Abstract
This study aims to explore the effect of various factors, including freezing duration, on frozen embryo transfer outcomes. We analyzed 2291 frozen-thawed cycles based on embryo freezing time (<4, 4–12, and >12 months). There were no significant differences in the rates of clinical pregnancy, live birth, miscarriage, or preterm labor between the groups. The intergroup difference in the birth weight of singletons was significant (P = .012). Following cleavage-stage embryo transfer, maternal age at frozen embryo transfer (odds ratio [OR], 95% confidence interval [CI] = 0.813 [0.674–0.981], P = .030), (OR [95% CI] = 0.779 [0.635–0.955], P = .016), and number of embryos transferred (OR [95% CI] = 1.527 [1.172–1.989], P = .002), (OR [95% CI] = 1.688 [1.270–2.242], P < .001) were associated with the clinical pregnancy and live birth rates, respectively. Following blastocyst-stage embryo transfer, cycle number of transplantation (OR [95% CI] = 0.570 [0.369–0.881], P = .011), (OR [95% CI] = 0.565 [0.366–0.871], P = .010), and number of embryos transferred (OR [95% CI] = 1.734 [1.296–2.322], P < .001), (OR [95% CI] = 1.951 [1.460–2.606], P < .001) were associated with the clinical pregnancy and live birth rates, respectively. Ovarian stimulation protocol (OR [95% CI] = 1.511 [1.006–2.268], P = .047) was associated only with the clinical pregnancy rate. Embryo cryopreservation duration did not affect the clinical pregnancy or live birth rates regardless of embryo stage. Older patients could consider blastocyst embryo culture and transplantation to improve clinical pregnancy and live birth rates.
Keywords: cohort study, cryopreservation, embryo transfer, treatment outcome, vitrification
1. Introduction
The development of in vitro fertilization (IVF) and other derived technologies as a last resort in infertility treatment has been a remarkable breakthrough in the field of reproductive medicine. In addition to fresh embryo transfer, there are various medical and social factors, such as the need for preimplantation genetic diagnosis and prevention of ovarian hyperstimulation, that make it impossible for cultured embryos to be transferred to the uterus immediately. Therefore, thawing and transferring frozen embryos has become increasingly important in IVF treatment,[1] making the freeze-all strategy necessary in the implementation of IVF technology.
Embryo freezing technology has advanced from the traditional slow-freezing methods to current vitrification technologies. Vitrification causes less damage to embryos and results in higher embryo survival rates.[2,3] Continuous advancements in technology used in embryology have led to an increase in embryo survival rates and indications for embryo freezing, thereby contributing to a global increase in the number of frozen and preserved embryos.[4]
Recently, with the prevalence of blastocyst culture technology, the success rate of IVF treatment has greatly improved. However, poor-quality embryos still limit the adoption of conservative cleavage.[5] Cleavage-stage and blastocyst-stage embryos are sequentially transferred in the same treatment cycle to improve the success rate.[6] However, this method has increased the incidence of multiple births[7] and the associated costs. Furthermore, the benefits are still controversial.[8,9] Therefore, thawing and transplantation of cleavage embryos is still an important strategy to assist pregnancy. Several studies have reported on the effect of transplantation time interval on the outcome of frozen embryo transplantation.[1,10,11] Although the conclusions differed owing to differences in samples in these studies, no embryos were at cleavage or blastocyst stage.
We conducted a retrospective study of the treatment outcomes of 2291 thawed cycles of transplantation using the whole embryo transfer technique to investigate factors affecting the success rate of embryo transfer, including the embryo cryopreservation duration.
2. Materials and methods
2.1. Study design
This single-center cohort study included patients who underwent embryo transfer via IVF at the Reproductive Medicine Center of Huzhou Maternal and Child Health Care Hospital between January 2016 and December 2023. The study protocol was approved by the Medical Ethics Committee at Huzhou Maternity and Child Health Care Hospital (approval number: 2024-J-007; approval date: January 22, 2024) and was performed in accordance with the ethical principles outlined in the Declaration of Helsinki. The ethics committee waived the requirement for informed consent owing to the retrospective nature of the study. We included women who underwent thawing and transfer of frozen embryos after the freeze-all strategy. Treatment outcomes were followed up. We excluded patients who underwent sequential embryo transfer, those without transferable embryos after thawing, and those with missing data. All patients underwent embryo transfer via either IVF or intracytoplasmic sperm injection. Preimplantation genetic diagnosis was not performed, and donor eggs or sperm were not used. Embryos were classified into 3 groups based on their embryo cryopreservation duration: <4 months (group 1), 4–12 months (group 2), and > 12 months (group 3).
