Is double frozen blastocyst transfer better than sequential single frozen blastocyst transfers in womenover >35 years of age?

Authors

Abstract

AimThis study aimed to assess live birth rates after double blastocyst transfer versus sequential single blastocyst transfers following freeze-all cycles in women aged over 35 years.
MethodsBetween January 2016 and December 2018, we conducted a retrospective analysis of n = 296 women over 35 years of age who had all their embryos frozen and subsequently transferred. Inclusion criteria were women over 35 years of age with their entire cohort of embryos frozen at the blastocyst stage for different indications based on physician preference. All patients had at least two good-quality embryos in their frozen cohort. Preimplantation genetic testing cycles were excluded. Demographic data were extracted from patient files and electronic databases. Women were categorized based on their mode of embryo transfer (D-FBT vs SS-FBT). The couple decided to adopt one or more strategies after consultation. The study’s primary outcome was the live birth rate (LBR) per woman after one double versus two sequential single embryo transfers.
ResultsOverall, 296 women underwent 362 embryo transfer cycles (D-FBT = 186; SS-FBT = 176). When adjusted for female age, the cumulative LBR per woman was similar in D-FBT (46.2%) and SS-FBT (58.2%) (p=0.054, aHR=1.62, 95% CI: 1.00-2.60). While one monozygotic twin delivery was observed in the SS-FBT group, 22 of 86 (25.6%) live births after D-FBT were twins.
ConclusionFollowing a freeze-all cycle, SS-FBT yielded similar live birth rates (LBR) as D-FBT in women aged over 35 years.

Keywords

Introduction

In the early years of In Vitro Fertilization (IVF), multiple embryos were transferred into the uterus to compensate for low implantation rates. However, advances in IVF laboratories—including improved culture media, advanced incubators, and particularly embryo vitrification—together with the recognition of complications from multiple pregnancies, reduced the number of embryos recommended for transfer. This paradigm shift occurred concurrently with advances in embryo freezing techniques. Embryo vitrification results in excellent post-thaw survival and implantation rates that are equivalent, if not superior, to fresh transfers.¹ Freezing all embryos for later transfer quickly became a widespread practice to prevent ovarian hyperstimulation syndrome, mitigate adverse effects of ovarian stimulation on implantation, and correct incidental endometrial pathologies encountered during ovarian stimulation.^2,3
Currently, it is customary to transfer a single embryo, especially if the quality is good and the patient does not have adverse prognostic factors such as advanced age or previous implantation failures. Vitrification directly contributes to the global acceptance of elective single embryo transfer (eSET), which is considered the best choice for preventing maternal/fetal complications associated with multiple pregnancies.^4,5,6 Defining embryo quality for successful eSET is fundamental.
According to Turkish IVF legislation, single embryo transfer is mandatory in women under 35 years of age, after which no more than two embryos are allowed. However, many patients and physicians prefer embryos to be transferred individually, regardless of age. Although retrospective and prospective studies have compared fresh sequential single embryo transfer versus double embryo transfer, there is limited data regarding outcomes of freeze-all cycles.
This study aimed to assess live birth rates after double blastocyst transfer versus sequential single blastocyst transfers following freeze-all cycles in women aged > 35 years.

