Abstract
Background:
The number of oocytes retrieved is a key determinant of in-vitro fertilization (IVF) success, positively associated with fertilization rate, blastocyst formation, and cumulative live birth outcomes. Optimal results are observed in cycles retrieving 16–20 oocytes, with no decline in cumulative live birth rate at higher yields.
Objective:
This study aimed to compare embryological outcomes between vitrified and fresh oocytes in the same patients with diminished ovarian reserve.
Methods:
This retrospective study included 114 women with diminished ovarian reserve (DOR) who underwent oocyte accumulation using both vitrified and fresh oocytes between June 2021 and January 2025. A total of 400 vitrified and 291 fresh oocytes were analyzed. The survival rate, fertilization rate, cleavage rate, and day-2 embryo quality were compared. Subgroup analysis was performed according to POSEIDON groups 3 (<35 years) and 4 (≥35 years). Pregnancy outcomes were preliminarily evaluated in 52 patients who underwent frozen embryo transfer using embryos derived from both oocyte types.
Results:
The mean survival rate was 93.1% ± 14.6%. No significant differences were observed between vitrified and fresh oocytes in fertilization rate (79.7% vs. 86.0%, p = 0.41), cleavage rate (90.8% vs. 93.9%, p = 0.31), or day-2 embryo quality (p > 0.05). Embryological outcomes were comparable in both POSEIDON 3 and 4 groups. In group 4, the proportion of grade 3 embryos was higher in the vitrified oocyte group than in the fresh group (16.0% vs. 9.5%), but this difference was not statistically significant. The overall pregnancy and clinical pregnancy rates were 51.9% and 36.5%, respectively.
Conclusion:
Oocyte vitrification is a feasible and effective strategy for oocyte accumulation in women with DOR, yielding comparable embryological outcomes to fresh oocytes. This approach may offer a valuable option for improving reproductive outcomes in low-prognosis patients.
Keywords: vitrification, diminished ovarian reserve, embryo quality, POSEIDON classification, oocyte accumulation
1. BACKGROUND
The number of oocytes retrieved is a key determinant of in-vitro fertilization (IVF) success, positively associated with fertilization rate, blastocyst formation, and cumulative live birth outcomes. Optimal results are observed in cycles retrieving 16–20 oocytes, with no decline in cumulative live birth rate at higher yields (1). However, this target is often unachievable in patients with diminished ovarian reserve (DOR), particularly those classified in POSEIDON group 4, where both oocyte quantity and quality are severely impaired (2). To address this limitation, the strategy of oocyte accumulation via multiple stimulation and vitrification cycles has been proposed to increase the total pool of mature oocytes, potentially enhancing embryo yield and improving pregnancy prospects in low-prognosis patients.
The clinical rationale for this approach lies in the proven efficacy of oocyte vitrification. Compared to slow freezing, vitrification offers significantly higher survival rate—up to 94.5% (3). Using vitrified oocytes, studies have shown that fertilization rate, embryo development, and clinical pregnancy outcomes are comparable to those achieved with fresh oocytes (4–6). Furthermore, no significant differences have been observed in the rates of aneuploidy, mosaicism, or euploid embryos between fresh and vitrified oocytes (5, 7). A recent systematic review involving 4,159 offspring conceived from vitrified oocytes reported no increase in congenital anomalies or adverse outcomes in birthweight and neurodevelopmental parameters up to six years of age compared with natural conceptions (8).
Given these reassuring findings, oocyte vitrification has been widely adopted in assisted reproductive technology (ART), particularly in patients with DOR, poor ovarian response, advanced maternal age, or ovarian-compromising conditions, as a strategy to maximize the number of mature oocytes available for fertilization. Nevertheless, the effectiveness of oocyte accumulation in this population remains debated. Cobo et al. (2012) reported improved embryo availability and reduced cycle cancellation with accumulation strategies (9), whereas Lee et al. (2023) found no significant improvement in cumulative live birth rates despite increased oocyte numbers, potentially due to elevated miscarriage rates in vitrified-oocyte cycles (10).
While oocyte vitrification has been extensively studied, most comparative data between vitrified and fresh oocytes come from donor cycles, where oocyte quality is presumed to be almost optimal (11). In contrast, evidence from autologous IVF cycles—particularly in patients with diminished ovarian reserve—is limited, as these individuals typically exhibit reduced oocyte yield and compromised oocyte quality.
