Abstract
Purpose
To explore if a day 7 blastocyst is predictive of the reproductive potential of sibling day 5 or day 6 blastocysts?
Methods
Retrospective cohort of autologous frozen embryo transfers (FET), February 2019 to April 2022. Cycles divided into groups 1 to 5, according to the day of embryo cryopreservation and the presence of a day 7 blastocyst sibling within the cohort: group 1/group 2—day 5 blastocyst without/with a day 7 sibling, group 3/group 4—day 6 blastocyst without/with a day 7 sibling, group 5—day 7 blastocyst. Clinical, ongoing pregnancy and miscarriage rates, cycle, and patient characteristics are reported. Multivariable generalized estimating equations (GEE) logistic regression analysis accounts for confounders and assesses the effect of a sibling day 7 blastocyst on ongoing pregnancy rates of day 5 or day 6 blastocyst FETs.
Results
Ongoing pregnancy rates are 38.4%, 59.5%, 30.8%, 32.7%, and 4.4% in groups 1–5, respectively. When correcting for maternal age, number of oocytes retrieved and discarded per cohort, and ploidy, embryos cryopreserved on either day 6 or day 7 have reduced odds of ongoing pregnancy after FET compared to day 5 blastocysts (OR = 0.76, IQR [0.61–0.95], p-value = 0.01). However, the presence of a day 7 sibling does not significantly affect odds of ongoing pregnancy of day 5 or day 6 blastocysts compared to the same-day blastocyst without a day 7 sibling (p-value = 0.20 and 0.46, respectively). This finding is consistent within both the Preimplantation Genetic Testing for Aneuploidy (PGT-A) unscreened and screened (euploid) embryo subgroups.
Conclusions
Day of embryo cryopreservation significantly affects ongoing pregnancy rates. However, day 7 embryos within a cohort do not affect the reproductive potential of sibling day 5 and day 6 blastocysts, suggesting that slow embryo development is an embryo-specific trait.
Keywords: Blastocyst, Day 5, Day 6, Day 7, Oocyte
Introduction
A blastocyst suitable for transfer, vitrification, or biopsy is determined by the morphology of the inner cell mass, trophectoderm, and stage of expansion [1]. Embryos cultured in vitro most commonly reach the fully expanded blastocyst stage 5 to 6 days after fertilization. Yet, in laboratories that follow embryo development beyond day 6, 2–8% of embryos do not fully blastulate until day 7 [2].
Current trends in assisted reproductive technologies favor embryo transfer of a blastocyst-stage embryo over a cleavage-stage embryo, although more evidence is needed to confirm the benefit to cumulative pregnancy rates [3]. When more than one embryo develops within a cohort of retrieved oocytes, and more than one embryo is available for cryopreservation and future transfer, clinicians and embryologists need to prioritize which embryo to transfer. Current evidence attests that the highest pregnancy rates can be achieved after the transfer of a day 5 blastocyst [4]. However, there are contradicting reports regarding the reproductive potential of slower-developing blastocysts that do not reach full blastulation until days 6 and 7 after fertilization compared to day 5 [5–7].
A recent meta-analysis reviewed the reproductive potential of day 7 blastocysts and found a substantial reduction of reproductive potential in terms of clinical pregnancy rate (CPR), live birth rates (LBR), and euploid rates when compared to the transfer of blastocysts that expanded and were cryopreserved earlier. The major limitations in meta-analyzing information in this field are the small sample size of the group of day 7 frozen and transferred blastocysts compared to the day 5 and day 6 groups, the heterogeneity in patients’ characteristics, clinical features, and laboratory procedures/strategies among the various studies reflected in low quality of many of the studies included [8].