Materials and Methods

Study Population and ParticipantsThis study is a retrospective analysis of 362 frozen embryo transfer (FET) cycles in n = 296 couples undertaken in a single tertiary care private hospital-assisted reproduction center between January 2016 and December 2018. Patients provided consent for anonymous analysis and publication of the gathered data for scientific purposes.
We screened 860 patient files in which the cohort of embryos was cryopreserved at the blastocyst stage for various indications. Of these, 296 women aged 35–45 years who had at least two good-quality blastocysts vitrified on the fifth day were selected. The current Turkish legislation in effect since 2010 allows the transfer of two embryos only after 35 years of age, which is why an older cohort was chosen for analysis.⁷ Couples undergoing pre-implantation genetic testing and patients with > 2 prior implantation failures were excluded. Patients were categorized and analyzed based on the mode of embryo transfer [one double frozen blastocyst transfer (D-FBT) versus two sequential single frozen blastocyst transfers (SS-FBT)] (Figure 1). Demographic and clinical data were extracted from patient files and electronic records.
InterventionsOvarian Stimulation, Oocyte Retrieval, Fertilization, Vitrification, and Warming
Ovarian stimulation was performed using recombinant follicle-stimulating hormone (FSH) alone or in combination with human menopausal gonadotropin (HMG). Premature luteinizing hormone (LH) surge was suppressed using a gonadotropin-releasing hormone (GnRH) antagonist in a fixed protocol. Final maturation of oocytes was induced with 250 μg recombinant human chorionic gonadotropin (hCG) or leuprolide acetate in the event of overstimulation. Oocyte retrieval was performed under general anesthesia 35–36 h after the ovulation trigger, using a double-lumen needle. Intracytoplasmic sperm injection (ICSI) was used to fertilize the oocytes. All included patients had at least two day 5 embryos with grade 3BB and above that were vitrified.⁸
Correction of Uterine Pathology
Patients with endometrial polyps discovered during ovarian stimulation underwent hysteroscopic removal prior to vitrified/warmed embryo transfer.
Endometrial Preparation for Vitrified/Warmed Embryo Transfer
All vitrified-warmed embryo transfers were performed in an artificial cycle without downregulation. On menstrual cycle day 2 or 3, a baseline transvaginal ultrasound examination was carried out to rule out the presence of a corpus luteum, follicles > 10 mm in size, or uterine pathology. If all conditions were met, estradiol valerate tablets (2 mg three times daily) were started. Endometrial thickness, echogenicity, and ovarian activity were checked after 10–12 days. Embryo transfer was planned if the endometrial thickness was ≥ 8 mm with a triple-line appearance, and there were no follicles measuring ≥ 10 mm. Embryo transfer was performed on the 6th day after once-daily progesterone gel administration, with dosage increased to twice daily. All embryo transfers were performed under ultrasound guidance using a Wallace or Cook catheter.
Before embryo transfer, clinicians counseled couples regarding the success and risks of transferring more than one embryo and encouraged single embryo transfer. The number of embryos transferred was determined based on a shared decision between the patient and attending physician.
Ethical ApprovalEthics Committee approval for the study was obtained.
Statistical EvaluationData distribution was assessed using the Kolmogorov-Smirnov test. Continuous variables were defined as median (25th–75th percentile), and categorical variables as frequency and rate. The two groups were compared using the Mann-Whitney U test for continuous data and Chi-Square or Fisher’s exact tests for categorical data. The primary outcome was the live birth rate per initiated cycle, and the secondary outcome was the multiple birth rate. A generalized estimating equation model and logistic regression analysis were performed to adjust for confounding factors. Confounders with biological plausibility derived from the literature were selected. Statistical significance was set at two-tailed p<0.05.

Results

A total of 362 embryo transfer cycles (176 single and 186 double blastocyst transfers) in n = 296 women aged over 35 years were analyzed (Figure 2). The mean age of the women was 39 years (range: 36–41). The indications for IVF were male factor in 121, female factor in 97, and unexplained in 78 couples. Freeze-all indications were physician preference (n = 219), overstimulation and/or serum progesterone level > 1.5 ng/mL on the day of hCG administration (n = 61), and endometrial polyps discovered during ovarian stimulation (n = 16). Descriptive characteristics were comparable between the D-FBT and sequential SS-FBT groups (Table 1).
Gonadotrophin dose, estradiol level on the day of trigger, number of oocytes collected, number of Metaphase II (MII) oocytes, fertilization rate, and number of vitrified blastocysts on day 5 of embryo culture did not differ between the groups (Table 2).
Of the 186 women who had transferred two vitrified-warmed blastocysts, 119 (64%) had a positive pregnancy test result, 86 pregnancies resulted in live births (46.2%), and 33 had preclinical and clinical pregnancy losses (Table 2). Of the 110 women who had transferred a single blastocyst, 52 (47.3%) had a positive pregnancy test result, and 44 pregnancies resulted in live births (40.0%). Sixty-six women (58 who failed to conceive and 8 who had a pregnancy loss) underwent a second single blastocyst transfer and achieved a pregnancy rate of 37.9% (25/66) and a live birth rate of 30.3% (20/66).
Implantation rates (ultrasound verified gestational sac) were 37.9% in the D-FBT, 44.5% in the first SS-FBT, and 33.3% in the second SS-FBT cycle. While only one monozygotic twin delivery was observed in the SS-FBT group, 22 of 86 deliveries (25.6%) in the D-FBT group were twins. The cumulative live birth rate per initiated cycle was higher in the SS-FBT group (58.2%) than in the D-FBT group (46.2%). However, when adjusted for female age (the only variable associated with the live birth rate in multivariate analysis), the likelihood of a live birth per cycle initiated was similar between the two groups (p=0.054, aHR=1.62, 95% CI: 1.00–2.60).

