Abstract

Objective: This study aimed to evaluate the effectiveness of elective single embryo transfer (eSET) versus double embryo transfer (DET) in frozen embryo transfer cycles following in vitro fertilization (IVF) treatment in good prognosis patients. The outcome would provide medical data for the multiple pregnancy rate reduction in IVF treatment.


Methods: This multicenter retrospective cohort study was performed in patients undergoing the first frozen embryo transfer (FET) cycles at IVF centers which belonged to the IVFMD Group, Vietnam, from January 2018 to May 2020. Patients were divided into four groups, based on the number of embryos transferred, as follows: Group 1: one good quality day-3 embryo (eSET D3), Group 2: one good quality day-5 embryo (eSET D5), Group 3: two good quality day-3 embryos (DET D3), and Group 4: two good quality day-5 embryos (DET D5). The primary outcome of the study was live birth rates (LBR) after the first FET. Secondary outcomes were also analyzed, including pregnancy outcomes (β-hCG positive, clinical pregnancy, miscarriage < 12 weeks, ongoing pregnancy 12 weeks, miscarriage < 20 weeks, and multiple birth rates [MBR]), as well as neonatal outcomes (birth weight and gestational age at birth).


Results: There were 819 patients, of which 819 FET cycles were analyzed, including 132 eSET D3, 278 eSET D5, 140 DET D3, and 269 DET D5. LBR and MBR values were significantly lower in the eSET D3 group than in the DET D3 group (LBR: 22.7% vs 39.3%, p = 0.002; MBR: 3.3% vs 29.1%, p < 0.001, respectively). MBR was also significantly lower in eSET D5 compared with DET D5 (9.6% vs 38.3%, p < 0.001), while LBR was comparable between the two groups (41.4% vs 42.8%, p < 0.74). Birth weight and gestational age at birth were similar between eSET and DET, regardless of day-3 or day-5 embryo transfer.


Conclusions: Among infertile, good prognosis women undergoing FET, the eSET significantly decreased multiple birth rates compared with double embryo transfer, while still sustaining an acceptable rate of live birth as well as pregnancy and neonatal outcomes.


INTRODUCTION

The success of an in vitro fertilization (IVF) cycle is to achieve a healthy baby. Due to the advancements of IVF techniques and the improvement of policies for embryo transfer, live birth rate (LBR) have continuously improved since 1978. According to the United States (US) national data, from 1995 to 2013, LBR in fresh embryo transfer cycles increased from 15% to 30%1. In addition to the improvement of LBR, the status of multiple pregnancies commonly occurred following IVF treatment, accounting for about 31 — 41%, which was higher than those in natural conception, around 3.4% (according to the U.S. Centers for Disease Control and Prevention from 2013 to 2016)2, 3. Multiple pregnancies induced many consequences on the health and psychology for both mother and children due to higher risks of miscarriage, preterm birth, low birth weight, very low birth weight, and so on4, 5. Therefore, it has been necessary to ensure the safety of IVF treatment by controlling the multiple pregnancy rate.

The main cause of multiple pregnancies following IVF treatment is the transfer of more than one embryo into the women’s uterus6. Therefore, reducing multiple pregnancy rate by decreasing the number of transferred embryos is one of the goals to achieve better efficacy and safety of IVF treatment. In recent years, many IVF centers all over the world have considered using eSET rather than DET in potential patients in order to reduce the multiple pregnancy rate7, 8, 9, 10, 11, 12, 13. Indeed, eSET was first recommended in 2004 by the American Society for Reproductive Medicine (ASRM) and the Society for Assisted Reproductive Technology (SART) as an alternative to replace DET for reducing multiple pregnancy rates from transfer cycles of fresh embryos in good prognosis patient 14. Good prognosis patients are typically defined as having: maternal age < 35 years old, the first IVF cycle or successful pregnancy during previous IVF treatment, good-quality embryos according to morphological evaluation, and good quality embryos used in transfer or frozen down14. According to the US national data, many IVF centers in the US have implemented eSET in patients < 38 years of age to reduce multiple pregnancies, but the cumulative live birth rate has not been significantly different15. In 2017, ASRM/SART recommended IVF center to limit the number of transferred embryos depending on the embryo developmental stage and patient prognosis in frozen embryo transfer (FET) cycles. By this guideline, SET was applied to good prognosis patients (maternal age < 38 years old, having at least one good quality embryo, and having euploid embryos, and having the first IVF treatment or successful pregnancy during previous treatment)11.

