Comparative Embryological Outcomes of Vitrified and Fresh Oocytes in 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. 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. 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 ). 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. 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 ( 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. 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%.

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. 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. 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 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. 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. 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 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. 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. 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.

This study aimed to compare embryological outcomes between vitrified and fresh oocytes in the same patients with diminished ovarian reserve.

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.

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.

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.