuoles in the cortical region (arrows), (C) an uneven organelle distribution and partially darkened cytoplasm (arrow), and (D) a swollen membrane or disrupted outer boundary (arrow)
The total dosage of follicle-stimulating hormone (FSH) was significantly lower in the MWP group than in the CWP and fresh groups (2206 ± 315 IU vs. 2410 ± 386 IU vs. 2400 ± 351 IU, P < 0.05). The basal levels of FSH and luteinizing hormone (LH) were consistent across the 3 groups. However, the basal level of estradiol (E2) was the highest in the CWP group (81 ± 86 pg/mL). On the trigger day, the 3 groups exhibited similar levels of LH, E2, and progesterone (P4). These findings suggested significant between-group differences in protocol preference and hormone levels, indicating the importance of tailored strategies for oocyte donors. These differences further highlighted the need for adjusting the aforementioned factors in subsequent analyses.
Survival and fertilization rates for donor oocytes
The vitrified mature oocytes were warmed using either the MWP (n = 980) or the CWP (n = 8506). After a 2 h culture period, the MWP and CWP groups exhibited similar rates of survival (93.9% vs. 93.7%). The vitrified-warmed oocytes were subjected to fertilization through ICSI. The outcomes were compared with those of fresh oocytes fertilized either through ICSI or conventional insemination (Table 2). The rates of oocyte degeneration after ICSI were similar between the vitrified-warmed oocytes and fresh oocytes (2.7–3.4% vs. 2.8%). However, the unfertilization rate was lower for the vitrified-warmed oocytes than for the fresh oocytes (7.6–8.0% vs. 10.9%, P < 0.05). By contrast, the rate of abnormal fertilization was significantly higher for the vitrified-warmed oocytes than for the fresh oocytes (9.1–10.1% vs. 3.3%), with elevated rates of 1-pronucleus (1PN) embryonic formation (7.3% vs. 2.2%) and 3-pronucleus embryonic formation (1.8–2.8% vs. 1%). Consequently, the rate of normal fertilization was significantly higher for the fresh oocytes than for the vitrified-warmed oocytes (83% vs. 79.5–79.6%, P < 0.05). In addition, fresh oocytes fertilized through conventional insemination exhibited a lower rate of normal fertilization than did those fertilized through ICSI (69.2% vs. 83.0%, P < 0.05, Table 2).
Blastocyst formation from normally fertilized donor oocytes
Blastocyst formation on day 5 or 6 was assessed in normally fertilized embryos derived from vitrified-warmed and fresh oocytes (Table 2). For oocytes fertilized through ICSI, the rate of blastocyst formation was lower in the CWP group than in the MWP and fresh groups (57.5% vs. 77.3% vs. 69.2%, P < 0.05). The rate of usable blastocyst formation in the MWP group was similar to that in the fresh group but significantly higher than that in the CWP group (51.4% vs. 48.5% vs. 35.4%). On days 5 and 6, the MWP group exhibited significantly higher rates of usable blastocyst formation than did the CWP group (day 5: 27.3% vs. 17.2%; day 6: 24% vs. 18.2%; P < 0.05). In addition, the rates of blastocyst formation and usable blastocyst formation were lower for fresh oocytes fertilized through conventional insemination than for those fertilized through ICSI (64.4% vs. 69.2% and 44.5% vs. 48.5%, respectively, P < 0.05, Table 2).