It was previously shown that sibling embryos that develop to the blastocyst stage are predictive of the reproductive potential of a transferred cleavage stage embryo [9]. In addition, fresh embryo transfer results are predictive of the success of subsequent blastocyst frozen embryo transfers [10]. These studies suggest sibling embryos share quality traits and reproductive potential of the oocyte cohort, possibly resulting from similar in vivo developmental environments and hormonal exposures. Yet, questions still remain regarding the reproductive potential of embryos from the same cohort, reaching blastulation on different days, and its prognostic value for sibling embryos. This study aims to expand the existing body of knowledge regarding the reproductive potential of day 7 blastocysts and also to specifically explore the effect of slow embryo development, day 7 blastocyst cryopreservation within a cohort, on sibling blastocysts by analyzing ongoing pregnancy rates of day 5 and 6 blastocyst transfers originating from the same oocyte cohort.
Materials and methods
Study design and population
This is a retrospective cohort study of autologous frozen embryo transfers (FET) occurring between February 2019 and April 2022. Non-blastocyst embryo transfers, gestational carriers, donor egg cycles, frozen embryos transferred during a controlled ovarian stimulation-fresh IVF cycle, and transfers of mosaic and inconclusive DNA analysis embryos are not included in the study.
Ovarian stimulation protocols
Cycles include GnRH antagonist and agonist standard protocols; gonadotropin stimulation includes a combination of rFSH (Gonal-F, EMD Serono) with or without rLH (Luveris, EMD Serono) or uFSH/LH (Menopur, Ferring) and start on day 3 of menses with the dose determined according to patient age and ovarian reserve parameters. Patients are brought in for dose adjustment on cycle day 7, except those on doses of FSH ≥ 300IU, who are brought in on cycle day 11. In antagonist cycles, the antagonist is started once E2 is greater than 2000 pmol/L, or there is a dominant follicle measuring ≥ 14 mm in size, or by cycle day 9 if neither of these criteria is met. Triggering of final oocyte maturation is achieved by hCG (Ovidrel, EMD Serono), GnRH agonist (Superfact, Xediton), or both (Dual trigger) according to cycle and patient characteristics and clinician’s choice, when at least three follicles reach 17 mm in size. Oocyte retrieval is performed 36 h after the trigger injection. Cumulus-oocyte complexes (COCs) were inseminated by conventional IVF, or intracytoplasmic sperm injection (ICSI) was performed when indicated. Semen samples with fair to good parameters (> 5.0 million total motile count; TMC) were processed by 40%/80% density gradient with subsequent wash. Poor quality samples were processed by single wash. For conventional insemination, COCs were inseminated individually in 20 µl wells with Global Fertilization media (CooperSurgical, USA) and 100,000 motile sperm/ml. For ICSI, oocyte denudation was performed approximately 2 h after oocyte retrieval and injection approximately 2 h later. Embryo culture was carried out at 37 °C, 7.5%CO2/6.0%O2 in Global 20 µl wells with Global Total media (CooperSurgical, USA).
Fertilization is assessed approximately 16–18 h after insemination [11]. Embryo progression is assessed on days 1, 2, 5, 6 and 7 after fertilization according to international standards [12]. Our center’s criteria for blastocyst vitrification or biopsy are blastocyst expansion grades 3–6, inner cell mass, and trophectoderm development of at least grade B. Day 7 blastocyst is defined as an embryo reaching full expansion and meeting vitrification/biopsy criteria within 168 h after insemination. The indications for trophectoderm biopsy for Preimplantation Genetic Testing for Aneuploidy (PGT-A) are advanced reproductive age, prior pregnancy loss, recurrent implantation failure, or patient preference. The biopsies are performed before blastocyst cryopreservation.