To address this gap, we conducted a retrospective study directly comparing embryological outcomes between vitrified and fresh oocytes in patients with DOR, and evaluated clinical pregnancy outcomes following frozen embryo transfer.
2. OBJECTIVE
This study aimed to compare embryological outcomes between vitrified and fresh oocytes in the same patients with diminished ovarian reserve.
3. MATERIAL AND METHODS
Ethical approval
This study was approved by the Institutional Review Board of Hanoi Medical University (Certificate of Approval of Ethical Aspects No. 849/GCN-HDDDNCYSH-TDHYHN, 2023). All patients provided written informed consent for the use of their anonymized clinical data for research purposes.
Study design and participants
This retrospective cross-sectional study was conducted at Hanoi Medical University Hospital from June 2021 to January 2025. A total of 114 patients with diminished ovarian reserve (DOR) who underwent in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) were included. Eligible patients were classified as POSEIDON group 3 or 4, with anti-Müllerian hormone (AMH) levels <1.2 ng/mL and/or antral follicle count (AFC) <5 (2). They underwent at least two oocyte retrieval cycles, including at least one cycle involving oocyte vitrification, and had both fresh and vitrified oocytes used for embryo creation. Patients were excluded if their partners had severe male-factor infertility (e.g., oligoasthenoteratozoospermia (WHO 2021) or surgically retrieved sperm), uterine abnormalities (e.g., adhesions, malformations, hydrosalpinx, cesarean scar defect), severe endometriosis, large fibroids, or a history of recurrent miscarriage.
Study procedures
A total of 114 eligible patients with both vitrified oocytes (accumulated from prior retrieval cycles) and fresh oocytes (from the final retrieval cycle) were included. Intracytoplasmic sperm injection (ICSI) was performed on both types of oocytes to create embryos. Post-thaw survival rate was assessed. All oocytes injected oocytes were cultured in individual microdroplets. Fertilization rate, cleavage rate, and cleavage-stage embryo quality were compared between fresh and vitrified oocytes within the same patient. All embryos were vitrified (day 2 or day 5). Among them, 52 patients who underwent frozen embryo transfer were followed to assess clinical outcomes.
Ovarian stimulation and oocyte retrieval
Ovarian stimulation was performed with recombinant FSH (150–300 IU/day), using follitropin alpha (Gonal F®, Merck Serono), follitropin beta (Puregon®, MSD), or Follitrope® (LG Chem), starting on menstrual cycle days 2–4. The FSH dose was individualized based on age,, anti-Müllerian hormone (AMH), body mass index (BMI), and antral follicle count (AFC). LH surge was suppressed using either a GnRH antagonist protocol (cetrorelix acetate, Cetrotide® or ganirelix, Orgalutran® at 0.25 mg/day from stimulation day 5–6), or with a progestin-primed ovarian stimulation (PPOS) protocol using dydrogesterone (Duphaston®, 30 mg/day) starting concurrently with gonadotropins. Final oocyte maturation was triggered with 250 µg recombinant hCG (Ovitrelle®, Merck Serono) or a dual trigger with 250 µg recombinant hCG and 0.2 mg triptorelin (Diphereline®, Merck Serono). Oocyte retrieval was performed 36–38 hours later under ultrasound guidance.
Oocyte vitrification and warming
Mature metaphase II (MII) oocytes were vitrified using an open system with Vitrification Kit 101 (Cryotech, Japan). Warming was performed using Cryotech Warming Solution Set 205 according to the manufacturer’s protocol. Post-thaw survival oocyte was still structurally intact zona pellucida and plasma membrane compared to pre-vitrification state.
ICSI and embryo culture
Thawed and fresh oocytes from the final retrieval cycle were performed ICSI. Thawed oocytes were cultured in G-IVF medium (Vitrolife, Sweden) for 2 hours before ICSI. Following injection, oocytes were cultured individually in 20 µL droplets of G-TL medium (Vitrolife, Sweden) in 35 mm diameter dishes. Fertilization and cleavage embryo development were assessed according to standard protocols. Embryo quality was assessed on day 2 in accordance with the 2011 Istanbul consensus. All embryos were vitrified using Cryotec embryo vitrification kits (Japan) on either day 2 or day 5.