Association between warming protocols and usable blastocyst formation
Univariate Poisson regression revealed the potential variables that significantly influenced the rate of usable blastocyst formation (Supplementary Table 1). The incidence rate ratio (IRR) for usable blastocyst formation increased with the fresh protocol (fresh vs. CWP, IRR = 1.335, 95% confidence interval [CI] = 1.190 to 1.498, P < 0.001) and the MWP (MWP vs. CWP, IRR = 1.477, 95% CI = 1.284 to 1.699, P < 0.001). Other factors significantly influencing the IRR for usable blastocyst formation were as follows (Supplementary Table 1): donor AMH levels (IRR = 1.029, 95% CI = 1.009 to 1.048), trigger methods (GnRHa vs. dual triggers, IRR = 1.132, 95% CI = 1.022 to 1.255), fertilization methods (ICSI vs. half-ICSI, IRR = 0.780, 95% CI = 0.695 to 0.875), basal E2 levels (IRR = 0.999, 95% CI = 0.998 to 1.000), P4 levels on the trigger day (IRR = 1.035, 95% CI = 1.000 to 1.071), and the number of 2-pronucleus (2PN) embryos (IRR = 1.057, 95% CI = 1.051 to 1.063). After being adjusted for the aforementioned factors, the multivariate Poisson regression model confirmed the associations between the warming protocols and the IRR for usable blastocyst formation. The IRR for usable blastocyst formation was significantly higher in the MWP group than in the CWP group (IRR = 1.423, 95% CI = 1.268 to 1.597, Table 3).
Table 3.
Assessing associations between variables and usable blastocysts using multivariate Poisson regression
| Variables | IRR | Significance | 95% CI | |
|---|---|---|---|---|
| Lower | Upper | |||
| Donor AMH | 0.997 | 0.740 | 0.979 | 1.015 |
| Trigger methods: GnRHa vs. dual trigger* | 1.027 | 0.556 | 0.939 | 1.124 |
| Trigger methods: hCG vs. dual trigger* | 0.994 | 0.944 | 0.850 | 1.163 |
| Fertilization methods: ICSI vs. half ICSI* | 0.627 | < 0.001 | 0.513 | 0.768 |
| Basal E2 | 1.000 | 0.679 | 0.999 | 1.000 |
| P4 (trigger day) | 0.973 | 0.252 | 0.929 | 1.061 |
| 2PN numbers | 1.054 | < 0.001 | 1.047 | 1.061 |
| Fresh vs. CWP* | 1.216 | 0.034 | 1.015 | 1.458 |
| MWP vs. CWP* | 1.423 | < 0.001 | 1.268 | 1.597 |
The abbreviations “IRR”, “CI”, “AMH”, “GnRHa”, “hCG”, “ICSI”, “E2”, “P4”, “2PN”, “MWP”, and “CWP” denoted the incidence rate ratio, confidence interval, anti-Mullerian hormone, gonadotropin-releasing hormone agonist, human chorionic gonadotropin, intracytoplasmic sperm injection, estradiol, progesterone, 2-pronucleus embryos, the modified warming protocol, and the conventional warming protocol, respectively. *indicated the reference group.
Pregnancy outcomes of different warming protocols
This pregnancy dataset excluded patients who had experienced at least three consecutive implantation failures with donor oocytes. The analysis involved 638 embryo transfer cycles, including 29 cycles with embryos of uncertain ploidy and 609 cycles with embryos of known ploidy. The average embryo number per transfer was 1.6 ± 0.6. Patients were transferred with selected day 5 (66.6%, 425/638), day 6 (18.5%, 118/638), or both days 5 and 6 (14.9%, 95/638) blastocysts based on embryo morphology and/or embryonic ploidy status. Endometrial preparation for the recipients was accomplished using either the artificial cycle protocol (98.1%, 626/638) or the modified natural cycle protocol (1.8%, 12/638). The pregnancy outcomes of different warming protocols were revealed in Table 4. The MWP group had the highest rates of implantation, clinical pregnancy, and ongoing pregnancy/live birth (64.2%, 75%, and 66.7%) as compared to the CWP (47.2%, 64.6%, and 50.4%) and fresh groups (52.5%, 58.4%, and 50.8%). Several factors were found to differ between groups of embryo transfer cycles, including BMI, endometrial preparation methods, preimplantation genetic testing for aneuploidy (PGT-A), embryo transfer day, and embryo transfer numbers (Table 4). Logistic regression analysis was therefore applied to discover the potential variables that significantly influenced the probabilities of ongoing pregnancy/live birth. Backward elimination was used to identify the potential confounders until the remaining variables had a P value < 0.2 (Supplementary Table 2). After controlling for embryo day and transferred embryo numbers, this study confirmed a positive association between MWP and ongoing pregnancy/live birth (MWP vs. CWP, adjusted odds ratio [OR] = 1.899, 95% CI = 1.002 to 3.6, P < 0.05, Supplementary Table 2).