Endometrial preparation protocols
FET endometrial preparation protocols included both hormone medicated and ovulatory cycles, determined according to patient characteristics and physician clinical judgment. Medicated cycles consist of daily 17-β estradiol (Estrace, Acerus Pharmaceuticals Corporation) 4 mg twice daily either orally or vaginally, beginning on day 2 of the cycle. On days 12–15 of medication, a transvaginal ultrasound is performed to assess the endometrial lining (target: ≥ 8 mm and “triple line” appearance). If the endometrial lining is not thick enough, a 17-β estradiol 100 mcg transdermal patch (Estradot, Novartis Pharmaceuticals) every 2 days is added. Once the endometrium lining has achieved the target thickness, progesterone treatment is initiated: either micronized progesterone (Prometrium, Organon) 200 mg vaginally three times daily, progesterone vaginal inserts (Endometrin, Ferring) 100 mg vaginally three times daily, or progesterone vaginal gel (Crinone, EMD Serono) 90 mg vaginally once daily. In the ovulatory cycle, the patient is followed until detection of ovulation by serial hormonal and sonographic measurements. hCG (Ovidrel, EMD Serono) is administrated on the day of ovulation, and progesterone is initiated, either micronized progesterone (Prometrium, Organon) 200 mg vaginally twice daily, progesterone vaginal inserts (Endometrin, Ferring) 100 mg vaginally twice daily, or progesterone vaginal gel (Crinone, EMD Serono) 90 mg vaginally twice daily. On day 6 of progesterone administration, the embryo is thawed and transferred under ultrasound guidance. The number of blastocyst(s) transferred is based on the clinic’s protocol, national and provincial regulations, and/or the staff’s clinical judgment. Following embryo transfer, in the hormone-supported protocol, estrogen (tablets and/or patch) is reduced by half, and progesterone is continued at the same dose. Estrace and progesterone are continued until 10 weeks gestation, if pregnant, or until 8 weeks gestation in ovulatory FET protocol, or until a negative pregnancy test.
Data collection
Electronic documentation in our clinic started in February 2019; thus, 773 transferred day 5 and day 6 embryos were created before the study period and lacked some retrievable embryology cycle information, including the day of cryopreservation of sibling oocytes. However, the practice of extended blastocyst culturing to 7 days coincided with the beginning of the study period; thus, cycle information regarding all day 7 embryos is complete and does not impair the analysis as no day 7 blastocysts were created before the study period.
FET cycles are divided into five groups according to the day of embryo cryopreservation and the presence of a sibling day 7 blastocyst within the cohort: group 1/group 2—day 5 blastocyst transfer without or with a day 7 sibling, group 3/group 4—day 6 blastocyst transfer without or with a day 7 sibling, group 5—day 7 blastocyst transfer (Fig. 1).
Fig. 1.
Study design
Outcomes measured
The primary outcome of interest is the ongoing pregnancy rate per embryo transfer, defined as the proportion of appropriately developing pregnancies beyond the first trimester. Secondary outcomes are clinical pregnancy rate, defined as a pregnancy diagnosed by ultrasonographic visualization of one or more gestational sacs; biochemical pregnancy rate, pregnancies diagnosed only by the detection of beta hCG in serum or urine that did not progress to clinical pregnancy; and miscarriage rate, defined as pregnancy loss subsequent to ultrasound confirmation of viable implantation [13]. Patient and cycle characteristics are reported.
Statistical analysis
Characteristics of patients and cycles are summarized using mean (SD) or median [IQR] for continuous variables and number, percent for categorical ones. Formal comparisons are performed using ANOVA, Kruskal-Wallis, Chi-squared, and Fisher’s exact tests. Generalized estimating equations (GEE) logistic regression models with exchangeable correlation covariance are used to account for multiple cycles per subject. Multivariable models are adjusted for potential confounders: maternal age, the number of oocytes retrieved and discarded within the cohort, and euploid status (if known) of the transferred embryo tested the effect of day of cryopreservation on ongoing pregnancy rates and the presence of day 7 blastocyst on the reproductive potential of sibling day 5 and day 6 blastocysts. The multivariable models are created on the subgroup of transfers that had complete cycle information on all cycle characteristics. We also evaluated the interaction between ploidy status and the study group to determine whether euploid status is an effect modifier for the association of the group and ongoing pregnancy. A p-value < 0.05 is considered significant. Analysis is executed with the R.4.1.2 statistical program (R Foundation for Statistical Computing, Vienna, Austria).
