Abstract
RESEARCH QUESTION:
Day of cryopreservation, inner cell mass (ICM) grade, trophectoderm (TE) grade and blastocyst expansion grade have been associated with differences in live birth rate in frozen embryo transfer (FET) cycles. We sought to examine the likelihood of live birth and whether blastocyst morphologic grade is more or equally useful in FET cycles among PGT-A tested and untested blastocysts.
DESIGN:
Retrospective cohort study of 6,271 vitrified-warmed, autologous, single embryo transfer cycles among patients undergoing in vitro fertilization (IVF) from July 2013 to December 2017 at a single, university-affiliated infertility practice. The primary outcome was live birth, calculated by generalized estimating equation.
RESULTS:
Among PGT-A tested embryos, we observed inferior ICM grade was associated with lower chance of live birth (ICM grade B vs. A aRR: 0.91; 95%CI: 0.84–0.99). Among untested blastocysts there was a lower live birth rate in blastocysts cryopreserved day 6 versus day 5 (aRR: 0.87; 95% CI: 0.78–0.96), inferior pre-vitrification TE grade (TE grade B Vs. A aRR: 0.86; 95% CI: 0.79–0.94). Blastocysts with a higher pre-vitrification expansion grade (grade 5 vs. 4 aRR: 1.1; 95% CI: 1.01–1.2) were but ICM grade was not associated with chance of live birth (grade B vs. A aRR: 0.93 95%; CI: 0.86–1.02).
CONCLUSIONS:
Among PGT-A untested blastocysts, assessing embryo quality by day of cryopreservation, TE grade and expansion grade may help identify embryos with the highest likelihood of live birth. Identifying euploid embryos by PGT-A appears to homogenize the cohort, making blastocyst morphologic grade and the day of cryopreservation less important.
Keywords: frozen embryo transfer, blastocyst morphology, trophectoderm, inner cell mass, PGT-A
INTRODUCTION
Vitrification and advances in embryo culture conditions have closed the historical gap in live birth rate between frozen embryo transfer (FET) and fresh embryo transfer cycles (Chen et al., 2016; Roque et al., 2013; Wong et al., 2017). FET largely eliminates the risk of ovarian hyperstimulation syndrome and has been associated with a lower incidence of ectopic pregnancy, preterm birth, and low birthweight (Ishihara et al., 2011; Maheshwari et al., 2018; Sha et al., 2018). While fresh embryos encounter a variably stimulated endometrium, vitrified-warmed embryos benefit from a more physiologic uterine environment, thereby allowing for managed synchronization with the window of implantation through endometrial programming (Casper and Yanushpolsky, 2016). FET also allows for preimplantation genetic testing for aneuploidy (PGT-A) via trophectoderm (TE) biopsy without time constraints to obtain a result. In addition, there is data to suggest that an elevated serum progesterone on the day of trigger is detrimental to fresh cycle outcomes (Bosch et al., 2010; Venetis et al., 2013). For these reasons and others, some clinicians advocate a “freeze-all” strategy, culturing embryos and vitrifying them with or without TE biopsy after they reach the mature blastocyst stage (Basile and Garcia-Velasco, 2016).
The advent of single embryo transfer (SET), in the setting of multiple available frozen blastocysts, has led to the clinical conundrum of which blastocyst to transfer first. From an available cohort of frozen blastocysts, many characteristics have been proposed to select those of the highest quality and with the maximum chance of a live birth. Blastocyst morphologic grade remains the most commonly used criteria for blastocyst selection. Some retrospective studies suggest blastocysts vitrified on culture day 5 (D5) may have improved clinical outcomes, including a higher live birth rate, when compared to those vitrified on day 6 (D6) (Ferreux et al., 2018; Wang et al., 2016). A 2010 systematic review and meta-analysis found a higher live birth rate in D5 versus D6 frozen blastocysts; however, slow freezing was used in eight out of nine studies. When limited to studies that used vitrification, the ongoing pregnancy and live birth rates in D5 and D6 blastocysts were comparable (Sunkara et al., 2010). A 2011 study by Ahlström et al. investigated the influence of blastocoele expansion, TE, and inner cell mass (ICM) grades on clinical outcomes in FET. They found that TE grade was the strongest indicator of success (Ahlström et al. 2011). In contrast, Du et al.’s similar retrospective analysis suggested blastocoele expansion degree was the morphologic parameter best associated with live birth (Du et al., 2016). Attention also has been given to TE morphology and embryonic progression, with some evidence of improved clinical pregnancy and live birth rates among faster-growing embryos and those with higher grade TE morphology (Ahlström et al., 2011; Cimadomo et al., 2018; Irani et al., 2018; Kaing et al., 2018; Kroener et al., 2012; Rienzi et al., 2019). These observations support a growing body of literature that suggest the morphological grade of blastocysts may be better indicators than day of vitrification alone. However, these data have largely been limited to PGT-A untested blastocysts.