2.2. Clinical treatment procedures
All couples with infertility underwent routine physical examinations to ensure that they met the inclusion criteria prior to IVF treatment. After formalizing the technique, all patients underwent an ovarian stimulation program with superovulation. The GnRHa ultra long protocol was performed with a gonadotropin-releasing hormone (GnRH) agonist (Beyaz; Shanghai Livzon, Shanghai, China) and gonadotropins (human menopausal gonadotropin [HMG]; Shanghai Livzon or Gonal-f; Merck Serono [Merck KGaA], Coinsins, Vaud, Switzerland; or Li shen bao; Shanghai Livzon). The long GnRHa protocol was performed with a GnRH agonist (Diphereline; Ferring, Saint-Prex, Vaud, Switzerland) and gonadotropins (HMG; Shanghai Livzon or Gonal-f; Merck or Shanghai Livzon). The GnRH antagonist protocol was performed with an antagonist (Cetrotide; Merck Serono, Coinsins, Vaud, Switzerland) and gonadotropins (HMG; Shanghai Livzon or Gonal-f; Merck or Shanghai Livzon). Other protocols, including microstimulation, were performed with an estrogen modulator (Clomid; Codal Synto Ltd., Limassol, Cyprus), gonadotropins (HMG; Shanghai Livzon), and natural cycles. Following dynamic monitoring, which considered the size and number of the patients’ follicles and sex hormone levels, gonadotropin was discontinued, a pharmacological trigger was administered by 5.000 to 10.000 IU of human chorionic gonadotrophin (Shanghai Livzon), and the eggs were retrieved vaginally under ultrasound after 34 to 36 hours. Insemination was performed 4 to 6 hours after egg retrieval.
Embryos were frozen at either the cleavage or blastocyst stage, depending on their number and quality. Before freezing, morphological scoring was performed to assess embryo quality. Cleavage-stage embryos were graded according to the Istanbul Consensus 201,[12] whereas blastocysts were classified using the Gardner Blastocyst Classification Scale.[13] For the embryo vitrification procedure, embryos with cryopreservation values were selected and transferred into vitrification solution equilibration solution (KITAZATO, Fuji-shi, Shizuoka, Japan) for 8 minutes. Thereafter, they were transferred into vitrification solution (KITAZATO) for 1 minute. The embryos were then loaded into freezing carriers within 2 minutes. The carriers with embryos were promptly placed into liquid nitrogen, loaded into plastic sleeves and thereafter into stents, and placed into liquid nitrogen storage tanks at −196°C after confirming the accuracy of embryo information.
Preparation for endometrial synchronization is necessary before embryo transfer to ensure optimal endometrial tolerance on the day of transfer, and thawed embryos are prioritized based on their grade scores. The frozen embryos were thawed as follows: the embryos were quickly washed in thawing solution 1 (KITAZATO) for 1 minute at 37°C, transferred into diluent solution (KITAZATO) for 3 minutes. Then the specimens were rinsed twice in WS droplets (5 minutes each) to remove residual cryoprotectants. The embryos were transferred to pre-equilibrated culture medium (G2-PLUS; Vitrolife, Gothenburg, Sweden) and incubated for 30 to 60 minutes at 37°C/5% CO2 for 1 to 2 hours before transfer. Embryos were transferred to EmbryoGlue (Vitrolife, Gothenburg, Sweden) dishes 10 to 30 minutes before transfer.
2.3. Follow-up
After the patients had undergone thawed embryo transfer, they received regular follow-up visits from day 7 after the transfer until pregnancy termination or interruption to record treatment outcomes. All data were registered with the Clinical Reproductive Medicine Management System (Nanjing Difei, Jiangsu, China).
2.4. Evaluation indicators
The primary outcomes were clinical pregnancy and live birth rates. The secondary outcomes were miscarriage, premature labor, pregnancy-related complications, low birth weight, and macrosomia.