Furthermore, eSET was applied primarily to the blastocyst rather than the cleavage-stage embryo; this was because eSET resulted in lower clinical pregnancy and live birth rates at the cleavage-stage compared to blastocyst stage16, 17, 18. Some studies showed that eSET at the blastocyst stage reduced multiple pregnancy rates from 10 to 20-fold compared with DET, while the results of pregnancy and live birth were not significantly different in the groups18, 19, 20. Moreover, birth weight following fresh blastocyst eSET was significantly different from DET in young patients (< 35 years old) (3226.2 g vs 2832.2 g, p = 0.001)19.

Up to now, reports of the perinatal and neonatal outcomes of the eSET strategy have been very limited. In Vietnam, there have been many studies on the efficacy of various embryo transfer strategies. A randomized controlled trial (RCT) by Lan et al. (2018) compared the efficiency of frozen embryo versus fresh embryo transfer at My Duc Hospital. The results of ongoing pregnancy and live birth rates were not significantly different in frozen versus fresh embryo groups (36.3% vs 34.5%, RR = 1.05, 95% CI: 0.87 – 1.27, p = 0.65, respectively, for the ongoing pregnancy group and 33.8% vs 31.5%, RR = 1.07, 95% CI: 0.88 – 1.31, p = 0.54, respectively, for the live birth rates group)21. According to another retrospective study in 2018, the findings showed that the cumulative live birth rate of day-3 transferred embryo was not different from those of day-522. A retrospective study by Phuong et al. (2019) was performed to compare the IVF outcomes following single embryo transfer (SET) with or without pre-implantation genetic testing for aneuploidy (PGT-A) in advanced maternal age women. The study showed that PGT-A could improve the ongoing pregnancy rate and reduce the number of transferred embryos and multiple pregnancy rates in advanced age patients23. Until now, there have been no studies on the efficacy of SET, which can provide evidence-based information on the number of transferred embryos in Vietnamese IVF patients and for best IVF practices.

Currently, the strategy of elective single embryo transfer (eSET) is proposed for good prognosis patients to ensure pregnancy outcome and control multiple pregnancies in the IVF centers belonging to My Duc Hospital (IVFMD). This study was performed to evaluate the effectiveness of eSET strategy compared with double embryo transfer (DET) of cleavage-stage embryo or blastocyst.

MATERIALS & METHODS

Patient selection and study design

This was a multicenter retrospective cohort study performed on FET cycles at IVF centers belonging to IVFMD, Vietnam, from January 2018 to May 2020. This study was approved by the Medical Ethics Committee of My Duc Hospital, Ho Chi Minh City, Vietnam (22.1/19/ĐĐ-BVMD). Personal information was coded to ensure patient privacy.

Patients were included in this study if they met the following criteria: ≤ 35 years of age, number of retrieved oocyte cycles ≤ 2, having at least four grade-1 or grade-2 embryos on day 3, and having undergone FET with at least one grade-1 or grade-2 embryo on day 3 or day 5. Patients undergoing in vitro maturation (IVM), oocyte donation, pre-implantation genetic testing (PGT), artificial oocyte activation (AOA), and/or having surgical sperm, uterine, or pelvic abnormalities were excluded. Patients were divided into four groups, based on the number of embryos transferred: Group 1: one good quality day-3 embryo (eSET D3), Group 2: one good quality day-5 embryo (eSET D5), Group 3: two good quality day-3 embryos (DET D3), and Group 4: two good quality day-5 embryos (DET D5).

Sperm preparation

Semen samples were collected by the method of masturbation directly into sterile containers and were left for 15 – 30 minutes for the liquefaction process. Samples were prepared by discontinuous density gradient centrifugation with 40% density top layer and a 80% density lower layer (40%, 80% PureCeption-SAGE). This centrifugation let motile spermatozoa swim through the gradient materials to form a soft pellet at the bottom of the tube. After that, the soft pellet was collected and washed with 3 ml of Sperm Preparation medium (Origio, Denmark). The washed spermatozoa were concentrated in 0.2 – 0.3 mL for use in intracytoplasmic perm injection (ICSI)24.

Ovarian stimulation and oocyte retrieval

All the patients underwent controlled ovarian hyperstimulation according to the protocol for the use of follicle-stimulating hormones (FSH) and gonadotropin-releasing hormone antagonists. The dose of recombinant follicle-stimulating hormone was dependent on the woman’s age, antimüllerian hormone levels (AMH), and response to FSH in any prior IVF cycle. Follicular development was monitored by ultrasonography estradiol (E2) and progesterone (P4) levels were evaluated. When the mean diameter of at least two leading follicles was 17 mm, recombinant human chorionic gonadotropin (hCG) or diphereline was administered to trigger oocyte maturation, and oocyte retrieval was performed 36 hours later.