Recent studies have shown significantly higher live birth and lower miscarriage rates in select populations when transferring vitrified-warmed blastocysts deemed euploid by PGT-A (Chen et al., 2015; Irani et al., 2018; Levy et al., 2013). Blastocyst biopsy for PGT-A is not thought to impair the reproductive potential, and is not associated with reduction in live birth (Scott et al., 2013). It is also well accepted that although morphology and aneuploidy are linked at the blastocyst stage, the association is weak, and consequently, morphologic analysis cannot be relied on to ensure transfer of chromosomally normal blastocysts (Alfarawati et al., 2011). On a daily basis, clinicians and patients are faced with selecting a single blastocyst from a frozen cohort, whether PGT-A tested or untested. In this study, we sought to identify characteristics of cryopreserved blastocysts that were associated with the highest likelihood of live birth in FET cycles and to examine whether blastocyst morphology is more useful in PGT-A tested compared to untested blastocyst transfers.
MATERIALS AND METHODS
Inclusion criteria
We conducted a retrospective cohort study of patients who underwent autologous in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles at a single university-affiliated infertility practice and had an oocyte retrieval from July 1, 2013 to December 31, 2017. For each oocyte retrieval, we included all of the subsequent SET cycles with embryos that were cultured to the blastocyst stage and vitrified. If PGT-A was performed, we excluded cleavage-stage biopsy and included only those that underwent aneuploidy testing by TE biopsy.
Stimulation, transfer, blastocyst culture and vitrification
Patients underwent ovarian stimulation, vaginal oocyte retrieval and subsequent blastocyst vitrification using antagonist and agonist protocols at the discretion of the treating physician. These protocols included subcutaneous doses of gonadotropins; monitoring with transvaginal ultrasound, serum estradiol, luteinizing hormone and progesterone levels; and follicle maturation with human chorionic gonadotropin (hCG), gonadotropin releasing hormone agonist, or both, as described previously (Murphy et al., 2019; Vaughan et al., 2017). For PGT-A embryos, laser was used on day 3 to facilitate hatching on the day of trophectoderm biopsy. This was achieved by pulsing the zona pellucida for 250 μsec. If PGT-A was performed, only those blastocysts that were deemed euploid by next generation sequencing were transferred. At our center, more than 90% of TE biopsies were analyzed by a single laboratory. The infertility practice was not provided with embryonic mosaicism data.
Blastocyst cryopreservation occurred on D5 or D6 after reaching expansion grade 3 or greater and any ICM or TE grade combination of A and B, according to the Gardner scoring criteria (Gardner et al., 2000; Sakkas and Gardner, 2012). This selection criteria was based on internal data and a previous published study (Ahlström et al., 2013). Blastocysts not meeting these criteria (3BB or better) were also frozen outside of protocol on a case-by-case basis. This was only performed for a small amount of cases and involved a specific clinician or patient request when no other blastocysts were available for the patients. All blastocysts, except for those that had undergone biopsy for PGT-A, where collapsed by laser prior to vitrification. Blastocysts were vitrified using the FUJIFILM Irvine Scientific vitrification system (Santa Ana, CA). To ensure standardized blastocyst grading the clinic has a rigid training protocol which is completed by each embryologist prior to the performance of any grading activities. Our IVF clinic is also part of the American Association of Bioanalysts Proficiency Testing for Embryo Grading, and all embryologists take part in a biannual grading test of blastocyst morphology.
FET was performed according to provider preference. Treatment protocols included natural cycle or modified natural cycle with clomiphene or letrozole, which have been demonstrated to have equivalent rates of live birth (Mackens et al., 2020). In these cycles, either hCG trigger was used for final follicular maturation or the patient was monitored until a serum luteinizing hormone surge was detected. Programmed hormone replacement cycles also were performed during the study period with both oral and transdermal estradiol used, while progesterone was administered intramuscularly, vaginally, or both. Of note, success rates at our center for both natural and programmed hormone replacement FET cycles are equivalent, which is consistent with the existing literature (Ghobara et al., 2017).