Ethical approval for retrospective review of patient information was obtained from the institutional review board; approval number 22-0087-C. Patient information was de-identified before the analysis.
Results
Three thousand and seventy-five frozen embryo transfers of 3246 blastocysts in 2096 patients are included during the study period. Group 1 (day 5 blastocyst without a day 7 sibling) includes 1765 FETs in 1151 patients (median age 36 y [33, 38]), group 2 (day 5 blastocyst with a day 7 sibling) includes 42 FETs in 33 patients (median age 36 y [32, 39]), group 3 (day 6 blastocyst without a day 7 sibling) includes 1123 FETs in 788 patients (median age 37 y [34, 39]), group 4 (day 6 blastocyst with a day 7 sibling) includes 55 FETs in 47 patients (median age 34 y [33, 38]), and group 5 (day 7 blastocyst) includes 90 FETs in 77 patients (median age 37 y [34, 39]).
Two thousand nine hundred and four (94.4%) of the cycles are single frozen embryo transfers, and 171 (5.6%) are double embryo transfers. Overall, 808 (26.2%) embryo transfer cycles are with euploid embryos, and the rest (2267; 73.7%) are with chromosomally untested embryos.
The following cycle characteristics differed between the groups: the number of eggs retrieved per IVF cycle (17.03 ± 8.00, 18.03 ± 7.53, 14.02 ± 6.38, 15.49 ± 7.25, 13.05 ± 6.42, for groups 1, 2, 3, 4, and 5, respectively, p-value < 0.001), the total number of blastocysts developed per IVF cycle (6.10 ± 3.44, 6.52 ± 3.17, 3.82 ± 2.06, 3.95 ± 2.04, 2.74 ± 1.33, for groups 1, 2, 3, 4, and 5, respectively, p-value < 0.001), and attrition rate, defined as the cumulative number of oocytes, zygotes, and embryos discarded divided by the total number of oocytes retrieved in the cycle (60.3%±17.18, 59.9%±15.87, 68.7%±16.61,70.7%±15.69, 74.2%±17.69, for groups 1, 2, 3, 4, and 5, respectively, p-value < 0.001).
The proportion of embryos transferred that are known to be euploid differed among groups: 26.6%, 57.1%, 22.0%, 63.6%, and 41.1% in groups 1, 2, 3, 4, and 5, respectively, p-value < 0.001 (Table 1).
Table 1.
Patient and cycle characteristics
| Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | P-value*** | |
|---|---|---|---|---|---|---|
| FET (N) | 1765 | 42 | 1123 | 55 | 90 | |
| Patients (N) | 1151 | 33 | 788 | 47 | 77 | |
| Age, year (median [IQR]) | 36 [33, 38] | 36 [32, 39] | 37 [34, 39] | 34 [33, 38] | 37 [34, 39] | < 0.001 |
| Partner age, year (median [IQR]) | 37 [34, 41] | 37.5 [35, 39] | 38 [35, 42] | 38 [34, 39] | 39 [36, 43] | < 0.001 |
| BMI, kg/m2 (median [IQR]) | 24.03 [21.46, 27.80] | 23.08 [21.16, 24.11] | 24.28 [21.63, 28.33] | 23.20 [20.98, 27.08] | 24.80 [22.44, 28.12] | < 0.035 |