Embryo culture

The oocytes were denuded from cumulus cells by hyaluronidase (SAGE, Denmark) in combination with the mechanical force of the pipette. The mature oocytes (metaphase II) were injected with sperm by ICSI at 39 – 41 hours after hCG injection. After ICSI, the oocytes were incubated at 37 °C, 5% CO2, 5% O2 until the time check of fertilization and the cleavage-stage embryo (day 3) or blastocyst (day 5).

Evaluation of embryo quality

Fertilization was evaluated at 16 - 18 hours after ICSI. The quality of embryos was morphologically classified according to the guideline of the IVFMD (based on Alpha Scoring System, 201125). Day-3 embryos were evaluated at 66 - 68 hours post-ICSI based on the number of blastomeres, blastomere symmetry and fragmentation (% ratio of fragment cell to embryo volume). Accordingly, a good grade 1 embryo contained 8 blastomeres with equal size, fragmentation ≤ 10% and absence of abnormal factors like vacuoles, and blastomere multinucleation (MNB) (Figure 1a). A grade 2 embryo typically contained 6 to 7 or more than 8 blastomeres with unequal size and fragmentation ≤ 25% (Figure 1b). The other were grade 3 embryos.

The blastocysts were evaluated at 112 – 116 hours after ICSI (day 5) based on the expansion of the embryo cavity, the inner cell mass (ICM) characteristics, and the trophectoderm (TE) cell layer. Accordingly, good blastocysts (grade 1 and grade 2) — which had full cavity-enlarged, compacted, large ICM — and many TE cells were assigned as priority choices for transfer or freezing (Figure 1c, d).

Figure 1 . Day-3 embryos and day-5 embryos (20X, scale bar A-A: 50 μm) ( a ) Day-3 embryo grade 1, ( b ) Day-3 embryo grade 2, ( c ) Day-5 embryo grade 1, and ( d ) Day-5 embryo grade 2.

Embryo cryopreservation

The patients were consulted about the options of embryo freezing. All good embryos were proposed to be frozen. All embryos were frozen by vitrification kit method (Cryotech, Japan). The frozen embryos were stored in liquid nitrogen (-196 °C).

Frozen embryo transfer

The patients underwent the process of endometrium preparation before embryo transfer. The frozen embryos were thawed using Warming kit (Cryotech, Japan). The embryos were recorded for their quality and survival rate post thawing. The thawed embryos were cultured in Global Total LP medium (LifeGlobal, US) and were monitored throughout the steps of assisted hatching. Then, embryo were transferred into the uterus under ultrasound guidance.

Determination of clinical outcomes

The primary outcome was LBR after the first frozen embryo transfer cycle. Live birth was defined as the birth of at least one newborn after 24-week gestation that exhibits any sign of life.

The secondary outcome were other pregnancy outcomes (beta-positive, clinical pregnancy, < 12-week miscarriage, 12-week ongoing pregnancy, < 20-week miscarriage, multiple deliveries [2 babies]), and neonatal outcomes (birth weight and gestational age at birth). These clinical outcomes were monitored and recorded in the electronic medical record of each patient.

Serum beta-hCG level was measured at 2 weeks after embryo transfer. If beta-hCG was positive (≥ 25 IU ml), a clinical pregnancy was confirmed at 6 weeks after embryo transfer through ultrasonography of the gestational sac. Ongoing pregnancy was defined as pregnancy with a detectable heartbeat after 12-week of gestation. Miscarriage < 12 weeks or miscarriage < 20 weeks were defined as complete clinical abortion in 12-week or 20-week gestation. Multiple births were twins born with vital signs of life after 24-week gestation. Moreover, the birth weight and gestational age at birth were recorded.

Statistical analysis

The baseline characteristics of the patients were described by descriptive statistics for each group. The data were expressed as mean ± standard deviation (SD) for continuous variable and number (%) for binary variables. P-values were estimated using the Student’s t-test for continuous variables and the Chi-square or Fisher’s exact test was used for binary variables. Factors affecting live birth rates were evaluated by univariate logistic regression and multivariate logistic regression. All data in the study was processed by statistical software R version 3.3.3 (R Foundation for Statistical Computing, Vienna, Austria).

Results

From January 2018 to May 2020, there were 819 patients included in the study, with a total of 819 FET cycles analyzed, including 132 eSET D3, 278 eSET D5, 140 DET D3, and 269 DET D5. All of the baseline characteristics of the patients (eSET and DET group) were recorded including age, body mass index (BMI), AMH, duration of infertility, type of infertility, number of IVF cycles, and IVF indications. There were no statistically significant differences between the two groups (p > 0.05) (Table 1).

Table 1.