Statistical Analysis
The primary outcome was live birth. Secondary outcomes included positive pregnancy test, clinical pregnancy, and miscarriage. Data are presented as median and interquartile range (IQR) or n (%). Risk ratios (RR), adjusted RRs (aRR) and 95% confidence intervals (CI) were calculated using generalized estimating equations to account for multiple cycles per patient and multiple oocytes retrieved per cycle. All models were adjusted for age at oocyte retrieval, number of oocytes retrieved, number of prior IVF cycles, expansion grade pre-vitrification, ICM grade, TE grade, and day of blastocyst vitrification. Although outcomes are reported as incidence, the terms live birth rate, clinical pregnancy rate, and miscarriage rate are used to be consistent with the literature. Live birth rate also was compared in the 12 blastocyst quality combinations with the greatest number of live births, using blastocysts cryopreserved day 5 with ICM grade A and TE grade A (D5 4AA) as the reference group.
Ethical approval
This study was reviewed and approved by the Beth Israel Deaconess Medical Center Committee for Clinical Investigation (Protocol #: 2015P-000122; approved May 2015).
RESULTS
During the study period, 6,271 FETs met inclusion criteria. These blastocysts were retrieved in 4,761 cycles from a total of 4,338 patients. Of the blastocysts transferred, 2,478 (40%) were deemed euploid by PGT-A, and 3,793 (60%) were untested (Table I). The group that had undergone PGT-A testing had a median maternal age at transfer of 36.2 vs 33.7 for the group with PGT-A untested blastocysts. There was a lower proportion of diagnosis of unexplained infertility among the PGT-A tested group, and ICSI was performed in 55% of PGT-A tested blastocysts and 41% of untested blastocysts. Baseline characteristics of each group are shown in Table I.
Table I.
Baseline patient and cycle characteristics for all vitrified-warmed single blastocyst transfers, stratified by use of PGT-A testing
| PGT-A tested n=2478 | Untested n=3793 | |
|---|---|---|
| Age at retrieval (years) | 36.2 (33.4 – 38.9) | 33.7 (31.2 – 36.1) |
| Age at transfer (years) | 36.6 (33.8 – 39.3) | 34.6 (32.1 – 37.1) |
| BMI (kg/m2) | 24.1 (21.6 – 27.6) | 25.1 (22.1 – 29.6) |
| Smoking at cycle start | 18 (0.7) | 32 (0.8) |
| Primary infertility | 1799 (72.6) | 2402 (63.3) |
| Reason for in vitro fertilization | ||
| Gravidity | ||
| Parity | ||
| Prior miscarriage | ||
| Oocytes retrieved in fresh cycle | 15 (10 – 22) | 15 (10 – 21) |
| Number of prior cycles | 0 (0 – 2) | 1 (1 – 2) |
| Endometrial thickness | 9.2 (8.1 – 10.8) | 9.5 (8.2 – 11.0) |
| Intracytoplasmic sperm injection | 1360 (54.9) | 1565 (41.3) |
| Natural frozen embryo transfer | 288 (11.6) | 504 (13.3) |
Data presented as median (interquartile range) or n (%).
A lower proportion of PGT-A tested blastocysts was cryopreserved on D5 (58%) compared to untested blastocysts (75%). Nearly all tested (99%) and untested blastocysts (99%) were of high morphologic quality, defined as a score of 3BB or higher, reflecting our cryopreservation protocol. Blastocyst characteristics are shown in Table II.
Table II.
Blastocyst quality per transfer, stratified by use of PGT-A testing
| PGT-A tested n=2478 | untested n=3793 | |
|---|---|---|
| Blastocyst expansion grade | ||
| 3 | 752 (30.3) | 721 (19.0) |
| 4 | 857 (34.6) | 2273 (59.9) |
| 5 | 520 (21.0) | 727 (19.2) |
| 6 | 349 (14.1) | 72 (1.9) |
| Inner cell mass grade | ||
| A | 1208 (48.7) | 1955 (51.5) |
| B | 1260 (50.8) | 1822 (48.0) |
| C | 10 (0.4) | 16 (0.4) |
| Trophectoderm grade | ||
| A | 1069 (43.1) | 1651 (43.5) |
| B | 1389 (56.1) | 2111 (55.7) |
| C | 20 (0.8) | 31 (0.8) |
| Day of blastocyst vitrification | ||
| Day 5 | 1425 (57.5) | 2847 (75.1) |
| Day 6 | 1053 (42.5) | 946 (24.9) |
Data presented as n (%)
PGT-A tested blastocysts appeared to have lower rates of miscarriage (6.5%) than PGT-A untested blastocysts (9.1%). Tested blastocysts also had higher rates of positive pregnancy tests (73% vs. 66%), clinical pregnancy (63% vs. 52%), and live birth (56% vs. 42%) relative to untested blastocysts (data not shown).