| Oocytes retrieved (mean, SD) | 17.03 ± 8.00 | 18.03 ± 7.53 | 14.02 ± 6.38 | 15.49 ± 7.25 | 13.05 ± 6.42 | < 0.001 |
| Total blastocysts (mean, SD) | 6.10 ± 3.44 | 6.52 ± 3.17 | 3.82 ± 2.06 | 3.95 ± 2.04 | 2.74 ± 1.33 | < 0.001 |
| Oocytes/zygotes/embryos discarded (mean, SD) | 10.12 ± 6.61 | 10.71 ± 5.68 | 9.25 ± 5.77 | 11.05 ± 6.88 | 10.02 ± 6.06 | 0.025 |
| Attrition rate per FET (mean, SD) | 60.26% ± 17.18 | 59.92% ± 15.87 | 68.74% ± 16.61 | 70.65% ± 15.69 | 74.18% ± 17.69 | < 0.001 |
| Biopsied and euploid embryos*, N (%) | 469 (26.57) | 24 (57.14) | 247 (21.99) | 35 (63.64) | 37 (41.11) | < 0.001 |
| Infertility diagnosis, N (%)** | ||||||
| Endometriosis | 115 (6.6) | 2 (4.8) | 89 (8.0) | 2 (3.6) | 5 (5.6) | 0.461 |
| Advanced reproductive age | 223 (12.8) | 11 (26.2) | 194 (17.4) | 8 (14.5) | 26 (28.9) | < 0.001 |
| Ovulatory dysfunction | 214 (12.3) | 6 (14.3) | 147 (13.2) | 6 (10.9) | 7 (7.8) | 0.625 |
| Uterine factor | 64 (3.7) | 2 (4.8) | 38 (3.4) | 2 (3.6) | 4 (4.4) | 0.974 |
| Male factor | 403 (23.1) | 11 (26.2) | 278 (24.9) | 11 (20.0) | 33 (36.7) | 0.047 |
| Diminished ovarian reserve | 148 (8.5) | 4 (9.5) | 176 (15.8) | 3 (5.5) | 10 (11.1) | < 0.001 |
| Oncology fertility preservation | 12 (0.7) | 0 (0.0) | 10 (0.9) | 0 (0.0) | 1 (1.1) | 0.864 |
| Social fertility preservation | 3 (0.2) | 0 (0.0) | 1 (0.1) | 0 (0.0) | 0 (0.0) | 0.961 |
| Repeat pregnancy loss | 18 (1.0) | 0 (0.0) | 26 (2.3) | 0 (0.0) | 1 (1.1) | 0.051 |
| Unexplained infertility | 333 (19.1) | 16 (38.1) | 223 (20.0) | 17 (30.9) | 17 (18.9) | 0.008 |
| Tubal factor | 146 (8.4) | 1 (2.4) | 99 (8.9) | 1 (1.8) | 3 (3.3) | 0.077 |
| Preimplantation genetic testing | 169 (9.7) | 7 (16.7) | 89 (8.0) | 7 (12.7) | 4 (4.4) | 0.071 |
*Euploid embryo transfers: 2 double embryo transfers (DET) in group 1, 1 DET in groups 3 and 5
**Each patient can have multiple infertility diagnoses
***ANOVA, Kruskal-Wallis, or Chi-squared tests
FET, frozen embryo transfer; BMI, body mass index
Reproductive outcomes are summarized in Table 2: Ongoing pregnancy rates are 38.4%, 59.5%, 30.8%, 32.7%, and 4.4% in groups 1, 2, 3, 4, and 5, respectively, p-value < 0.001. Clinical pregnancy rates are 45.8%, 61.9%, 37.7%, 38.2%, and 12.2% in groups 1, 2, 3, 4, and 5, respectively, p-value < 0.001. Biochemical pregnancy rates are 9.0%, 9.5%, 10.9%, 7.3%, and 7.8% in groups 1, 2, 3, 4, and 5, respectively, p-value = 0.443. Spontaneous miscarriage rates are 7.5%, 2.4%, 6.9%, 5.5%, and 7.8% in groups 1, 2, 3, 4, and 5, respectively, p-value = 0.706.
Table 2.