Table 4.
Clinical outcomes in embryo transfer cycles using vitrified-warmed or fresh donor oocytes
| Groups | MWP | CWP | Fresh |
|---|---|---|---|
| Cycles | 48 | 393 | 197 |
| Recipient age | 43.5 ± 4.4 | 42.8 ± 5.5 | 43.3 ± 5.7 |
| Recipient BMI | 24.3 ± 3.7ab | 22.7 ± 3.5a | 23.0 ± 3.7b |
| Donor age | 26.5 ± 4.0 | 25.4 ± 3.7 | 25.6 ± 3.3 |
| Sperm with OAT, % (n) | 8.3% (4/48) | 12.2% (48/393) | 13.2% (26/197) |
| Preparation of endometrium, % (n) | |||
| Artificial cycle | 93.8% (45/48)a | 97.7% (384/393)b | 100% (197/197)ab |
| Modified natural cycle | 6.3% (3/48) | 2.3% (9/393) | 0% (0/197) |
| PGT-A, % (n) | 83.3% (40/48)a | 98.2% (386/393)ab | 92.9% (183/197)b |
| Embryo day, % (n) | |||
| Day 5 | 66.7% (32/48)ab | 50.9% (200/393)ac | 98.0% (193/197)bc |
| Day 5 and Day 6 | 20.8% (10/48)a | 20.9% (82/393)b | 1.5% (3/197)ab |
| Day 6 | 12.5% (6/48)ab | 28.2% (111/393)ac | 0.5% (1/197)bc |
| Embryo numbers | 1.7 ± 0.5a | 1.7 ± 0.6b | 1.4 ± 0.5ab |
| Implantation, % (n) | 64.2% (52/81)a | 47.2% (324/687)a | 52.5% (146/278) |
| Clinical pregnancy, % (n) | 75.0% (36/48)a | 64.6% (254/393) | 58.4% (115/197)a |
| Ongoing pregnancy/live birth, % (n) | 66.7% (32/48)ab | 50.4% (198/393)a | 50.8% (100/197)b |
a, b, c Chi-square and Kruskal-Wallis statistics were used to identify significant differences in the row. The abbreviations “CWP”, “MWP”, “BMI”, “OAT”, and “PGT-A” denoted conventional warming protocol, modified warming protocol, body mass index, oligoasthenoteratozoospermia, and preimplantation genetic testing for aneuploidy, respectively.
Discussion
To streamline workflow and reduce embryologist fatigue, in vitro fertilization (IVF) researchers have focused on modifying the warming protocols for vitrified embryos [5]. The ultrafast protocol eliminates intermediary steps, considerably reducing handling time to only 1 min of incubation in TS. This simplified procedure alleviates stress during frozen blastocyst transfer while maintaining survival, re-expansion, and clinical outcomes (e.g., implantation and pregnancy rates) similar to those of the CWP [7, 8, 12]. However, rapid warming leads to necrosis and excessive rehydration [12]. Besides, after vitrification with a shortened 2 min protocol, an ultrafast protocol was developed for warming immature human oocytes by substituting TS with DS [5, 13]. Although warming in DS with 0.5 M sucrose preserves the oocytes’ ability to resume nuclear meiotic activity and supports post-thaw survivability, the effect of this ultrafast protocol on the developmental potential of thawed oocytes remains to be explored and elucidated.