Among tested blastocysts deemed euploid by PGT-A, the crude RR suggested live birth rate was significantly lower in blastocysts cryopreserved D6 compared with D5 (RR 0.92; 95% CI: 0.86 – 0.99), though this difference was attenuated and no longer significant in our final model (aRR 0.98; 95% CI: 0.90 – 1.1). Similarly, the adjusted effect of TE grade also was attenuated and not associated with live birth (aRR 0.96; 95% CI: 0.89 – 1.04 comparing TE grade B vs. A, aRR 0.90; 95% CI: 0.55 – 1.5 comparing TE grade C vs. A). Pre-vitrification expansion grade in tested blastocysts was not associated with live birth before or after adjusting for covariates. However, ICM grade did remain significant for live birth after adjusting (ICM grade B vs. grade A aRR: 0.91; 95% CI: 0.84–0.99). Vitrified blastocyst quality and chance of live birth among PGT-A embryos are shown in Table III.
Table III.
Vitrified-warmed blastocyst quality and chance of live birth among PGT-A tested and untested cycles
| Live birth | Crude RR (95% CI) | p | Adjusted RR* (95% CI) | p | |
|---|---|---|---|---|---|
| Day of vitrification | |||||
| PGT-A tested | |||||
| 5 (n=1425) | 818 (57.4) | Reference | Reference | ||
| 6 (n=1053) | 559 (53.1) | 0.93 (0.86 – 0.99) | 0.03 | 0.98 (0.90 – 1.1) | 0.58 |
| Untested | |||||
| 5 (n=2847) | 1278 (44.9) | Reference | Reference | ||
| 6 (n=946) | 325 (34.4) | 0.77 (0.69 – 0.85) | <0.001 | 0.87 (0.78 – 0.96) | 0.007 |
| Pre-vitrification expansion grade | |||||
| PGT-A tested | |||||
| 3 (n=752) | 432 (57.4) | 1.04 (0.96 – 1.1) | 0.30 | 1.1 (0.97 – 1.2) | 0.17 |
| 4 (n=857) | 471 (55.0) | Reference | Reference | ||
| 5 (n=520) | 301 (57.9) | 1.05 (0.96 – 1.2) | 0.29 | 1.1 (0.96 – 1.2) | 0.27 |
| 6 (n=349) | 173 (49.6) | 0.90 (0.80 – 1.02) | 0.10 | 0.93 (0.82 – 1.1) | 0.28 |
| Untested | |||||
| 3 (n=721) | 261 (36.2) | 0.85 (0.77 – 0.95) | 0.004 | 0.93 (0.83 – 1.03) | 0.18 |
| 4 (n=2273) | 965 (42.4) | Reference | Reference | ||
| 5 (n=727) | 347 (47.7) | 1.13 (1.03 – 1.2) | 0.01 | 1.1 (1.01 – 1.2) | 0.04 |
| 6 (n=72) | 30 (41.7) | 0.98 (0.74 – 1.3) | 0.90 | 1.1 (0.79 – 1.4) | 0.70 |
| Inner cell mass grade | |||||
| PGT-A tested | |||||
| A (n=1208) | 713 (59.0) | Reference | Reference | ||
| B (n=1260) | 660 (52.4) | 0.89 (0.83 – 0.95) | <0.001 | 0.91 (0.84 – 0.99) | 0.03 |
| C (n=10) | 4 (40.0) | 0.68 (0.32 – 1.5) | 0.32 | 0.78 (0.35 – 1.7) | 0.53 |
| Untested | |||||
| A (n=1955) | 924 (47.3) | Reference | Reference | ||
| B (n=1822) | 677 (37.2) | 0.79 (0.73 – 0.85) | <0.001 | 0.93 (0.86 – 1.02) | 0.13 |
| C (n=16) | 2 (12.5) | 0.26 (0.07 – 0.99) | 0.048 | 0.40 (0.10 – 1.6) | 0.20 |
| Trophectoderm grade | |||||
| PGT-A tested | |||||
| A (n=1069) | 623 (58.3) | Reference | Reference | ||
| B (n=1389) | 745 (53.6) | 0.92 (0.86 – 0.99) | 0.02 | 0.96 (0.89 – 1.04) | 0.40 |
| C (n=20) | 9 (45.0) | 0.77 (0.49 – 1.2) | 0.27 | 0.90 (0.55 – 1.5) | 0.69 |
| Untested | |||||
| A (n=1651) | 815 (49.4) | Reference | Reference | ||
| B (n=2111) | 782 (37.0) | 0.75 (0.70 – 0.81) | <0.001 | 0.86 (0.79 – 0.94) | 0.001 |
| C (n=31) | 6 (19.4) | 0.39 (0.20 – 0.77) | 0.007 | 0.67 (0.34 – 1.3) | 0.25 |
Data presented as n (%) across the rows, or risk ratio (RR) with 95% confidence interval (CI)
Adjusted for patient age at oocyte retrieval, number of oocytes retrieved, number of prior IVF cycles, expansion grade pre-vitrification, inner cell mass, trophectoderm grade, and day of vitrification