Endometrial preparation, luteal support, and pregnancy outcomes
Endometrial preparation was performed with oral estradiol valerate (Valiera®, Abbott; 4–16 mg/day) starting on day 2–3 of the menstrual cycle. When endometrial thickness reached ≥8 mm, progesterone supplementation was initiated using a combination of estradiol (4–16 mg/day), micronized vaginal progesterone (800 mg/day; Utrogestan®, Besins Healthcare), and dydrogesterone (20 mg/day; Duphaston®, Abbott). Embryo transfer was scheduled based on embryo stage (day 3 or day 5). Pregnancy was determined by serum β-hCG testing: 12 days after day-3 embryo transfer or 10 days after day-5 transfer. If β-hCG level ≥25 IU/L was considered positive. Clinical pregnancy was confirmed by transvaginal ultrasound showing a gestational sac with a fetal heartbeat two weeks later.
Outcome measures
The primary outcomes were the survival rate, fertilization rate, cleavage rate, and day-2 embryo quality, compared between vitrified and fresh oocytes within the same patient. Survival rate was defined as the proportion of oocytes surviving post-warming over the total number of vitrified oocytes. Fertilization rate was calculated as the number of two-pronuclear (2PN) zygotes per total number of injected oocytes. Cleavage rate referred to the proportion of fertilized oocytes that developed into day-2 embryos. Embryo quality was assessed based on day-2 morphology and categorized into grade 1, 2, or 3; the proportion of each grade was calculated per total cleavage-stage embryos.
Comparisons between vitrified and fresh oocytes were conducted intra-patient to control for inter-individual variability. Subgroup analyses were performed according to POSEIDON groups 3 (<35 years) and 4 (≥35 years) to assess age-related differences in embryological outcomes.
Secondary outcomes included the analysis of laboratory outcomes stratified by POSEIDON group 3 and 4, pregnancy rate and clinical pregnancy rates in patients who underwent frozen embryo transfer using embryos derived from both vitrified and fresh oocytes.
Statistical analysis
Data were analyzed using STATA version 15 (UCLA, USA). Continuous variables were expressed as mean ± standard deviation, and categorical variables as frequency and percentage. Comparisons between vitrified and fresh oocytes within the same patients were conducted using the Wilcoxon signed-rank test. Multivariate linear regression was used to identify potential predictors of post-warming oocyte survival, including age, AMH, AFC, and the number of vitrified oocytes per patient. A two-sided p-value < 0.05 was considered statistically significant.
4. RESULTS
Baseline characteristics of patients with diminished ovarian reserve
Between June 2021 and January 2025, 114 patients with diminished ovarian reserve were eligible for the study. Baseline clinical and reproductive characteristics of the study population are summarized in Table 1. The mean age was 36.8 years. Ovarian reserve parameters showed a mean AMH level of 0.8 ng/mL and a mean antral follicle count (AFC) of 3.6 at the first stimulation cycle. On average, patients underwent 2.9 oocyte retrieval cycles (range, 2–6), with the GnRH antagonist protocol applied in 96.5% of cycles. A total of 691 mature oocytes were retrieved, corresponding to a mean of 6.1 ± 3.1 oocytes per patient and 2.2 ± 1.3 oocytes per retrieval cycle.
Table 1. Baseline characteristics of the study population.
| Variable (n = 114) | Value |
| Age (years) | 36.8 ± 5.8 |
| BMI (kg/cm2) | 21.6 ± 2,2 |
| Duration of infertility (years) | 3.0 ± 2.7 |
| Type of infertility Primary infertility Secondary infertility |
47 (39.8%) 71 (60.2%) |
| AMH (ng/ml) | 0.8 ± 0.5 |
| AFC | 3.6 ± 1.6 |
| Stimulation protocol GnRH antagonist PPOS |
110 (96.5%) 4 (3.5%) |
| Number of retrieval cyles 2 cycles 3 cycles 4 cycles 5 cycles 6 cycles |
2.9 ± 0,9 41 (36.0%) 44 (38.6%) 26 (22.8%) 2 (1.8%) 1 (0.9%) |
| Number of MII oocytes per patient | 6.1 ± 3.1 |
| Number of MII oocytes per cycle | 2.2 ± 1.3 |
| Values are presented as mean ± standard deiation or frequency (percentage). BMI, body mass index; AMH, anti-Müllerian hormone; AFC, antral follicle count; PPOS, progestin-primed ovarian stimulation. | |
The survival rate and associated factors in patients with diminished ovarian reserve
A total of 400 vitrified oocytes were analyzed, of which 368 survived following warming, resulting in the survival rate of 93.1 ± 14.6% (range, 40%–100%). Multivariate linear regression was performed to assess the association between survival rate and key clinical variables, including patient’s age, AMH, AFC, and the number of vitrified oocytes per patient. None of these variables demonstrated a statistically significant correlation with oocyte survival (all p > 0.05), as detailed in Table 2. Among them, age showed a non-significant positive trend (β = 2.46; 95% CI: –0.41 to 5.32; p = 0.09), indicating a slight increase in survival with advancing age.