The baseline characteristics of patients

Characteristic eSET (N = 410) DET (N = 409) P-value
Age (years) 29.8 ± 3.1 30.0 ± 3.1 0.20
BMI (kg/m 2 ) 21.0 ± 2.5 21.1 ± 2.6 0.60
AMH (ng/ml) 6.3 ± 3.5 6.2 ± 3.8 0.69
Duration of infertility (years) 4.1 ± 2.6 4.0 ± 2.6 0.66
Type of infertility – n (%)
Primary 259 (63.2) 247 (60.4) 0.46
Secondary 151 (36.8) 162 (39.6)
Number of IVF cycles – n (%)
1 343 (83.7) 341 (83.4) 0.99
2 67 (16.3) 68 (16.6)
IVF indication – n (%)
PCOS 101 (24.6) 95 (23.2) -
Ovulation disorder 31 (7.6) 31 (7.6) -
Tubal factor 78 (19.0) 76 (18.6) -
Diminished ovarian reserve 5 (1.2) 14 (3.4) -
IUI failure 16 (3.9) 28 (6.8) -
Male factor 93 (22.7) 75 (18.3) -
Other 26 (6.3) 12 (2.9) -
Unexplained 60 (14.6) 78 (19.1) -

The clinical IVF characteristics and embryology outcomes of the two patient groups are shown in Table 2. The results showed that the first dose of FSH and trigger by hCG in the SET group of patients were lower than those in the DET group (p < 0.05). Indeed, the duration of ovarian stimulation, serum progesterone (P4) and estrogen (E2) levels on day of triggering of oocyte maturation, types of trigger (hCG or diphereline), number of fertilized oocytes, total number of embryos and day-3 embryo rate in the SET group were significantly higher than those in the DET group (p < 0.001) (Table 2). Meanwhile, the total dose of FSH, endometrial thickness, number of oocytes retrieved, number of good day-3 embryos, and total number of frozen embryos in the two groups were similar (p > 0.05).

Table 2.

C linical IVF characteristics and embryology outcomes

Characteristic eSET (N = 410) DET (N = 409) P-value
First dose of FSH (IU) 219.9 ± 52.0 229.00 ± 54.5 0.02
Duration of stimulation (days) 9.1 ± 1.2 8.9 ± 1.2 0.04
Total dose of FSH (IU) 2309.4 ± 687.1 2344.3 ± 664.1 0.46
E2 levels on day of trigger (pmol/l) 16982.2 ± 1682.5 11621.2 ± 1280.2 <0.001
P4 levels on day of trigger (pmol/l) 1.4 ± 0.9 1.2 ± 0.8 0.01
Type of trigger – n(%)
hCG 80 (19.5) 190 (46.5) < 0.001
Diphereline 330 (80.5) 219 (53.5)
Endometrial thickness (mm) 12.9 ± 2.3 12.5 ± 2.2 0.15
No. of oocytes retrieved (n) 16.8 ± 7.2 16.3 ± 6.3 0.35
No. of two-pronuclear fertilized oocytes (n) 15.8 ± 7.2 14.2 ± 6.6 < 0.001
No. of day-3 embryos (n) 15.0 ± 7.4 13.2 (6.9) < 0.001
Day-3 embryo rate (%) 93.2 ± 15.6 87.1 ± 17.7 < 0.001
No. good day-3 embryos (n) 8.4 ± 3.6 8.3 ± 3.5 0.58
Total no. of embryos frozen (n) 5.9 ± 4.2 5.6 ± 3.5 0.26
OHSS – n (%) 3 (0.7) 5 (1.2) 0.72

The embryology and pregnancy outcomes were compared between eSET D3 and DET D3, and shown in Table 3. Pregnancy parameters such as β-hCG positivity, clinical pregnancy, and ongoing pregnancy after 12-week gestation in the DET group were all significantly higher than those in the eSET group (p < 0.01). In particular, LBR and MBR in the DET D3 group were significantly higher than those in the eSET D3 group (39.3% vs 22.7%, p < 0.01; 29.1% vs 3.3%, p < 0.001; respectively). However, the rate of implantation, miscarriage, ectopic pregnancy, and neonatal outcomes did not differ between the two groups (p > 0.05). The results of univariate logistic regression showed that the number of transferred embryos, type of trigger, and the number of oocytes retrieved affected the results of live birth after day-3 embryo transfer (Supplemental Table 1). However, the results of the multivariate logistic regression showed that there was no correlation among the analyzed variables to live birth.

Table 3.