Clinical pregnancy was defined as the detection of a gestational sac using ultrasonography 30 days after implantation. Live birth was defined as at least one live birth after 24 weeks of gestation. Miscarriage was defined as intrauterine demise of the fetus before 28 weeks of gestation. Preterm delivery was defined as a delivery occurring between 24 and 37 weeks of gestation. Pregnancy-related complications and congenital disabilities were defined according to the International Classification of Diseases: low birth weight was < 2000 g; macrosomia was birth weight ≥ 4000 g.
2.5. Data collection and statistical analysis
Data were obtained from the Clinical Reproductive Medicine Management System and processed using SPSS 27 (IBM Corp., Armonk). The continuous data, expressed as medians and quartiles [M(P25, P75)], were analyzed using Kruskal–Wallis tests, indicating a non-normal distribution based on Kolmogorov–Smirnov tests. Categorical variables were reported as proportions and percentages and compared using either the chi-square test or Fisher exact test. Univariate analysis was performed to determine significant associations between baseline variables and frozen embryo transfer (FET) cycle characteristics. In addition to the main observation index of embryo cryopreservation duration, according to literature review and clinical experience, we included age, female body mass index, infertility type, infertility duration, infertility cause, superovulation regimen, fertilization method, endometrial preparation regimen, number of embryos transferred, and days after embryo transfer as confounding factors in the study. For variables significantly associated on univariate analysis were included in multivariate logistic regression analysis to identify the effect of baseline and FET cycle characteristics on the outcomes, defined as the clinical pregnancy and live birth rates. Associations were reported as odds ratios (OR) and 95% confidence intervals (CI) as appropriate. All tests were two-tailed, and P < .05 was considered significant.
3. Results
Overall 2291 female patients underwent embryo transfer via a freeze-all strategy. Significant differences were observed between the groups in terms of maternal and paternal age at FET, infertility duration, endometrial thickness, endometrial preparation program, and developmental stage of embryos transferred (Table 1).
Table 1.
Comparison of patient baseline characteristics between different embryo freezing durations.
| Group 1 | Group 2 | Group 3 | P value | |
|---|---|---|---|---|
| Number of FET cycles | 853 | 1175 | 263 | |
| Female age at FET (yrs) | 30.0 (27.0–34.0) | 31.0 (28.0–35.0) | 32.0 (29.0–35.0) | <.001 |
| Female age at pre-IVF (yrs) | 30.0 (27.0–34.0) | 30.0 (27.0–35.0) | 30.0 (27.0–34.0) | .109 |
| Male age at FET (yrs) | 32.0 (29.0–36.0) | 32.0 (29.0–36.0) | 33.0 (30.0–37.0) | <.001 |
| Infertility duration(yr) | 3.4 (2.1–5.4) | 3.9 (2.5–6.0) | 5.8 (4.1–7.9) | <.001 |
| Female body mass index (kg/m2) | 22.0 (20.0–24.5) | 22.3 (20.3–24.8) | 22.0 (20.1–24.6) | .179 |
| Basal FSH (mIU/mL) | 6.4 (5.4–7.4) | 6.4 (5.5–7.7) | 6.2 (5.5–7.2) | .320 |
| Endometrial thickness (mm) | 8.0 (8.0–9.0) | 8.0 (8.0–9.0) | 8.0 (7.0–9.0) | <.001 |
| Infertility type, n (%) | .419 | |||
| Primary infertility | 396 (46.4) | 529 (45.0) | 110 (41.8) | |
| Secondary infertility | 457 (53.6) | 646 (55.0) | 153 (58.2) | |
| Infertility causes, n (%) | .655 | |||
| Female factor | 664 (77.8) | 941 (80.1) | 221 (81.0) | |
| Male factor | 83 (9.7) | 101 (8.6) | 19 (7.0) | |
| Couple factor | 69 (8.1) | 93 (7.9) | 25 (9.2) | |
| Unexplained infertility | 37 (4.3) | 40 (3.4) | 8 (2.9) | |
| Ovarian stimulation protocol, n (%) | .762 | |||
| Long GnRHa | 387 (45.4) | 519 (44.2) | 131 (49.8) | |
| GnRHa ultra long | 171 (20.0) | 239 (20.3) | 49 (18.6) | |
| GnRH antagonist | 105 (12.3) | 147 (12.5) | 33 (12.5) | |
| Other protocols | 190 (22.3) | 270 (23.0) | 50 (19.1) | |
| Fertilization method, n (%) | .283 | |||
| IVF | 667 (78.2) | 951 (80.9) | 213 (81.0) | |
| ICSI | 186 (21.8) | 224 (19.1) | 50 (19.0) | |
| Endometrial preparation program, n (%) | <.001 | |||
| Hormonal replacement cycle | 453 (53.1) | 894 (76.1) | 185 (70.3) | |
| Natural cycle | 376 (44.1) | 212 (18.0) | 56 (21.3) | |
| Stimulated cycle | 21 (2.8) | 69 (5.9) | 22 (8.4) | |
| Development stage of embryos transferred, n (%) | .012 | |||
| Cleavage stage | 437 (51.2) | 662 (56.3) | 159 (60.5) | |
| Blastocyst | 416 (48.8) | 513 (43.7) | 104 (39.5) | |
FET = frozen embryo transfer, GnRH = gonadotropin-releasing hormone, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization.