Table 2. Association between clinical factors and survival rate.
| Variable | The survival rate | ||
|---|---|---|---|
| β | 95% CI | p-value | |
| Age | 2.46 | -0.41 to 5.32 | 0.09 |
| AMH | 16.72 | -24.53 to 57.98 | 0.42 |
| AFC | -2.69 | -11.65 to 6.27 | 0.55 |
| Number of MII vitrified oocyte | -1.92 | -7.88 to 4.04 | 0.52 |
| β, regression coefficient; CI, confidence interval; AMH, anti-Müllerian hormone; AFC, antral follicle count; MII, metaphase II. | |||
Comparison embryological outcomes of vitrified and fresh oocytes
Comparison of embryological outcomes between vitrified and fresh oocytes in the same patients with diminished ovarian reserve showed no significant differences in fertilization (79.7% vs. 86.0%) or cleavage rates (90.8% vs. 93.9%). Most day-2 embryos were usable, comprising mainly grade 1 and grade 2 embryos, with no significant difference in the distribution of embryo quality (grades 1, 2, and 3) between the two groups (Table 3).
Table 3. Embryological outcomes of vitrified and fresh oocytes.
| Variable (n = 52) | Value |
| Day of embryo transfer (%) | |
| Day 3 | 50.9 |
| Day 4-5 | 49.1 |
| Number of transferred embryos (x̄ ± sd) | 1.7 ± 0.5 |
| Pregnancy rate (%) | 51.9 |
| Clinical pregnancy rate (%) | 36.5 |
| Biochemical pregnancy rate (%) | 9.6 |
| Miscarriage rate (%) | 5.8 |
| Values are presented as percentages or mean ± standard deviation as appropriate. | |
The number of vitrification cycles was approximately twice that of fresh oocyte cycles. There was a trend toward a higher number of vitrified oocytes and injected oocytes per patient compared to fresh oocytes, although this difference did not reach statistical significance (p = 0.07 and p = 0.23, respectively). The mean number of day-2 embryos generated was 2.5 ± 1.7 in the vitrified group and 2.1 ± 1.9 in the fresh group, with no significant difference observed.
Values are presented as mean ± standard deviation. Comparisons were made within the same patients using paired statistical tests.
Subgroup analysis by POSEIDON classification (Table 4)
Table 4. Comparison embryological outcomes of vitrified vs. fresh oocytes in POSEIDON groups.
| Variable | POSEIDON 3 GROUP (< 35 years) | POSEIDON 4 GROUP (≥ 35 years) | p13 | p24 | ||||
|---|---|---|---|---|---|---|---|---|
| Vitrified oocyte (1) | Fresh oocyte (2) | p12 | Vitrified oocyte (3) | Fresh oocyte (4) | p34 | |||
| (x̄ ± sd) | (x̄ ± sd) | (x̄ ± sd) | (x̄ ± sd) | |||||
| Number of MII oocytes per patient | 3.0 ± 1.5 | 3.3 ± 2.6 | 0.42 | 3.4 ± 2.7 | 2.2 ± 2.2 | 0.02 | 0.94 | 0.02 |
| Number of injected oocytes per patient | 3.0 ± 1.5 | 3.3 ± 2.5 | 0.41 | 3.4 ± 2.6 | 2.2 ± 2.2 | 0.02 | 0.98 | 0.02 |
| Fertilization rate (%) | 74.2 ± 26.7 | 85.1 ± 20.3 | 0.85 | 82.8 ± 24.1 | 86.7 ± 20.0 | 0.44 | 0.81 | 0.60 |
| Cleavage rate (%) | 92.5 ± 27.8 | 95.2 ± 17.9 | 0.34 | 89.8 ± 21.7 | 92.9 ± 21.9 | 0.79 | 0.84 | 0.76 |
| Number of D2 embryos | 2.3 ± 1.4 | 2.6 ± 2.1 | 0.74 | 2.6 ± 1.9 | 1.7 ± 1.8 | 0.16 | 0.86 | 0.02 |
| Embryo quality (%) | ||||||||
| Grade 1 | 27.4 ± 31.7 | 35.7 ± 31.2 | 0.81 | 29.3 ± 44.8 | 27.8 ± 36.9 | 0.46 | 0.87 | 0.15 |
| Grade 2 | 60.0 ± 34.8 | 50.9 ± 33.1 | 0.38 | 66.1 ± 38.1 | 62.7 ± 38.5 | 0.24 | 0.47 | 0.08 |
| Grade 3 | 12.6 ± 27.3 | 13.4 ± 29.1 | 0.77 | 16.0 ± 52.5 | 9.5 ± 25.7 | 0.58 | 0.82 | 0.20 |
| Values are presented as mean ± standard deviation. Comparisons were made within (p12, p34) and between POSEIDON groups (p13, p24) using appropriate statistical tests. | ||||||||
When stratified by POSEIDON group, there were no statistically significant differences between vitrified and fresh oocytes in terms of fertilization rate, cleavage rate, or day-2 embryo quality in either age group. In POSEIDON group 3 (<35 years), the number of mature oocytes, fertilization rate, cleavage rate, and embryo quality on day 2 were comparable between vitrified and fresh oocytes (all p > 0.05).