Day -3 embryo transfer cycles outcomes

Outcome eSET D3 (N = 132) DET D3 (N = 140) Relative risk [95%CI] Absolute differences (95%CI)* P-value
Age (years) 29.9 ± 3.1 30.5 ± 3.1 - 0.16
BMI (kg/m 2 ) 21.3 ± 2.9 21.3 ± 2.4 - 0.84
AMH (ng/ml) 5.7 ± 3.0 5.1 ± 3.5 - 0.12
No. of oocytes retrieved (n) 12.1 ± 4.8 13.6 ± 4.4 - 0.01
No. of two-pronuclear fertilized oocytes (n) 11.7 ± 4.9 10.3 ± 4.5 - 0.02
No. of day-3 embryos (n) 11.7 ± 4.9 9.0 ± 4.6 - < 0.001
No. good day-3 embryos (n) 7.2 ± 3.2 6.6 ± 2.9 - 0.07
Live birth – n (%) 30 (22.7) 55 (39.3) 0.45 [0.27-0.77] < 0.05
β-hCG positive – n (%) 44 (33.3) 81 (57.9) 0.36 [0.22-0.60] < 0.001
Implantation (%) 28.0 ± 46.8 30.3 ± 34.8 -2.3 (-12.2, 7.6) 0.64
Clinical pregnacy – n (%) 36 (27.2) 68 (48.6) 0.40 [0.24-0.66] <0.001
Miscarriage < 12 weeks – n (%) 4 (3.0) 8 (5.7) 0.52 [0.15-0.75] 0.28
Ectopic pregnacy – n (%) 2 (1.5) 4 (2.9) 0.52 [0.09-2.90] 0.45
Ongoing pregnacy 12-week gestation– n (%) 32 (24.2) 59 (42.1) 0.44 [0.26-0.74] < 0.05
Miscarriage < 20 weeks – n (%) 2 (1.5) 2 (1.4) 1.06 [0.15-7.65] 0.95
Gestational age at birth (weeks) 36.4 ± 4.6 36.5 ± 4.1 -0.1 (-2.0, 1.8) 0.93
Multiple birth rate – n (%) 1 (3.3) 16 (29.1) 0.06 [0.01-0.45] < 0.001
Birth weight of singleton (g) 3086.7 ± 325.1 2898.2 ± 598.3 188.5 (-9.6, 386.5) 0.06
Birth weight of twins (g) 2000.0 ± NA 2290.6 ± 446.2 - -

The embryology and pregnancy outcomes in the eSET D5 and DET D5 groups are shown in Table 4. The results showed that the implantation rate of eSET D5 was significantly higher than that of DET D5 (54.3% ± 57.3% vs 34.2% ± 37.2%; Absolute difference = 20.1, 95% CI: 12.0, 28.2 p < 0.001), while most pregnancy and neonatal outcomes did not differ between the two groups (p > 0.05). Interestingly, MBR in the eSET D5 group decreased significantly compared to that in the DET D5 (9.6% vs 38.3%, p < 0.001). Thus, eSET or DET with good quality embryos on day 5 did not affect pregnancy or neonatal outcome, while eSET minimized MBR in these patients.

The embryology and pregnancy outcomes between the eSET D3 and eSET D5 groups are shown in Table 5. LBR in the eSET D3 group was significantly lower than that of eSET D5 (22.7% vs 41.4%, p < 0.001). Similarly, other embryology and pregnancy outcomes (such as β-hCG positivity, implantation, clinical pregnancy, and ongoing 12-week pregnancy) were significantly lower in the eSET D3 group (p < 0.001). Meanwhile, the rates of miscarriage < 12 weeks, ectopic pregnancy, MBR, and gestation age at birth did not differ between the two groups (p > 0.05). Furthermore, birth weight of singleton in the eSET D3 group was significantly higher than that of the eSET D5 group (3086.7 g ± 325.1 g vs 2859.1 g ± 620.3 g, p = 0.01). However, this outcome was within the normal range (2500 – 4000 g), as referenced by WHO standards. The birth weight of twins was lower in the eSET D3 than eSET D5 group, but remained within the normal range. Thus, good embryos of eSET D3 group resulted in significantly lower LBR compared to that of eSET D5, but MBR of eSET D5 group was higher than that of eSET D3.

Table 4.