Discussion

Our results show that when adjusted for female age, SS-FBT yields a similar cumulative live birth rate as one D-FBT in freeze-all cycles. Importantly, while only one twin delivery was observed with SS-FBT (1.6%), 22 of 86 (25.6%) live births after D-FBT were twins.
IVF success is ideally measured by the rate of a healthy singleton infant delivered with normal weight.⁵ Elective single embryo transfer (eSET) is becoming more common due to recognition of risks associated with multiple pregnancies.⁹ In IVF patients with good prognosis—specifically women younger than 37 years in their first or second cycle with good-quality embryos—the Practice Committee of the American Society for Reproductive Medicine recommends eSET.¹⁰
eSET results in a higher chance of delivering a term singleton live birth than double embryo transfer (DET). Although pregnancy rates are lower in fresh cycles compared to DET, this difference is nearly overcome by an additional single transfer cycle. The multiple pregnancy rate after eSET is comparable to spontaneous pregnancies.¹¹ However, for couples with prolonged infertility, definitions of success may differ. eSET can increase costs, lengthen time to pregnancy, and reduce the chance of a second child in older couples.¹² Despite risks, women over 35 years often prefer twin pregnancies.¹³ Notably, nearly 30% of clinicians decide embryo number without consulting patients, and more than one-third of patients are unaware of their preference for singleton versus twin gestation.¹⁴
Recent studies highlight the importance of embryo stage and patient age. One study suggested that in cleavage-stage freeze-all cycles, eSET should be offered to women aged 35–40 with favorable or average prognosis, as twinning rates can exceed 20% in these groups; DET should be reserved for poor prognosis cases.¹⁵ In our study, despite patients being > 35 years, the twinning rate was 25.6% in the D-FBT group, reflecting favorable characteristics such as the presence of at least two good-quality blastocysts on day five.
Most data comparing single versus double transfers come from fresh cycles. As freeze-all policies gain momentum, it is crucial to assess whether sequential single transfers yield equivalent or higher pregnancy rates with lower multiple birth rates than double transfers.
Eum et al. compared DET versus sequential SET in fresh and vitrified cycles.¹⁶ In women aged 35 years, pregnancy rates were similar (52.0% vs 54.3%, p=0.77), and live birth rates showed no significant difference (38.0% vs 50.0%, p=0.12). However, multiple pregnancy rates were significantly higher in DET (p=0.005; p=0.002). Park et al. categorized vitrified blastocyst transfers in women > 35 years and concluded that single good-quality blastocyst transfers are recommended to reduce multiple pregnancies.^17,18
Long et al. analyzed 24,422 frozen-thawed cycles and found single-blastocyst transfer to be the best choice across all age groups.¹⁹ Monteleone et al. compared sequential SET (n=237) versus DET (n=373) in 610 cycles, reporting similar success rates (45.9% vs 46.6%) but significantly lower multiple pregnancy rates in sequential SET (6.7% vs 32.2%).²⁰ However, their cohort included both fresh and frozen transfers, and patients were younger (33.6–33.9 years) than in our study.
Cost considerations also play a role. Crawford et al. showed that sequential SET in women < 35 years resulted in higher treatment costs than DET, but overall expenses were lower due to reduced pregnancy/infant-associated medical costs.²¹ For advanced-aged patients, where twinning rates are lower, cost-effectiveness analyses are still needed.¹⁵

Conclusion

Our results show that over 35 years of age, SS-FBT has similar LBR success as D-FBT in a freeze-all environment. Thus SS-FBT rather than D-FBT should be offered to women aged > 35 years in the presence of good-quality blastocysts available for transfer. The strengths of this study include the homogenous patient population and the adjustment for all potential confounders. Limitations are the relatively small number of patients analyzed, the fact that only patients aged > 35 years were included, the unavailability of cost-effectiveness analysis, and the retrospective nature of the study. Future studies should analyze double versus sequential single embryo transfers from the perspective of cost-effectiveness. More conclusive evidence can only be obtained in properly designed large-scale randomized studies.

Declarations

References

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About This Article

Received:

February 25, 2023

Accepted:

March 23, 2023

Published Online:

March 24, 2023

Printed:

March 25, 2023