Our initial analysis revealed that vitrified in vitro matured oocytes warmed using the ultrafast protocol exhibited poor morphological features such as vacuoles and inflated membranes (Fig. 1), indicating that excessive rehydration may cause post-thaw damage, consistent with the findings of another study [12]. This result could be attributed to the fact that we used the conventional vitrification protocol, which included an extended period (12–15 min) of equilibration solution. In this circumstance, the vitrified-warmed oocytes were more sensitive to the ultrafast warming protocol [5, 13]. To avoid drastic osmotic changes, in the present study, the CWP was modified by eliminating the WS step, thereby developing a 2-step warming protocol, that is, an MWP. This modification reduced the manipulation time from 9 to 4 min. Oocytes warmed using the MWP exhibited an intact cell membrane with few morphological abnormalities. An analysis of 980 mature donor oocytes warmed using the MWP revealed that the MWP yielded survival and fertilization outcomes similar to those achieved with the CWP (Table 2). The rates of oocyte degeneration after ICSI remained low for the vitrified-warmed (2.7–3.4%) oocytes, consistent with the Vienna consensus benchmark (< 5%) [14], as compared to the fresh group (2.8%)(Table 2).
In the present study, the rate of normal fertilization was significantly lower for the vitrified-warmed oocytes than for the fresh oocytes (79.5–79.6% vs. 83.0%, Table 2), likely because of the higher incidence rates of abnormal fertilization in the MWP and CWP groups (9.1–10.1%), as observed through time-lapse monitoring. Studies have highlighted that oocytes contain components essential for the remodeling of sperm chromatin [15, 16]. Vitrification may alter the oocyte cytoplasm [17, 18], thereby preventing the decondensation of sperm chromatin and the formation of pronuclei [19]. Vitrification and warming may also alter oocytes’ metabolic properties—for example, by increasing reactive oxygen species levels [20, 21], further hindering sperm decondensation [22]. These findings are consistent with our finding of an increased rate of 1PN embryonic formation from vitrified-warmed oocytes. Besides, cryopreservation may damage the meiotic spindle, limiting oocytes’ ability to coordinate fertilization processes [23, 24]. Vitrified-warmed oocytes exhibit delayed recovery of the metaphase II plate structure and error-prone kinetochore-microtubule attachments [24, 25]. These aberrations in the meiotic spindle may cause abnormal extrusion of the second polar body, resulting in fertilization failures or abnormalities observed in the present study.
For oocytes fertilized through ICSI, the MWP group exhibited the highest rates of blastocyst formation and usable blastocyst formation; these rates were higher than those of the CWP group and similar to those of the fresh group (77.3% vs. 57.5% vs. 69.2% and 51.4% vs. 35.4% vs. 48.5%, respectively, Table 2). The multivariate Poisson regression model adjusted for various confounders (such as donor AMH levels, trigger methods, fertilization methods, basal E2 levels, P4 levels on the trigger day, and 2PN embryo numbers) revealed that the MWP outperformed the CWP (adjusted IRR: 1.423; Table 3). Besides, the MWP group demonstrated higher rates of implantation, clinical pregnancy, and ongoing pregnancy/live birth (64.2% vs. 47.2%, 75% vs. 64.6%, and 66.7% vs. 50.4%, respectively) when compared to the CWP group (Table 4). After controlling for confounders, such as embryo days and transferred embryo numbers, the multivariate logistic regression model revealed that the MWP performed better than the CWP (adjusted OR: 1.899; Supplementary Table 2). The MWP was specifically designed to minimize severe osmotic stress and conduct all steps at 37 °C. This 37 °C warming strategy promotes the formation of blastocysts from vitrified-warmed oocytes. A retrospective case-control study reported that maintaining the temperature at 37 °C during embryonic warming increased clinical pregnancy and implantation rates in frozen-thawed embryo transfer cycles, particularly for blastocysts [6]. A randomized controlled trial indicated that rehydrating vitrified oocytes at 37 °C markedly improved post-thaw survival rates; the fertilization, implantation, and clinical pregnancy rates were slightly higher with the 37 °C warming strategy than with conventional methods, underscoring the potential benefits of this strategy for vitrified oocytes [26].