Day-5 embryo transfer cycles outcomes

Outcome eSET D5 (N = 278) DET D5 (N = 269) Relative risk [95%CI] Absolute differences (95%CI)* P-value
Age (years) 29.6 ± 3.1 29.8 ± 3.1 - 0.61
BMI (kg/m 2 ) 20.9 ± 2.3 21.1 ± 2.6 - 0.46
AMH (ng/ml) 6.5 ± 3.7 6.7 ± 3.8 - 0.55
No. of oocytes retrieved (n) 19.0 ± 7.1 17.8 ± 6.7 - 0.04
No. of two-pronuclear fertilized oocytes (n) 17.8 ± 7.3 16.2 ± 6.6 - 0.02
No. of day-3 embryos (n) 16.6 ± 7.8 15.3 ± 6.9 - 0.04
No. good day-3 embryos (n) 9.0 ± 3.7 9.2 ± 3.5 - 0.53
Live birth – n (%) 115 (41.4) 115 (42.8) 0.94 [0.67-1.33] 0.74
β-hCG positive – n (%) 180 (64.7) 175 (65.1) 0.99 [0.69-1.40] 0.94
Implantation (%) 54.3 ± 57.3 34.2 ± 37.2 20.1 (12.0, 28.2) <0.001
Clinical pregnacy – n (%) 140 (50.4) 139 (51.7) 0.95 [0.68-1.33] 0.76
Miscarriage <12 weeks – n (%) 17 (6.1) 18 (6.7) 0.91 [0.46-1.80] 0.78
Ectopic pregnacy – n (%) 5 (1.8) 5 (1.9) 0.97 [0.28-3.38] 0.96
Ongoing pregnacy 12-week gestation – n (%) 123 (44.2) 121 (45.0) 0.97 [0.69-1.36] 0.86
Miscarriage < 20 weeks– n (%) 8 (2.9) 5 (1.9) 1.56 [0.51-4.84] 0.43
Gestational age at birth (weeks) 35.8 ± 4.8 36.3 ± 4.1 -0.5 (-1.7, 0.6) 0.34
Multiple birth rate – n (%) 11 (9.6) 44 (38.3) 0.21 [0.11-0.42] < 0.001
Birth weight of singleton (g) 2859.1 ± 620.3 2834.2 ± 627.6 24.9 (-136.6, 186.3) 0.76
Birth weight of twins (g) 2275.0 ± 524.6 2428.2 ± 584.5 -153.2 (-535.6, 229.2) 0.41

Table 5.

Outcomes of eSET D3 and eSET D5 group

Outcomes eSET D3 (N = 132) eSET D5 (N = 278) Relative risk [95%CI] Absolute differences (95%CI)* P-value
Age (years) 29.9 ± 3.1 29.7 ± 3.2 - 0.40
BMI (kg/m 2 ) 21.3 ± 2.9 20.9 ± 2.3 - 0.1
AMH (ng/ml) 5.7 ± 3.0 6.5 ± 3.7 - 0.03
No. of oocytes retrieved (n) 12.1 ± 4.8 19.0 ± 7.1 - < 0.001
No. of two-pronuclear fertilized oocytes (n) 11.7 ± 4.9 17.8 ± 7.3 - < 0.001
No. of day-3 embryos (n) 11.7 ± 4.9 16.6 ± 7.8 - < 0.001
No. good day-3 embryos (n) 7.2 ± 3.1 9.0 ± 3.7 - < 0.001
Live birth – n (%) 30 (22.7) 115 (41.4) 0.42 [0.26-0.67] < 0.001
β-hCG positive – n (%) 44 (33.3) 180 (64.7) 0.27 [0.18-0.42] < 0.001
Implantation (%) 28.0 ± 46.8 54.33 ± 57.3 -26.3 (-36.8, -15.8) < 0.001
Clinical pregnacy – n (%) 36 (27.2) 140 (50.4) 0.27 [0.18-0.42] < 0.001
Miscarriage < 12 weeks – n (%) 4 (3.0) 17 (6.1) 0.48 [0.16-1.46] 0.19
Ectopic pregnacy – n (%) 2 (1.5) 5 (1.8) 0.84 [0.16-4.39] 0.84
Ongoing pregnacy 12-week gestation – n (%) 32 (24.2) 123 (44.2) 0.40 [0.25-0.64] < 0.001
Miscarriage < 20 weeks– n (%) 2 (1.5) 8 (2.9) 0.52 [0.11-2.48] 0.40
Gestational age at birth (weeks) 36.4 ± 4.6 35.8 ± 4.8 0.6 (-1.2, 2.4) 0.45
Multiple birth rate – n (%) 1 (3.3) 11 (9.6) 0.19 [0.02-1.45] 0.07
Birth weight of singleton (g) 3086.7 ± 325.1 2859.1 ± 620.3 227.6 (63.3, 391.9) 0.01
Birth weight of twins (g) 2000.0 ± NA 2275.0 ± 524.6 - -

DISCUSSION

This was the first study on the effectiveness and safety of elective single frozen embryo transfer in Vietnam. The results of our study showed that LBR following eSET D3 was significantly lower than that for DET D3 in FET cycles in good prognostic patients. Meanwhile, this rate was similar in the two groups of patients transferred day-5 embryo (eSET D5 and DET D5).