Among PGT-A untested blastocysts , we observed a significantly lower live birth rate in blastocysts cryopreserved D6 compared with D5 (aRR: 0.87; 95% CI: 0.78–0.96) and among embryos with pre-vitrification TE grade B compared with grade A (aRR: 0.86; 95% CI: 0.79–0.94). We also observed a higher live birth rate in PGT-A untested blastocysts with a higher expansion grade prior to cryopreservation (grade 5 vs 4 aRR: 1.1; 95% CI: 1.01–1.2). ICM grade was not significantly associated with the chance of live birth in our final model. Vitrified blastocyst quality and chance of live birth among PGT-A untested blastocysts are shown in Table III.
We then combined blastocyst quality parameters to compare live birth rate for each blastocyst subtype against the subtype with the greatest number of live births, D5 4AA, stratified by whether PGT-A was used. Among blastocysts deemed euploid by PGT-A, there was no significant difference in the likelihood of a live birth among other blastocyst quality subtypes, compared with D5 4AA blastocysts (Figure 1A, Supplemental Table 1). Among untested blastocysts, several embryo quality subtypes were significantly associated with a lower likelihood of live birth when compared with D5 4AA blastocysts (Day 5 3BB aRR 0.79; 95% CI: 0.59 – 0.84), Day 5 4BB aRR 0.85; 95% CI: 0.73 – 0.96), Day 6 4AB aRR 0.63; 95% CI: 0.45 – 0.90), Day 6 4BB aRR 0.65; 95% CI: 0.54 – 0.79), Day 6 3BB aRR 0.73; 95% CI: 0.54 – 0.99), Day 6 5BB aRR 0.76; 95% CI: 0.60 – 0.97), and this relationship was more pronounced than among PGT-A tested blastocysts (Figure 1B, Supplemental Table 1).
Figure 1A.
Embryo morphology and chance of live birth in PGT-A cycles.
Figure 1B.
Embryo morphology and chance of live birth in untested cycles
DISCUSSION
Among PGT-A untested blastocysts, our model indicates assessment of blastocyst quality by day of vitrification, TE grade, and expansion grade may help identify blastocysts with the highest likelihood of live birth in FET cycles. Among blastocysts deemed euploid by PGT-A, however, the cohort appears to be homogenized, making blastocyst morphologic grade and the day of cryopreservation less important for blastocyst selection.
Embryonic progression, day of cryopreservation, TE and ICM morphology all have been hypothesized to reflect blastocyst quality. Compared to D6, embryos cryopreserved on D5 achieve embryonic developmental milestones faster. In theory, this suggests that they may be of higher quality. Recent retrospective cohort studies using vitrification have demonstrated that the live birth rate may be slightly higher with D5 versus D6 vitrified-warmed blastocysts (Ahlström et al. 2011; Kroener et al. 2012). In addition, even slower growing embryos extended to culture day 7 (D7) may have lower euploidy rates compared to D5 and D6 blastocysts (Tiegs et al., 2019). While blastocysts vitrified D7 were shown to have favorable neonatal outcomes, they have also been associated with significantly lower rates of clinical pregnancy, implantation and live birth (Du et al., 2018). Maternal age is also known to impact blastocyst ploidy status and quality, so it would be expected that older patients would produce lower grade and slower growing embryos. A 2019 study by Irani et al. suggested otherwise, noting that the rate of blastocyst development and blastocyst grading does not vary with patient age (Irani et al., 2019). If a blastocyst is of high morphological grade, perhaps the rate at which that embryo developed is not as relevant. Among PGT-A untested blastocysts, our results support previous studies that supported the utility of day of cryopreservation in the assessment of blastocyst quality. Untested blastocysts cryopreserved D5 versus D6 were significantly associated with higher chance of live birth in our adjusted model. Our data on untested embryos and those from previous studies relating morphology and day of cryopreservation become less meaningful once a blastocyst has been deemed euploid by PGT-A.