In contrast, in POSEIDON group 4 (≥35 years), the number of mature oocytes (3.4 ± 2.7 vs. 2.2 ± 2.2; p = 0.02) and the number of oocytes injected per patient (3.4 ± 2.6 vs. 2.2 ± 2.2; p = 0.02) were significantly higher in the vitrified group compared to the fresh oocyte group. However, fertilization rate, cleavage rate, and embryo quality remained comparable (all p > 0.05). Notably, the proportion of grade 3 embryos tended to be higher in the vitrified group compared to the fresh group in older patients (16.0 ± 52.5% vs. 9.5 ± 25.7%).
When comparing patients by age group within each oocyte type, no significant differences were observed in the vitrified oocyte group between younger and older patients across all embryological parameters, including number of oocytes, injected oocytes, fertilization rate, cleavage rate, and embryo quality (all p > 0.05). However, in the fresh oocyte group, younger patients (<35 years) had significantly more oocytes and more day-2 embryos than older patients (p = 0.02), though fertilization rate, cleavage rate, and embryo quality did not differ significantly.
Values are presented as mean ± standard deviation. Comparisons were made within (p12, p34) and between POSEIDON groups (p13, p24) using appropriate statistical tests.
Clinical outcomes of frozen embryo transfer using embryos derived from both vitrified and fresh oocytes
Table 5 presents clinical outcomes of the first frozen embryo transfer (FET) cycle using embryos derived from both vitrified and fresh oocyte. Among the 114 patients, 52 underwent their first frozen embryo transfer (FET) cycle. Day-3 and day-4/5 embryo transfers were nearly equally distributed (50.9% vs. 49.1%). The mean number of embryos transferred per cycle was 1.7 ± 0.5. The overall pregnancy rate was 51.9%, with a clinical pregnancy rate of 36.5%, a biochemical pregnancy rate of 9.6%, and an early miscarriage rate of 5.8%.
Table 3. Embryological outcomes of vitrified and fresh oocytes.
| Characteristic | Vitrified oocyte | Fresh oocyte | p-value |
|---|---|---|---|
| (n=400) | (n=291) | ||
| Number of oocyte retrieval cycles | 218 | 114 | |
| Number of MII oocytes per patient | 3.5 ± 2.4 | 2.6 ± 2.4 | 0.07 |
| Number of injected oocytes per patient | 3.2 ± 2.3 | 2.6 ± 2.4 | 0.23 |
| Fertilization rate (%) | 79.7 ± 25.3 | 86.0 ± 20.0 | 0.41 |
| Cleavage rate (%) | 90.8 ± 24.0 | 93.9 ± 20.3 | 0.31 |
| Number of D2 embryos | 2.5 ± 1.7 | 2.1 ± 1.9 | 0.42 |
| Embryo quality (%) | |||
| Grade 1 | 28.6 ± 40.5 | 31.0 ± 34.7 | 0.38 |
| Grade 2 | 63.9 ± 36.9 | 58.0 ± 36.6 | 0.11 |
| Grade 3 | 14.8 ± 45.1 | 11.1 ± 27.0 | 1.00 |
| Values are presented as mean ± standard deviation. Comparisons were made within the same patients using paired statistical tests. | |||
5. DISCUSSION
This study focused on patients with diminished ovarian reserve (DOR) - a particularly challenging population in assisted reproduction due to their limited oocyte yield. In our cohort, the average number of mature oocytes retrieved per cycle was only 2.2, however, through oocyte accumulation over approximately 2.9 retrievals, the cumulative number of oocytes increased to 6.1 per patient. Although this oocyte yield remains suboptimal, it was sufficient to improve the chances of obtaining good-quality embryos. Shim et al. reported that at least 3–6 mature oocytes are needed to achieve 1–5 top-quality day-3 embryos (12). Moreover, Wang et al. (2023) demonstrated a positive association between cumulative live birth rates and oocyte yield regardless of age groups, with ≥6 oocytes needed to reach a cumulative live birth rate >60% in women aged ≤35 years (13). These data support the clinical utility of oocyte accumulation to optimize embryo availability and improve treatment outcomes in DOR patients.