When comparing between-group outcomes after FET treatment, the group with the shortest storage time had the highest clinical pregnancy and live birth rates, and the group with the longest storage time had the highest miscarriage rate; however, there were no significant differences between the groups (Table 2).
Table 2.
Comparing between-group outcomes of FET treatment in patients who underwent embryo transfer with different embryo freezing durations.
| Group 1 | Group 2 | Group 3 | P value | |
|---|---|---|---|---|
| Clinical pregnancy (%) | 429/853 (50.3) | 547/1175 (46.6) | 122/263 (46.4) | .217 |
| Miscarriage (%) | 69/429 (16.1) | 87/547 (15.9) | 27/122 (22.1) | .228 |
| Premature labor (%) | 68/353 (18.9) | 90/446 (20.2) | 15/90 (16.7) | .739 |
| Live birth rate (%) | 353 (41.4) | 446/1175 (38) | 90/263 (34.2) | .079 |
In between-group comparisons of maternal and neonatal outcomes after cleavage-stage transfer, we found that the proportion of male newborns was dominant in all 3 groups, and although the proportion of pregnancy complications was the highest in the group with the longest embryo cryopreservation time, there was no significant difference in the incidence among the 3 groups. This indicates no significant differences in the gestational age or mode of delivery between the groups. The difference in birth weight of singletons between the groups was significant; the group with the longest storage time had the lowest birth weight (Table 3).
Table 3.
Comparing between-group maternal and neonatal outcome of FET treatment with different embryo freezing durations.
| Group 1 | Group 2 | Group 3 | P | |
|---|---|---|---|---|
| 288 | 362 | 81 | ||
| Gestational age, wk | 38.0 (38.0–39.0) | 39.0 (38.0–39.0) | 38.0 (38.0–39.0) | .324 |
| Mode of delivery, n (%) | .145 | |||
| Vagina, n (%) | 105 (36.5) | 106 (29.3) | 25 (30.9) | |
| Cesarean section, n (%) | 183 (63.5) | 256 (70.7) | 56 (69.1) | |
| Birth weight of singletons | 3300.0 (3050.0–3600.0) | 3300.0 (3040.0–3650.0) | 3170.0 (2900.0–3450.0) | .012 |
| Gender of singletons | .977 | |||
| Male, n (%) | 160 (55.6) | 199 (55) | 44 (54.3) | |
| Female, n (%) | 128 (44.4) | 163 (45) | 37 (45.7) | |
| Pregnancy-related complication rates | 66 (22.1) | 83 (21.8) | 27 (33.3) | .071 |
| Low birth weight, n (%) | 3 (1.0) | 6 (1.7) | 2 (2.5) | .612 |
| Large birth weight, n (%) | 28 (9.7) | 26 (7.2) | 2 (2.5) | .085 |
After grouping the embryos according to days of development, separate univariate analyses performed for cleavage-stage embryos and blastocyst transfers of different storage durations (Tables 4 and 5). Variables showing significant differences in the univariate analysis, along with factors deemed clinically relevant, were included in the multivariate regression analysis. Multivariate regression analysis showed that embryo storage time did not affect the clinical pregnancy rate or live birth rate whether cleavage embryo or blastocyst. Maternal age at FET and number of embryos transferred were associated with the clinical pregnancy rate following cleavage embryo transfer. Moreover, maternal age at FET and number of embryos transferred were associated with the live birth rate. The embryo storage time was not a factor affecting the clinical pregnancy or live birth rates. The cycle number of transplantations, number of embryos transferred, and ovarian stimulation protocols were factors associated with the clinical pregnancy rate. The cycle number of transplantations and number of embryos transferred were factors associated with the live birth rate following blastocyst transfer (Figs. 1–4).