Reproductive outcomes of study groups
| Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | P-value* | |
|---|---|---|---|---|---|---|
| FET (N) | 1765 | 42 | 1123 | 55 | 90 | |
| Positive pregnancy, N (%) | 968 (54.84) | 30 (71.43) | 546 (48.62) | 25 (45.45) | 18 (20.00) | < 0.001 |
| Clinical, N (%) | 809 (45.84) | 26 (61.90) | 423 (37.67) | 21 (38.18) | 11 (12.22) | < 0.001 |
| Biochemical, N (%) | 159 (9.01) | 4 (9.52) | 123 (10.95) | 4 (7.27) | 7 (7.78) | 0.443 |
| SA, N (%) | 132 (7.48) | 1 (2.38) | 77 (6.86) | 3 (5.45) | 7 (7.78) | 0.706 |
| Ongoing, N (%) | 677 (38.36) | 25 (59.52) | 346 (30.81) | 18 (32.73) | 4 (4.44) | < 0.001 |
*Chi-squared or Fisher’s exact tests
FET, frozen embryo transfer; SA, spontaneous abortion
Reproductive outcomes for the euploid embryos subgroup are summarized in Table 3: Ongoing pregnancy rates are 54.4%, 75.0%, 46.7%, 31.4%, and 2.8% in groups 1, 2, 3, 4, and 5, respectively, p-value < 0.001. Clinical pregnancy rates are 61.7%, 75.0%, 52.4%, 37.1%, and 8.3% in groups 1, 2, 3, 4, and 5, respectively, p-value < 0.001. Biochemical pregnancy rates are 6.9%, 8.3%, 10.2%, 8.6%, and 11.1% in groups 1, 2, 3, 4, and 5, respectively, p-value = 0.591. Spontaneous miscarriage rates are 7.3%, 0%, 5.7%, 5.7%, and 5.5% in groups 1, 2, 3, 4, and 5, respectively, p-value = 0.643.
Table 3.
Reproductive outcomes within the euploid embryo transfer subgroup
| Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | P-value** | |
|---|---|---|---|---|---|---|
| FET cycles with biopsied and euploid embryo* (N) | 467 | 24 | 246 | 35 | 36 | |
| Positive pregnancy, N (%) | 320, 68.52% | 20, 83.33% | 154, 62.60% | 16, 45.71% | 7, 19.44% | < 0.001 |
| Clinical, N (%) | 288, 61.67% | 18, 75.00% | 129, 52.44% | 13, 37.14% | 3, 8.33% | < 0.001 |
| Biochemical, N (%) | 32, 6.85% | 2, 8.33% | 25, 10.16% | 3, 8.57% | 4, 11.11% | 0.591 |
| SA, N (%) | 34, 7.28% | 0, 0.00% | 14, 5.69% | 2, 5.71% | 2, 5.56% | 0.643 |
| Ongoing, N (%) | 254, 54.39% | 18, 75.00% | 115, 46.75% | 11, 31.43% | 1, 2.78% | < 0.001 |
*Euploid embryo transfers: 2 double embryo transfers (DET) in group 1, 1 DET in groups 3 and 5
**Fisher’s exact test
FET, frozen embryo transfer; SA, spontaneous abortion
In multivariable analysis presented in Table 4 (N = 1795), with group 1, day 5 blastocyst without a day 7 sibling as the reference group, while not statistically significant, group 2 is found to have higher odds of ongoing pregnancy compared to group 1: OR = 1.54 (IQR [0.79–2.99], p-value = 0.20) and group 4 is found to have lower odds of ongoing pregnancy: OR = 0.53 (IQR [0.27–1.05], p-value = 0.07). Group 3 is found to have lower odds of having an ongoing pregnancy: OR = 0.76 (IQR [0.61–0.95], p-value = 0.01). Group 5, day 7 blastocysts, had significantly lower odds of ongoing pregnancy compared to day 5 blastocysts: OR = 0.069 (IQR [0.026–0.181], p-value < 0.0001). A subgroup analysis comparing group 3, day 6 blastocyst without day 7 sibling as the reference group to group 4, accounting for confounders, found no significant difference in ongoing pregnancy rate: OR = 0.77 (IQR [0.39–1.53], p-value = 0.46).
Table 4.