Moreover, it is necessary to ensure safety in IVF treatment by controlling multiple pregnancies. Studies have recommended the implementation of eSET D3 and eSET D5 to reduce multiple pregnancy rate 15, 18, 26. In our study, eSET also demonstrated its safety because this strategy reduced MBR by 11-12% compared to DET. Specifically, MBR was reduced by 11% in eSET D3 group compared to DET D3, and by 12% in eSET D5 group compared to DET D5. Similarly, the analysis of data from 2004 to 2013 of the ASRM study (2017), which conducted the first fresh embryo transfer cycles in young patients (< 35 years old), showed that LBR in the eSET D3 decreased by 15% compared to that of DET D3, while eSET D5 decreased by 10% compared to DET D5. With eSET D5, multiple pregnancy rate decreased 22-47% compared to DET D5; with eSET D3, this rate decreased by 22-28% compared to DET D3 in patients under 38 years old 18.

In contrast, results of the largest sample size for retrospective study was reported by Racca et al. (2020), who showed that LBR between the SET and DET was similar following cleavage embryo transfer (13.1% vs 14.8%, p = 0.33, respectively) or blastocyst transfer (21.7% vs 23.4%, p = 0.4, respectively) while multiple delivery rates were significantly higher in women with DET compared to SET (16.7% vs 1.9%; p < 0.001)27. Their results of multivariate logistic regression adjusted for confounding factors also showed that the number of embryos transferred in the FET cycle was not related to LBR 27. Furthermore, other studies showed that eSET D5 did not reduce pregnancy and live birth rates compared to DET D515, 28. According to another study of Freeman et al. (2019), LBR between eSET D5 and DET D5 were similar (54 — 62% vs 54— 66%, p = 0.696 – 1,000) and MPR decreased significantly with eSET D5 compared to DET D5 (0-3% vs 24 – 65, p < 0.05) in good prognosis patients (under 38 years of age at oocyte collection, having at least two frozen blastocysts, and undergoing their first autologous FET cycle)29.

Our study showed that neonatal outcomes, such as birth weight and gestational age at birth for eSET, did not differ when compared to those for DET after day-3 or day-5 embryo transfer, but birth weight in the eSET D3 was significantly higher than that in eSET D5 (p = 0.01) (Table 5). According to Friedman et al., birth weight following eSET was significantly different compared with DET during the fresh blastocyst transfer cycles for patients < 35 years old19. The study by Martin et al. (2017) also found that singletons born after eSET did not have a high risk of adverse neonatal outcomes (preterm birth < 37 weeks, very preterm birth < 32 weeks, low birth weight < 2500 g, and very low birth weight < 1500 g), while singletons or twins born after DET had a higher risk compared with singletons of natural conception25. Analysis of data from three RCTs (publication dates of 2004, 2005, and 2006) also showed that gestational age at birth and birth weight of singletons born after the transfer of cleagave-stage embryos did not differ significantly versus blastocyst transfer30. Indeed, eSET was common in blastocysts rather than in cleavage-stage embryo due to the rates of clinical pregnancy, and LBR after single cleavage-stage embryo transfer was lower than that for single blastocyst transfer16, 17, 18.

The strategy of single day-3 embryo transfer has been considered in our center in recent years 16, 18. Our study herein showed that the rates of β-hCG-positivity, implantation, clinical pregnancy, ongoing pregnancy, and LBR (41.4% vs 22.7%, p < 0.001) of the eSET D5 group were significantly higher than those of eSET D3 group (Table 5).

Currently, embryo transfer at the blastocyst stage is being promoted over cleavage-stage embryo transfer by IVF centers all over the world due to many reasons. Firstly, blastocyst transfer optimizes physiological synchronization between the endometrium and embryo. Secondly, the gene expression in blastocysts is more complete, allowing self-selection of embryos with higher implantation potential to be transferred. Thirdly, evidence-based medicine has show that blastocyst transfer had better clinical outcomes than use of cleavage-stage embryos in some patient groups17, 31, 32. According to a systematic review of 10 RCTs, there were no differences in the rates of live birth, clinical pregnancy, and miscarriage between blastocyst-stage transfer versus cleavage-stage embryo transfer31. According to a previous study in IVFMD, the findings showed that cumulative LBR following day-3 embryo transfer did not differ from that of day-5 embryo transfer22. Once again, our study demonstrated that implantation rates of eSET D5 was highest (54.3% ± 57.3%) and was significantly different compared to DET D5 (34.2% ± 37.2%; Absolute difference: 20.1, 95% CI: 12.0, 28.2 p < 0.001), eSET D3 (28.0% ± 46.8%; Absolute difference: 26.3, 95% CI: 15.8, 36.8; p < 0.001).