Table 4.
Intergroup comparison of patients with different frozen cleavage stage embryo storage durations.
| Group 1 | Group 2 | Group 3 | P value | |
|---|---|---|---|---|
| Number of FET cycles | 437 | 662 | 159 | |
| Female age at FET (yrs) | 30.0 (28.0–35.0) | 31.0 (28.0–37.0) | 32.0 (29.0–37.0) | .001 |
| Female age at pre-IVF (yrs) | 30.0 (27.0–35.0) | 30.0 (27.0–36.0) | 30.0 (27.0–35.0) | .320 |
| Male age at pre-IVF (yrs) | 32.0 (29.0–37.0) | 33.0 (29.0–38.0) | 34.0 (30.0–38.0) | .012 |
| Infertility duration (yr) | 3.6 (2.3–5.8) | 4.0 (2.4–6.1) | 5.9 (4.2–7.8) | <.001 |
| Female body mass index (kg/m2) | 22.1 (19.9–24.6) | 22.1 (20.2–24.6) | 22.5 (20.4–24.8) | .496 |
| Basal FSH (mIU/mL) | 6.6 (5.7–7.7) | 6.6 (5.7–8.1) | 6.4 (5.5–7.6) | .228 |
| Endometrial thickness (mm) | 9.0 (8.0–10.0) | 8.0 (8.0–9.0) | 8.0 (8.0–9.0) | <.001 |
| Infertility type, n (%) | .234 | |||
| Primary infertility | 205 (46.9) | 286 (43.2) | 63 (39.6) | |
| Secondary infertility | 232 (53.1) | 376 (56.8) | 96 (60.4) | |
| Infertility causes, n (%) | .202 | |||
| Female factor | 323 (73.9) | 527 (79.6) | 123 (77.4) | |
| Male factor | 59 (13.5) | 64 (9.7) | 13 (8.2) | |
| Couple factor | 40 (9.2) | 49 (7.4) | 18 (11.3) | |
| Unexplained infertility | 15 (3.4) | 22 (3.3) | 5 (3.1) | |
| Ovarian stimulation protocol, n (%) | .590 | |||
| Long GnRHa | 186 (42.6) | 272 (41.1) | 74 (46.5) | |
| GnRHa ultra long | 63 (14.4) | 102 (15.4) | 29 (18.2) | |
| GnRH Antagonist | 58 (13.3) | 82 (12.4) | 15 (9.4) | |
| Other protocols | 130 (29.7) | 206 (31.1) | 41 (25.8) | |
| Fertilization method, n (%) | .045 | |||
| IVF | 314 (71.9) | 515 (77.8) | 126 (79.2) | |
| ICSI | 123 (28.1) | 147 (22.2) | 33 (20.8) | |
| Endometrial preparation program, n (%) | <.001 | |||
| Natural cycle | 214 (49) | 119 (18.0) | 35 (22.0) | |
| Hormonal replacement cycle | 209 (47.8) | 508 (76.7) | 114 (71.7) | |
| Stimulated cycle | 14 (3.2) | 35 (5.3) | 10 (6.3) | |
| A history of abortion, n (%) | .529 | |||
| Never | 316 (72.3) | 467 (70.5) | 112 (70.4) | |
| 1–2 | 111 (25.4) | 168 (25.4) | 43 (27.0) | |
| ≥3 | 10 (2.3) | 27 (4.1) | 4 (2.5) | |
| The number of embryos transferred, n (%) | .107 | |||
| 1 | 150 (34.3) | 200 (30.3) | 41 (25.8) | |
| 2 | 287 (65.7) | 462 (69.7) | 118 (74.2) | |
| Cycle of transplants, n (%) | <.001 | |||
| 1 | 144 (33.0) | 127 (19.2) | 11 (6.9) | |
| 2 | 213 (48.7) | 268 (40.5) | 33 (20.8) | |
| ≥3 | 80 (18.3) | 267 (40.3) | 115 (72.3) | |
FET = frozen embryo transfer, GnRH = gonadotropin-releasing hormone, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization.