Association of “day of embryo cryopreservation and presence of day7 sibling” groups with ongoing pregnancy rate (N = 1795)
| Univariable* | Multivariable* | |||
|---|---|---|---|---|
| OR [95%CI] | P-value | OR [95%CI] | P-value | |
| Group 2: Day 5 with day 7 sibling | 1.765 [0.897, 3.472] | 0.1 | 1.541 [0.793, 2.996] | 0.202 |
| Group 3: Day 6 without day 7 sibling | 0.664 [0.539, 0.818] | < 0.001 | 0.766 [0.613, 0.957] | 0.019 |
| Group 4: Day 6 with day 7 sibling | 0.608 [0.322, 1.145] | 0.123 | 0.537 [0.273, 1.056] | 0.071 |
| Group 5: Day7 | 0.062 [0.024, 0.16] | < 0.0001 | 0.069 [0.026, 0.182] | < 0.0001 |
| Maternal age | 0.965 [0.939, 0.991] | < 0.01 | 0.971 [0.943, 1] | 0.047 |
| The number of oocytes retrieved | 1.026 [1.013, 1.04] | < 0.001 | 1.042 [1.007, 1.078] | 0.018 |
| The number of oocytes discarded | 1.013 [0.997, 1.03] | 0.103 | 0.965 [0.926, 1.006] | 0.091 |
| Euploid embryo | 1.761 [1.435, 2.161] | < 0.0001 | 1.852 [1.49, 2.301] | < 0.0001 |
Logistic generalized estimating equations (GEE) model
OR, odds ratio; 95% CI, 95% confidence interval
Analyzing the effect of ploidy status by day of embryo cryopreservation shows no significant interaction effect.
Discussion
Slow embryo development, reflected by full blastulation and cryopreservation on days 6 and 7 after fertilization, is associated with decreased reproductive potential compared to embryos that reach full blastulation and are cryopreserved on day 5. In our study, blastocysts that were cryopreserved on day 6 and day 7 had significantly lower ongoing and clinical pregnancy rates as well as lower implantation rates compared to day 5 blastocysts. This finding is in accord with previous studies that reported lower pregnancy rates with day 6 vs. day 5 embryos [4, 14–18]. However, controversy remains as to whether the euploid status of an embryo can overcome the effect of slow blastulation, with reports of similar outcomes [19] versus poorer outcomes in euploid day 6 vs. day 5 embryos [20]. Our findings of lower pregnancy rates in day 7 blastocyst compared to day 6 and day 5 embryos were also previously reported by other groups [2, 20, 21]. Although, of note, our ongoing pregnancy rate in day 7 blastocytes is much lower than reported by others (4.4%).
A subanalysis of euploid days 6 and 7 embryos demonstrates lower pregnancy rates in slower-developing embryos, despite euploid status. This is consistent with the reports of lower pregnancy rates in euploid day 7 blastocysts [20, 21].
This is the first report addressing the effect of slow-developing, day 7 blastocysts on pregnancy rates of oocyte cohort sibling day 5 or day 6 frozen blastocyst transfers. We found that day 7 siblings do not impair pregnancy rates of day 5 and day 6 embryos in the same cohort. These findings suggest that slower embryo development is only associated with poorer reproductive outcomes for the specific day 7 blastocyst. Patients can be reassured that the poor prognostic features associated with slow embryo development reflect an embryo-specific trait and should not be extended to other embryos within the same cohort.
Although we are limited in our ability to test the quality of oocytes and embryos, PGT-A testing attests to the chromosomal composition of the transferred blastocysts to the limits of the platform used. Our study has found that euploid day 7 embryos have similarly poor performance as untested day 7 embryos, indicating that chromosomal competence is essential but insufficient to sustain embryonic competency and cannot compensate for abnormal post-fertilization events. These findings draw attention to the short but crucial period of early embryonic development associated with embryonic genome activation, mitochondrial function, energy, and metabolic processes required for successful preimplantation development, implantation, and pregnancy progression [22, 23].