A study demonstrated that the stability of the meiotic spindle in oocytes is dependent on the meiosis stage and temperature [27]. During metaphase II, oocytes are particularly vulnerable to spindle depolymerization at low temperatures [27, 28], which indicates the importance of temperature regulation during warming [29]. Regarding CPA toxicity, intracellular cryoprotectants can rapidly diffuse out of oocytes at 37 °C [30]. The MWP, which involves rapid dilution into TS and DS, likely reduces residual CPA levels within or around the oocytes, mitigating CPA toxicity. To the best of our knowledge, this study is the first to demonstrate that the MWP promotes the in vitro development and pregnancy outcomes of donor oocytes by shortening the warming process and rehydrating the oocytes at 37 °C.
This study has some limitations. First, its retrospective design may have introduced biases such as selection and randomization biases, and the sample sizes appeared to vary between groups. For example, based on the patient’s preferences or the results of consultations, our clinic offers fresh or vitrified-warmed donor oocytes. Patients who have experienced previous IVF failures or recurrent miscarriages are more likely to prefer fresh donor oocytes, which may contribute to the experimental bias. Second, several factors related to in vitro development and pregnancy outcomes significantly varied across the MWP, CWP, and fresh groups, despite the use of donor oocytes (Tables 1 and 4). Although a multivariate Poisson or logistic regression analysis was conducted to adjust for potential confounders and confirm the IRR for usable blastocyst formation or the OR for ongoing pregnancy/live birth with the MWP, the findings remain to be validated through randomized controlled trials. Finally, because this study relied on donor oocytes, further research is required to determine the applicability of the MWP to various clinical settings. Comprehensive evaluation and validation across clinical settings and laboratory conditions are also necessary before the implementation of the MWP in routine practice.
Conclusion
This study reveals that vitrified donor oocytes warmed using the MWP exhibit higher rates of blastocyst formation and pregnancy outcomes than those warmed using the CWP. Thus, the MWP holds promise as a superior alternative to the CWP in clinical practice. Our findings suggest that reducing the rehydration duration and maintaining the warming temperature at 37 °C can help improve clinical outcomes.
Methods
Study design and cohort
This retrospective study was conducted using relevant clinical data from women who had undergone donor cycles at Lee Women’s Hospital, Taiwan, between January 2019 and August 2024. The study protocol was approved by the Institutional Review Board of Chung Shan Medical University, Taichung, Taiwan (approval number: CS1-23027). The requirement for informed consent was waived by the institutional review board because of the retrospective nature of this study.
The women included in this study had undergone donor cycles for various reasons, such as diminished ovarian reserve, advanced maternal age, and multiple IVF failures. Recipients and anonymous donors were matched by blood type. All oocyte donors were aged under 35 years, had a BMI of 18 to 30 kg/m2, had a serum AMH level of 2.0 ng/mL or higher, and exhibited no signs of mental disorders or abnormalities in general physiological and ultrasonographic examinations. Women with infectious diseases (e.g., syphilis, gonorrhea, hepatitis B, and chlamydia) or hereditary conditions (e.g., chromosomal abnormalities, spinal muscular atrophy, fragile X syndrome, epilepsy, hemophilia, thalassemia, diabetes, and color blindness) were excluded from the donation program.