Table 5.
Intergroup comparison of patients with different frozen blastocyst stage embryo storage durations.
| Group 1 | Group 2 | Group 3 | P value | |
|---|---|---|---|---|
| Number of FET cycles | 416 | 513 | 104 | |
| Female age at FET (yrs) | 30.0 (27.0–33.0) | 31.0 (28.0–34.0) | 32.0 (29.0–34.5) | .001 |
| Female age at pre-IVF (yrs) | 30.0 (27.0–33.0) | 30.0 (27.0–33.0) | 30.0 (27.0–32.0) | .353 |
| Male age at FET (yrs) | 31.0 (28.0–34.0) | 32.0 (29.0–35.0) | 32.0 (30.0–36.0) | .001 |
| Infertility duration (yr) | 3.3 (2.0–5.0) | 3.8 (2.6–5.8) | 5.8 (3.9–8.2) | <.001 |
| Female body mass index (kg/m2) | 21.9 (20.2–24.5) | 22.6 (20.4–25.1) | 21.5 (19.7–24.3) | .043 |
| Basal FSH (mIU/mL) | 6.2 (5.2–7.1) | 6.2 (5.2–7.1) | 6.1 (5.3–7.0) | .969 |
| Endometrial thickness (mm) | 8.0 (8.0–9.0) | 8.0 (8.0–9.0) | 8.0 (7.0–9.0) | .005 |
| Infertility type, n (%) | .868 | |||
| Primary infertility | 191 (45.9) | 243 (47.4) | 47 (45.2) | |
| Secondary infertility | 225 (54.1) | 270 (52.6) | 57 (54.8) | |
| Infertility causes, n (%) | .660 | |||
| Female factor | 341 (82.0) | 414 (80.7) | 88 (84.6) | |
| Male factor | 24 (5.8) | 37 (7.2) | 6 (5.8) | |
| Couple factor | 29 (7.0) | 44 (8.6) | 7 (6.7) | |
| Unexplained infertility | 22 (5.3) | 18 (3.5) | 3 (2.9) | |
| Ovarian stimulation protocol, n (%) | .285 | |||
| Long GnRHa | 201 (48.3) | 247 (48.1) | 57 (54.8) | |
| GnRHa ultra long | 108 (26) | 137 (26.7) | 20 (19.2) | |
| GnRH Antagonist | 47 (11.3) | 65 (12.7) | 18 (17.3) | |
| Other protocols | 60 (14.4) | 64 (12.5) | 9 (8.7) | |
| Fertilization method, n (%) | .941 | |||
| IVF | 353 (84.9) | 436 (85.0) | 87 (83.7) | |
| ICSI | 63 (15.1) | 77 (15.0) | 17 (16.3) | |
| Endometrial preparation program, n (%) | <.001 | |||
| Natural cycle | 162 (38.9) | 93 (18.1) | 21 (20.2) | |
| Hormonal replacement cycle | 244 (58.7) | 386 (75.2) | 71 (68.3) | |
| Stimulated cycle | 10 (2.4) | 34 (6.6) | 12 (11.5) | |
| A history of abortion, n (%) | .883 | |||
| never | 260 (62.5) | 331 (64.5) | 66 (63.5) | |
| 1–2 | 142 (34.1) | 169 (32.9) | 36 (34.6) | |
| ≥3 | 14 (3.4) | 13 (2.5) | 2 (1.9) | |
| The number of embryos transferred, n (%) | <.001 | |||
| 1 | 336 (80.8) | 305 (59.5) | 56 (53.8) | |
| 2 | 80 (19.2) | 208 (40.5) | 48 (46.2) | |
| Cycle of transplants, n (%) | <.001 | |||
| 1 | 76 (18.3) | 70 (13.6) | 2 (1.9) | |
| 2 | 292 (70.2) | 223 (43.5) | 22 (21.2) | |
| ≥3 | 48 (11.5) | 220 (42.9) | 80 (76.9) | |
FET = frozen embryo transfer, GnRH = gonadotropin-releasing hormone, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization.