The low number of retrieved oocytes makes it difficult to determine how many cycles are needed to achieve an optimal yield. Lee et al. estimated that up to 8.6 retrievals may be needed to obtain ≥15 oocytes in POSEIDON groups 3 and 4, posing substantial financial and psychological burdens (10). In our study, even after six retrievals, the highest number of oocytes accumulated was only nine. Therefore, individualized strategies such as mild stimulation, progestin-primed ovarian stimulation (PPOS), or double stimulation should be considered to optimize outcomes while minimizing costs and patient stress. Ultimately, the decision on the number of cycles should balance clinical goals with the patient's resources and preferences.
Oocyte survival following vitrification is a critical factor influencing the success of accumulation strategies. In our cohort of patients with diminished ovarian reserve, the mean post-warming survival rate was 93.1% ± 14.6%, which was higher than the 85.7% reported by Lee et al. in a similar POSEIDON group 3 and 4 population (10). According to the classification proposed by Gallardo et al., both rates fall within the “competence” category, indicating acceptable technical performance (14). To identify potential predictors of survival, we conducted multivariate linear regression including age, AMH, AFC, and the number of vitrified oocytes per patient. None of these variables were significantly associated with survival (all p > 0.05). Age showed a non-significant positive trend (β = 2.46; p = 0.09), while AMH was not predictive of outcome (β = 16.72; p = 0.42), oocyte survival may be more dependent on procedural consistency than on baseline patient characteristics. These findings suggest that our vitrification protocol at Hanoi Medical University Hospital provides standard and reliable outcomes even in low-prognosis patients.
When comparing embryological outcomes between vitrified and fresh oocytes within the same patients with diminished ovarian reserve, no significant differences were found in fertilization, cleavage, or day-2 embryo quality. These findings support existing evidence that oocyte vitrification, when properly executed, yields laboratory outcomes comparable to those obtained from fresh oocytes. Most published studies reporting comparable embryological results between vitrified and fresh oocytes have been conducted in donor oocyte cycles, where oocyte quality is presumed to be optimal. In contrast, our study provides additional data demonstrating similar outcomes in a poor-prognosis population—patients with both impaired oocyte quantity and quality—highlighting the robustness of vitrification technology in real-world autologous IVF settings (15, 16).
Although the vitrified group underwent multiple retrievals (1–5 cycles) for oocyte accumulation, the total number of mature oocytes and embryos was not significantly different from the fresh group, in which oocytes were collected from a single retrieval. This outcome may be explained by a higher yield in the final (fresh) cycle, possibly due to consecutive or dual stimulation protocols to maximize number of oocyte obtained in patients with DOR. Importantly, oocyte survival after warming may represent a limiting factor; however, those that survive retain full developmental potential, as evidenced by comparable fertilization and cleavage outcomes. This is consistent with Gallardo et al. (2025), who found that vitrified oocytes, once survived, exhibit implantation and developmental capacities equivalent to their fresh counterparts (14).
Subgroup analysis by POSEIDON classification showed consistent embryological outcomes across oocyte types in both younger (<35 years) and older (≥35 years) patients. While the proportion of grade 3 embryos was slightly higher in vitrified oocytes among older women (16.0% vs. 9.5%), this was not statistically significant (p = 0.58). Previous studies have suggested that oocytes from older patients may be more vulnerable to cryopreservation-related stress (6). These findings reinforce the importance of considering both age and intrinsic oocyte quality when applying accumulation strategies.