Ovarian stimulation and oocyte vitrification
Controlled ovarian stimulation was performed following previously defined protocols, such as those involving GnRHa use, gonadotropin-releasing hormone antagonist use, or PPOS [31, 32]. Oocyte retrieval was scheduled and performed more than 36 h after ovulation was triggered. The retrieved donor oocytes were either fertilized in vitro or cryopreserved for future use in accordance with medical guidelines. Only mature oocytes with a defined first polar body at 37–38 h after ovulation were selected for cryopreservation in liquid nitrogen. All equipment and materials used for cryopreservation were obtained from Cryotec (Shinjuku, Tokyo, Japan). Vitrification was performed at 25 °C following the Cryotec Method [33]. Briefly, oocytes were equilibrated for 12 to 15 min in an equilibration solution. Subsequently, the oocytes were transferred to VS and incubated for 30 to 40 s. Next, they were transferred to another VS and incubated for 10 to 20 s. Finally, up to four oocytes were placed on a carrier device with a small volume of VS and immediately immersed in liquid nitrogen for cryopreservation.
Oocyte warming
Vitrified donor oocytes were thawed using either an MWP or a CWP. The CWP was conducted following the manufacturer’s (Cryotec) instructions and involved a 3-step dilution process: 2 min in TS at 37 °C, 2 min in DS at 25 °C, and 5 min in WS at 25 °C. Before incubation, the oocytes were washed 3 times in WS at 25 °C. By contrast, the MWP of this study involved a 2-step dilution process, excluding the WS step (i.e., 2 min in TS, followed by 2 min in DS). In both the TS and DS steps, the oocytes were maintained at 37 °C. Before incubation, the oocytes were washed 3 times in human tubal fluid (Kitazato Corporation, Shizuoka, Japan) at 37 °C. Finally, the vitrified-warmed oocytes were incubated in human tubal fluid droplets for 2 h at 37 °C in the presence of 90% nitrogen (N2), 5% oxygen (O2), and 5% carbon dioxide (CO2).
IVF and embryonic culture
In donor cycles involving fresh oocytes, IVF was performed through an ICSI approach for couples with oligoasthenoteratozoospermia or a half-ICSI approach (sibling oocytes subjected to either ICSI or conventional insemination) for those without oligoasthenoteratozoospermia. Sperm quality was evaluated in accordance with the guidelines outlined in the WHO Laboratory Manual for the Examination and Processing of Human Semen [34]. By contrast, in donor cycles involving vitrified-warmed oocytes, IVF was performed through the ICSI approach. The warmed oocytes were incubated for 2 h prior to ICSI in order to recover and assess their survivability. After fertilization, embryos were cultured in vitro for 6 days in sequential media containing 15% serum protein substitute (SAGE Biopharma, Bedminster, NJ, USA). A time-lapse culture system (EmbryoScope+; Vitrolife, Kungsbacka, Sweden) was used to maintain a stable environment with 5% O2, 5% CO2, and 90% N2 at 37 °C. Blastocyst quality was evaluated on day 5 or 6 on the basis of the criteria outlined by Gardner and Schoolcraft [35]. Usable blastocysts were defined as those with a diameter of 150 μm or more, considerable expansion (grade 4, 5, or 6), and acceptable inner cell mass and trophectoderm quality (grade AA, AB, BA, BB, or BC).
Embryo transfer
Embryo transfer was accomplished following our earlier publication [36]. Briefly, transferred blastocysts were prioritized based on PGT-A or embryo morphology. Endometrial preparation was conducted using either a modified natural cycle protocol or an artificial cycle protocol. In the modified natural cycle, follicular growth was monitored via ultrasound, and ovulation was triggered with hCG (Ovidrel; Merck Serono, Modugno, Italy) when follicle size reached ≥ 18 mm. Luteal-phase support (LPS) included oral dydrogesterone (Duphaston, Abbott Biologicals B.V., the Netherlands), vaginal progesterone (Crinone 8%, Merck Serono, Darmstadt, Germany), and estradiol valerate (Estrade, Synmosa, Taipei, Taiwan), administered from the first day after ovulation until the pregnancy test. Patients undergoing the artificial cycle received escalating doses of estradiol valerate, followed by LPS once endometrial thickness was adequate (≥ 7 mm). Embryo transfer was conducted on day 5 post-ovulation in the modified natural cycle or after progesterone administration in the artificial cycle. Following a positive pregnancy test, LPS was continued until 10 weeks of gestation. Clinical outcomes were assessed, including the rates of implantation (gestational sac per blastocyst transferred), clinical pregnancy (pregnancy with the fetal heartbeat after 8 weeks of gestation per transfer cycle), ongoing pregnancy (viable intrauterine pregnancy beyond clinical pregnancy at the time of follow-up per transfer cycle), and live birth (viable delivery after 24 weeks of gestation). This study combined ongoing pregnancies and live births for analysis because 27 cycles still reported ongoing pregnancies but no deliveries.