Figure 1.
Multivariate analysis of clinical pregnancy rate in thawed cleavage-stage embryo transfer. For women aged 35 and older, each additional year reduces the clinical pregnancy rate by 19.7%. Transferring 2 embryos increases the clinical pregnancy rate by 52.7% compared to transferring 1. CI = confidence interval, FET = frozen embryo transfer, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization, OR = odds ratio.
Figure 4.
Multivariate analysis of live birth rate in thawed blastocyst transfer. Transplanting 2 embryos, compared to 1, increases the clinical pregnancy rate by 95.1%, as illustrated in the forest map. CI = confidence interval, FET = frozen embryo transfer, GnRH = gonadotropin-releasing hormone, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization, OR = odds ratio.
Figure 2.
Multivariate analysis of live birth rate in thawed cleavage-stage embryo transfer. For women aged 35 and older, each additional year reduces the clinical pregnancy rate by 22.1%. Transferring 2 embryos increases the clinical pregnancy rate by 68.8% compared to transferring 1. CI = confidence interval, FET = frozen embryo transfer, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization, OR = odds ratio.
Figure 3.
Multivariate analysis of clinical pregnancy rate in thawed blastocyst. Transplanting 2 embryos, compared to 1, increases the clinical pregnancy rate by 73.4%, as illustrated in the forest map. CI = confidence interval, FET = frozen embryo transfer, GnRH = gonadotropin-releasing hormone, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization, OR = odds ratio.
4. Discussion
In this study, we compared treatment outcomes after transfer of frozen embryos with different cryopreservation durations and found no significant difference between the groups except for singleton birth weight.
All ongoing clinical trials evaluating the efficacy of embryo transfer strategies have delayed the first transfer by at least one menstrual cycle, based on clinical experience. A large retrospective study showed that the first FET can be performed as soon as possible, without waiting for more than 1 menstrual cycle and without compromising the success rate.[14] If patients’ endometrium status and hormone levels meet the conditions for transfer after the first or second menstrual cycle, embryo transfer can be performed immediately to achieve a more satisfactory therapeutic result.[15] Therefore, owing to cost considerations and the urgency of patients’ desire to have children, early transplantation can be performed when patient conditions are favorable.
Age is an important factor in assisted reproductive technology.[16,17] All tissues and organs of the female body physiologically decline with age. This decline leads to complications during pregnancy and childbirth, which increase the chances of adverse pregnancy outcomes.[18] IVF can compensate for the natural age-related decline in fertility; however, the negative reproductive effects of aging, such as a decline in the number of oocytes in the ovary and ongoing changes in the integrity of DNA within the oocytes, are permanent and irreversible.[19] In this study, after multivariate analysis of cleavage-stage and blastocyst-stage embryos, we found that maternal age at FET affected the clinical pregnancy rate and live birth rate following the transfer of cleavage-stage embryos but not blastocyst-stage embryos. Therefore, we speculate that the choice of blastocyst transfer in older women can achieve better outcomes than does cleavage-stage embryo transfer. Theoretical advantages of longer cell culture times include improved selection of higher-quality embryos, better embryo-endometrial synchrony, and reduced uterine contractility.[20,21] Increased maternal age may lead to abnormal cell chromosomes, increased endometrial collagen content, and decreased hormone receptors, thus, affecting the endometrial receptivity in women.[22] Moreover, the difference between the developmental stages of the transferred cleavage-stage embryo and reproductive tract may compromise embryonic viability and produce metabolic stress.[23] Because the embryo at the blastocyst stage matches the intimal implantation window, the blastocyst may better adapt to the uterine environment, even in older patients.
In addition to embryo recovery rate and quality, the success of vitrification-based FET depends on endometrial tolerance at the time of transfer.[24] Endometrial receptivity is an important factor in embryo implantation. At present, the initial preparation scheme before embryo transfer in our center includes natural cycle, hormone replacement, and stimulation cycle. The comparative effectiveness of different types of endometrial support during FET cycles is a controversial topic, with insufficient evidence to support one approach being superior to another in terms of clinical pregnancy rates or live birth rates.[25] Multiple studies showed no difference in treatment outcomes between intimal alternatives.[26,27] Similarly, the protocol in this study was not a factor affecting the treatment outcome.