Maternal age is well known to be a major factor affecting oocyte quality [24]. Interestingly, despite minimal variation in patient age within the study groups, embryology cycle characteristics are statistically different in groups 2, 4, and 5: cycles with slow embryo development and blastulation with cryopreservation on day 7. These cycles are characterized by fewer oocytes retrieved and a higher attrition rate reflected by fewer usable blastocysts per cycle. As our lab routinely follows embryos to the blastocyst stage and rarely performs day 3 embryo transfers, we can view the high attrition rate as a continuum of slow embryo development. Many of the oocytes in these cycles were fertilized but arrested before reaching blastulation and thus were discarded, leading to the high attrition rate reported above. This observation adds to the hypothesis that post-fertilization events play an important role in embryonic competence.
Additionally, cycles characterized by slow embryo development (groups 2, 4, and 5) have more embryos biopsied and a higher proportion of euploid embryos transferred. This is particularly intriguing; as in our clinic, patients do not have PGT-A routinely, and the decision to biopsy embryos is decided before egg retrieval and regardless of the day of blastulation; thus, the higher biopsy rate could not be prompted by the slow embryo development per se but rather by other patient and cycle characteristics. We speculate, but cannot prove, that there is a bias towards extended culture in cycles planned for PGT-A aiming to maximize the number of embryos available for biopsy and transfer. The higher proportion of PGT-A screened/euploid embryos among the transferred embryos in groups 2 and 4 may also explain the higher ongoing pregnancy rate in these groups.
The strength of this study is the large cohort of single embryo frozen transfers of day 7 blastocysts, adding to the limited numbers in the literature [8]. In addition, this is the first study to explore the effects of slow embryo development within an oocyte cohort and the predictive value of day 7 blastocyst on sibling embryo transfers. The statistical analysis accounted for major confounders such as maternal age, ploidy status, and the number of oocytes retrieved and discarded in the cycle.
The findings of this study have direct clinical implications on embryo prioritization for transfer in situations where more than one embryo is available, favoring day 5 over day 6 over day 7 blastocysts. Moreover, these findings are reassuring regarding the reproductive outcomes of day 5 and day 6 blastocysts, regardless of the presence of sibling day 7 embryos.
This study contains several limitations. Following embryo development until day 7 became routine practice in our clinic in 2019, while some of the transferred embryos originated in prior cycles; hypothetically, if extended culture were to be employed in these cycles, some of the embryos in groups 1 and 3 could have had day 7 siblings. This is a hypothetical scenario, and the statistical analysis accounted for the missing information; we believe this does not affect the strength of our results. In the current dataset, the detailed sperm parameters were not available for subanalysis of the male contribution to embryo development dynamics.
We routinely follow patients in our clinic through the first trimester; unfortunately, not all patients report birth outcomes or are otherwise lost to follow-up. As there is up to a 2-year delay in birth outcomes data collection and reporting from the provincial birth database, we are unable to report updated live birth rates resulting from the transfers within the study period. Data was collected at the embryo transfer level and was de-identified with respect to individual patients; thus, we were unable to calculate cumulative pregnancy rates. Similarly, we could not address the scenarios of a day 7 transfer following a successful or failed day 5 or 6 embryo transfer.
To conclude, our study confirms that the day of embryo full blastulation and cryopreservation significantly affects ongoing pregnancy rates. Day 5 blastocysts have better reproductive potential compared to day 6 and day 7 blastocysts. However, the presence of a day 7 blastocyst within an embryo cohort does not affect the reproductive potential of sibling day 5 and day 6 blastocysts. Our findings suggest that slow embryo development, reflected by full blastulation on day 7, is an embryo-specific trait that is not associated with chromosomal composition. These findings call for further investigation of the early embryo development dynamics.
Funding
No funding was received for conducting this study or to assist with the preparation of this manuscript.
Data availability
Data presented in this study are available upon request from the corresponding authors and are subject to the ethical approval of the institutions involved.
Declarations
Ethics approval
Ethical approval for retrospective review of patient information was obtained from the institutional review board; approval number 22-0087-C. Patient information was de-identified before the analysis.
Conflict of interest
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data presented in this study are available upon request from the corresponding authors and are subject to the ethical approval of the institutions involved.