Statistical analysis
The cycles, oocytes, and normally fertilized embryos were categorized into 3 groups: a CWP group, an MWP group, and a fresh group. Between-group differences were analyzed using the Kruskal-Wallis test or the Pearson chi-square test, as appropriate. Poisson and logistic regression models within a generalized estimating equation were used to determine IRR for usable blastocyst formation and the OR for ongoing pregnancy/live birth. Multivariate models were applied to adjust the significant confounders (P < 0.05) identified in the univariate regression analysis. All statistical analyses were conducted using IBM SPSS Statistics version 26.0 (IBM, Armonk, NY, USA). Categorical variables are presented as percentages and counts, while continuous variables are presented as mean ± standard deviation. A P value less than 0.05 was considered statistically significant.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The authors are grateful to all of the staff at the Infertility Division of Lee Women’s Hospital.
Abbreviations
- MWP
Modified warming protocol
- CWP
Conventional warming protocol
- CPA
Cryoprotectant
- VS
Vitrification solution
- TS
Thawing solution
- DS
Dilution solution
- WS
Wash solution
- IVF
In vitro fertilization
- BMI
Body mass index
- AMH
Anti-Müllerian hormone
- GnRHa
Gonadotropin-releasing hormone agonist
- PPOS
Progestin-primed ovarian stimulation
- N2
Nitrogen
- O2
Oxygen
- CO2
Carbon dioxide
- ICSI
Intracytoplasmic sperm injection
- LPS
Luteal-phase support
- IRR
Incidence rate ratio
- OR
Odds ratio
- FSH
Follicle-stimulating hormone
- LH
Luteinizing hormone
- E2
Estradiol
- P4
Progesterone
- 1PN
1-pronucleus
- 2PN
2-pronucleus
- vs.
Versus
Author contributions
C.H.C., M.S.L., C.I.L., and H.H.C. formulated this study. C.I.L., H.H.C., S.H.L., C.C.H., T.H.L., P.Y.L. M.J.C., and C.H.C. collected and processed the data. C.H.C., C.I.L., and H.H.C. carried out analyses and wrote the manuscript. All authors reviewed the manuscript and provided editorial feedback.
Funding
This study was supported by a grant (NSTC 111-2218-E-040-001) from the National Science and Technology Council, Taiwan.
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
The Institutional Review Board of Chung Shan Medical University approved this study protocol (approval number CS2-24096), which granted a waiver regarding the requirement for written informed consent.
Human ethics and consent to participate declarations
not applicable.
Consent for publication
Not applicable.
Authors’ information
C.H.C. is a senior researcher in embryology and reproductive medicine. He is an embryologist at Lee Women’s Hospital in Taichung, Taiwan. His research interests include preimplantation embryo development and the use of artificial intelligence in the IVF field.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Chun-I Lee and Hsiu-Hui Chen contributed equally to this work.
Contributor Information
Maw-Sheng Lee, Email: msleephd@gmail.com.
Chien-Hong Chen, clonemail@gmail.com.
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Associated Data
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Supplementary Materials
Data Availability Statement
No datasets were generated or analysed during the current study.