Blastocyst culture promotes advantages such as self-selection of chromosomally normal embryos with better growth potential. Blastocyst transplantation can significantly improve the clinical pregnancy and live birth rates. However, in clinical practice, no additional benefit from blastocyst transfer compared with cleavage stage embryo transplantation has been noted. Therefore, the choice between cleavage-stage embryo transfer and blastocyst transplantation for a freeze-thaw embryo transfer cycle remains controversial.[28–30]
In our study, blastocysts obtained using an antagonist regimen yielded a better clinical pregnancy rate, which is similar to the findings of a previous study.[31] The use of the antagonist regimen is already quite common in most countries, having economic and time benefits and effective prevention of ovarian hyperstimulation syndrome. In the 2020 European Society of Human Reproduction and Embryology guidelines, antagonist regimens have become the first-line choice for ovulation induction regimens in various types of ovarian response populations.[32] The antagonist regimen is achieved by blocking the secretion of luteinizing hormone by GnRH antagonist, with the high expression of inflammatory factor-1 through tumor necrosis factor-alpha.[33,34] Therefore, antagonist regimens are more detrimental to endometrial tolerance,[35] but pre-cyclical endometrial repair of FET improves endometrial tolerance, which avoids the detrimental effects of fresh grafts.
This study has some limitations. Firstly, although the covariates included in this study were all established confounding factors from previous research, the absence of data on female metabolism may introduce bias in the conclusions. As this was a retrospective study, relevant data could not be retroactively obtained. Therefore, we plan to incorporate more comprehensive data in future prospective studies to further validate the robustness of these findings. Secondly, embryos frozen for more than 1 year were not grouped, as most thawing and transfers of frozen embryos are performed within 1 year after the implementation of the freeze-all strategy. Previously, several studies have been conducted on the outcome of embryo transfers with a long freezing time; however, no distinction on the developmental stage of the transplanted embryos has been made.
In conclusion, our findings confirmed that the duration of embryo preservation by vitrification had no association with the development of pregnancy-related complications or neonatal outcomes. Furthermore, patients with advanced age and poor ovarian reserve function, who require several rounds of ovulation induction and embryo freezing, should consider blastocyst embryo culture and transplantation whenever possible to improve the clinical pregnancy and live birth rates.
Acknowledgments
We gratefully acknowledge the financial support from the Huzhou Public Welfare Application Research Project (Grant No. 2023GYB08) and Zhejiang Province Traditional Chinese Medicine Science and Technology Project (Grant No. 2024ZL1030). We would like to acknowledge Editage (https://www.editage.com/) for English language editing.
Author contributions
Conceptualization: Liqun Lu.
Data curation: Liwen Shen.
Formal analysis: Liwen Shen.
Funding acquisition: Liwen Shen, Xiaoqin Pan.
Methodology: Liqun Lu.
Writing – original draft: Liwen Shen.
Writing – review & editing: Liqun Lu, Xiaoqin Pan.
Abbreviations:
- CI
- confidence interval
- FET
- frozen embryo transfer
- GnRH
- gonadotropin-releasing hormone
- HMG
- human menopausal gonadotropin
- IVF
- in vitro fertilization
- OR
- odds ratio
This work was supported by the Zhejiang Province Traditional Chinese Medicine Science and Technology Project [grant number 2024ZL1030] and Huzhou Municipal Science and Technology Bureau [grant number2023GYB08]. The funding sources had no role in the study design, the collection, analysis, or interpretation of the data; the writing of the report; nor in the decision to submit this article for publication.
The study protocol was approved by the Medical Ethics Committee at Huzhou Maternity and Child Health Care Hospital (approval number: 2024-J-007; approval date: January 22, 2024) and was performed in accordance with the ethical principles outlined in the Declaration of Helsinki. The ethics committee waived the requirement for informed consent owing to the retrospective nature of the study.
The authors have no conflicts of interest to disclose.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
How to cite this article: Lu L, Shen L, Pan X. Maternal age at frozen embryo transfer, not cryopreservation duration, is more important in the transfer of cleavage-stage embryos than blastocysts: A single-center retrospective study. Medicine 2026;105:10(e47922).
Contributor Information
Liqun Lu, Email: fbyluliqun@126.com.
Xiaoqin Pan, Email: cunzai1101@163.com.
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