Interestingly, age-related differences in oocyte yield were observed only in the fresh group, where younger patients produced more oocytes and day-2 embryos (p = 0.02). In contrast, outcomes in the vitrified group remained consistent across age groups, suggesting that optimized vitrification protocols may help mitigate age-related declines in oocyte availability and developmental potential.
The clinical outcomes observed following frozen embryo transfer using embryos derived from both oocyte types were encouraging. Among 52 patients undergoing transfer, the pregnancy rate and clinical pregnancy rate were 51.9% and 36.5%, respectively. When compared with the study by Lee et al. (2023), which evaluated frozen embryo transfers from vitrified oocytes alone in a similar population, our clinical pregnancy rate was higher (36.5% vs. 27.5%), and the miscarriage rate among clinical pregnancies was lower (29.6% vs. 41.4%) (10). Several factors may account for these differences: a) Our study did not separate embryos based on oocyte origin, thus reflecting the cumulative effect of combining vitrified and fresh oocytes per patient; b) All patients in our study underwent frozen embryo transfer, while Lee’s study underwent fresh transfer. These favorable outcomes may be attributed to the combination of fresh and vitrified oocytes for embryo creation and subsequent frozen embryo transfer may represent an appropriate and effective strategy for patients with diminished ovarian reserve in our center.
Nonetheless, this study has several limitations. First, as a retrospective observational analysis, it is subject to selection bias and confounding. Second, embryo development was assessed only to day 2, as extended culture was not routinely performed due to low embryo numbers. Third, the origin of embryos (vitrified vs. fresh) was not tracked during transfer, precluding analysis of their individual clinical performance. Future prospective studies are warranted to evaluate cumulative live birth rates and neonatal outcomes in patients undergoing this combined oocyte strategy.
6. CONCLUSION
This study highlights the clinical feasibility and effectiveness of combining vitrified and fresh oocytes to optimize embryo availability in women with diminished ovarian reserve. Despite the modest cumulative number of oocytes retrieved (mean 6.1 per patient), the integration of vitrified oocytes from earlier cycles with fresh oocytes from the final retrieval yielded high survival rates (93.1%) and comparable embryological outcomes between oocyte types. Notably, the clinical pregnancy rate following frozen embryo transfer was 36.5%, underscoring the potential of this strategy to enhance reproductive outcomes in low-prognosis patients.
Acknowledgments:
This study is part of the PhD thesis of Trinh Thi Ngoc Yen and was supported by Hanoi Medical University, Vietnam. The authors would like to thank the staff at the Center of IVF and Tissue engineering - Hanoi Medical University Hospital for their assistance with data collection and laboratory procedures.
Ethical Approval:
The study protocol was approved by the Institutional Review Board of Hanoi Medical University (approval number: 1065/GCN-HMUIRB, issued on 06 February 2024).
Availability of Data and Material:
The datasets generated and/or analysed during the current study are not publicly available due to privacy concerns but are available from the corresponding author on reasonable request.
Author’s contribution:
Conceptualization: T.T.N.Y., D.T.T.P., N.M.H.; Methodology: T.T.N.Y., N.P.H.; Data collection: N.T.H.Y., N.T.Q.A., P.H.P., N.N.M.; Formal analysis and interpretation: T.T.N.Y., N.P.H.; Visualization: T.T.N.Y.; Writing – original draft preparation: T.T.N.Y.; Writing – review and editing: N.P.H., D.T.T.P., N.M.H.; Supervision: D.T.T.P., N.M.H.; Final approval of the manuscript was made by the all authors.
Conflict of interest:
The authors declare that there is no conflict of interest.