However, extending embryo culture to the blastocyst stage is heavily influenced by external culture conditions. Therefore, the patient may face a higher risk of having no embryos to transfer 33, 34. According to the analysis of ASRM, the rate of no blastocyst on day 5 was significantly higher compared to cleavage-stage embryo (8.9% vs 2.8%; 16 RCTs: OR 2.85; 95% CI: 1.97 – 4.11), but this was no different in good prognosis patients (9 RCTs: OR 1.50; 95% CI: 0.79 – 2.84) 34. According to reported data, the blastocyst formation rate was approximately 35-45%35. Therefore, extended culture should only be performed when the IVF laboratory has a stable embryo culture system.

Besides, blastocyst transfer also has higher risk than cleavage-stage embryo transfer. These risks include preterm birth, monozygotic twinning (MZT), and imbalance sex ratio (e.g. male offspring higher than female offspring)32, 33, 34, 36, 37, 38. Furthermore, the use of eSET D3 and eSET D5 strategy combination with selected embryos transferred by time-lapse morphokinetics showed no difference in pregnancy outcomes (such as implantation rate, early ectopic, and live birth) and neonatal outcomes (such as preterm birth, gestational age, birth height, and birth weight). The rate of MZT in the eSET D5 group was significantly higher than that of eSET D3 (6.98% vs 0.00%, p < 0.05)39. Our study showed that when performing eSET, there was also MZT, and multiple birth rates of eSET D3 were lower but not statistically significant compared to those of eSET D5 (3.3% (1/30) versus 9.6% (11/115), respectively; RR = 0.19, 95% CI: 0.02 – 1.45, p = 0.07). Therefore, eSET D3 can be an option for embryo transfer for minimizing monozygotic twins. The results of this study provide scientific evidence for doctors or embryologists to consult the good prognosis patients about the strategies of eSET versus DET at cleavage-stage embryo or blastocyst stage.

The limitations of this study included its retrospective design, which only focused on analysis of pregnancy and neonatal outcomes in one patient group (good prognosis patients). Further studies with larger sample sizes and prospective design will be needed to increase the reliability of the efficacy of the eSET strategy in different patient groups.

CONCLUSIONS

Elective single embryo transfer in good prognosis patients should be a choice to minimize the risk of multiple pregnancies while achieving acceptable live birth and neonatal outcomes. The strategy of elective single blastocyst transfer for good prognosis patients was an optimal option that ensured a balance between live birth outcomes and minimizing the risk of multiple pregnancies. Besides, elective single day-3 embryo transfer had the lowest live birth rate but minimized the risk of multiple pregnancies. Therefore, the doctor or embryologist should consult the good prognosis patients about the efficiency and safety within each embryo transfer strategy. The results of this study can provide scientific evidence and rational to support controlling infertility treatment through embryo transfer strategy in Vietnamese good prognostic IVF patients.

Abbreviations

AMH: Anti-Muller hormone

ASRM: American Society for Reproductive Medicine

BMI: Body mass index

D3: Day 3

D5: Day 5

DET: Double embryo transfer

eSET: elective single embryo transfer

FET: Frozen embryo transfer

FSH: Follical stimulating hormone

ICM: Inner cell mass

ICSI: Intracytoplasmic sperm injection

IVF: In vitro fertilization

IVM: In vitro maturation

LBRs: Live birth rates

MBRs: Multiple brith rates

OHSS: Ovarian hyperstimulation syndrome

MPR: Multiple pregnancy rates

PCOS: Polycystic ovary syndrome

SART: Society for Assisted Reproductive Technology

Acknowledgments

This study was performed at IVFMD PN-My Duc Phu Nhuan Hospital, IVFMD-My Duc Hospital. The authors would like to thank the Board of Directors for their support. We are also thankful to our colleagues from IVFMD PN who positively assisted in completing this study.

Author’s contributions

All authors read and approved the final manuscript.THLT wrote the manuscript. NHD re-vised the manuscript. DQV planned and designed the experiments. DTL and PHH collected the data. NTL collected and analysed the data. NTTH supervised the study and finalized the manuscript. All authors read and confirmed the publication of the article.

Funding

None.

Availability of data and materials

Data and materials used and/or analysed during the current study are available from the corresponding author on reasionable request.

Ethics approval and consent to participate

This study was conducted in accordance with the amended Declaration of Helsinki. The institutional review board approved the study, and all participants provided written informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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