Financial support and sponsorship:
The authors received no specific funding for this work. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors
REFERENCES
- 1.Fanton M, Cho JH, Baker VL, Loewke K. A higher number of oocytes retrieved is associated with an increase in fertilized oocytes, blastocysts, and cumulative live birth rates. Fertil Steril. 2023 May;119(5):762–769. doi: 10.1016/j.fertnstert.2023.01.001. [DOI] [PubMed] [Google Scholar]
- 2.Humaidan P, Alviggi C, Fischer R, Esteves SC. The novel POSEIDON stratification of “Low prognosis patients in Assisted Reproductive Technology” and its proposed marker of successful outcome. F1000Res. 2016;5:2911. doi: 10.12688/f1000research.10382.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Pujol A, Zamora MJ, Obradors A, Garcia D, Rodriguez A, Vassena R. Comparison of two different oocyte vitrification methods: a prospective, paired study on the same genetic background and stimulation protocol. Human Reproduction. 2019 Jun 4;34(6):989–997. doi: 10.1093/humrep/dez045. [DOI] [PubMed] [Google Scholar]
- 4.Cornet-Bartolomé D, Rodriguez A, García D, Barragán M, Vassena R. Efficiency and efficacy of vitrification in 35 654 sibling oocytes from donation cycles. Hum Reprod. 2020 Oct 1;35(10):2262–2271. doi: 10.1093/humrep/deaa178. [DOI] [PubMed] [Google Scholar]
- 5.Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. 1. Vol. 99. Fertil Steril; 2013. Jan, pp. 37–43. [DOI] [PubMed] [Google Scholar]
- 6.Doyle JO, Richter KS, Lim J, Stillman RJ, Graham JR, Tucker MJ. Successful elective and medically indicated oocyte vitrification and warming for autologous in vitro fertilization, with predicted birth probabilities for fertility preservation according to number of cryopreserved oocytes and age at retrieval. Fertility and Sterility. 2016 Feb 1;105(2):459–466.e2. doi: 10.1016/j.fertnstert.2015.10.026. [DOI] [PubMed] [Google Scholar]
- 7.Tsai S, Johal J, Malmsten J, Spandorfer S. Embryo ploidy in vitrified versus fresh oocytes: Is there a difference? J Assist Reprod Genet. 2023 Oct;40(10):2419–2425. doi: 10.1007/s10815-023-02901-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Da Luz CM, Caetano MA, Berteli TS, Vireque AA, Navarro PA. The Impact of Oocyte Vitrification on Offspring: a Systematic Review. Reprod Sci. 2022 Nov;29(11):3222–3234. doi: 10.1007/s43032-022-00868-4. [DOI] [PubMed] [Google Scholar]
- 9.Cobo A, Garrido N, Crespo J, José R, Pellicer A. Accumulation of oocytes: a new strategy for managing low-responder patients. Reprod Biomed Online. 2012 Apr;24(4):424–432. doi: 10.1016/j.rbmo.2011.12.012. [DOI] [PubMed] [Google Scholar]
- 10.Lee KS, Lin MH, Hwu YM, Yang JH, Lee RKK. The live birth rate of vitrified oocyte accumulation for managing diminished ovarian reserve: a retrospective cohort study. Journal of Ovarian Research. 2023 Mar 3;16(1):49. doi: 10.1186/s13048-023-01128-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Chang CC, Shapiro DB, Nagy ZP. The effects of vitrification on oocyte quality. Biology of Reproduction. 2022 Feb 1;106(2):316–327. doi: 10.1093/biolre/ioab239. [DOI] [PubMed] [Google Scholar]
- 12.Shim YJ, Hong YH, Kim SK, Jee BC. Optimal numbers of mature oocytes to produce at least one or multiple top-quality day-3 embryos in normal responders. Clin Exp Reprod Med. 2020 Jul 21;47(3):221–226. doi: 10.5653/cerm.2019.03377. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Wang P, Zhao C, Xu W, Jin X, Zhang S, Zhu H. The association between the number of oocytes retrieved and cumulative live birth rate in different female age strata. Sci Rep. 2023 Sep 4;13(1):14516. doi: 10.1038/s41598-023-41842-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Gallardo M, Goncalves I, Redondo J, Soares AP, Garrido N, Metello JL. Assessing the effect of below-benchmark vitrified/ warmed donor-oocyte survival rates in subsequent laboratory and clinical outcomes. Fertility and Sterility. 2025 Mar 1;123(3):448–456. doi: 10.1016/j.fertnstert.2024.09.041. [DOI] [PubMed] [Google Scholar]
- 15.Cohen J, Chabbert-Buffet N, Darai E. Diminished ovarian reserve, premature ovarian failure, poor ovarian responder—a plea for universal definitions. J Assist Reprod Genet. 2015 Dec 1;32(12):1709–1712. doi: 10.1007/s10815-015-0595-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Jirge PR. Poor ovarian reserve. J Hum Reprod Sci. 2016;9(2):63–69. doi: 10.4103/0974-1208.183514. [DOI] [PMC free article] [PubMed] [Google Scholar]