Blastocyst expansion may not be a reliable indicator of quality, or indicative of the underlying chromosomal complement. Fragouli et al. assessed the ploidy status of over 1000 cleavage and blastocyst stage embryos by microarray and correlated this with embryonic progression and morphologic grade. In their study, grade 1–2 blastocysts had slightly higher rates of aneuploidy compared to grade 5–6. Overall, aneuploidy had little impact on morphology and there were many high-quality appearing blastocysts that were aneuploid. Our results fit with this understanding. Among euploid blastocysts, differences in expansion grade were not associated with differences in live birth. Among PGT-A untested blastocysts, expansion grade 5 was better than grade 4, but the association of expansion grade with live birth was not uniform across other grades. Further complicating this, Fragouli et al. confirmed that sex affected rate of development, as it has long been thought that male embryos may reach the expanded blastocyst stage prior to female embryos (Fragouli et al., 2014).
There is robust evidence that blastocyst morphologic grade, in particular ICM and TE grade, are associated with viability. ICM grade has been reported to differentiate among euploid blastocysts, with higher rates of ongoing pregnancy among excellent morphologic grade blastocysts when compared to those with good, average, or poor quality (Irani et al., 2017). Ahlström et al. performed a similar analysis but did not detect pre-vitrification ICM grade as a significant indicator for a higher chance of live birth. For each unit decrease in pre-vitrification TE grade, however, they did find that the live birth rate decreased by 29% (Ahlström et al., 2013). The lesser association between ICM morphology and live birth compared to TE morphology has been shown in a number of studies (Ahlström et al., 2011; Ebner et al., 2016; Honnma et al., 2012). Our ICM results are consistent with Irani et al., but more modest, with our adjusted model indicating ICM morphologic grade in euploid blastocysts is associated with higher live birth. Among untested embryos, our model is consistent with Ahlström, demonstrating grade A TE morphology remains significantly associated with higher chance of live birth. Capalbo et al. correlated higher morphologic grades with lower rates of aneuploidy among D5, D6 and D7 blastocysts, but also demonstrated that despite lower morphologic grades, ongoing implantation rates were similar among blastocysts as long as they had been deemed euploid by PGT-A (Capalbo et al., 2014). Overall, our own model confirms that among PGT-A untested blastocysts, assessment of blastocyst quality through TE morphologic grade, expansion grade, and day of cryopreservation offer useful guidance for selecting blastocysts with the highest chance of live birth in FETs. Among blastocysts 3BB or better and deemed euploid by PGT-A, the differences in morphologic grade and progression are not statistically significant.
The questions of how morphology assessment can be improved and how it relates to aneuploidy has long been a main goal of embryologists and clinicians. Rather than static images alone, time lapse morphological assessment holds promise to improve assessment of embryo quality. The use of deep learning and artificial intelligence to analyze videos of embryo development has begun to be evaluated on its ability to predict live birth in IVF cycles(Campbell et al., 2013; Tran et al., 2019). With validation, these methods may prove more useful for guiding blastocyst selection in future cryo-thaw cycles when compared to static scoring, as is currently utilized. Looking forward, metabolic imaging seems to be a promising, non-invasive blastocyst selection tool (Sanchez et al., 2019, 2017).
A limitation of this study is the paucity of low-quality blastocyst transfers. Among both PGT-A and untested blastocysts, 99% were 3BB or better, attendant to the clinical cryopreservation protocol in our center. However, there were no significant differences in live birth rate among blastocysts with grade C TE or ICM, with or without PGT-A, when compared to those with grade A parameters (Table III). It would be expected that blastocysts of very poor quality would have poorer outcomes, but our dataset lacks the sample size to detect this difference. Blastocysts within the study were graded using the Gardner Criteria, which may limit direct application of these findings to other IVF programs. However, these grades can be translated efficiently to other blastocyst morphology assessment systems, including those published by the Istanbul consensus (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). Blastocyst euploidy rate, expansion rate and blastocyst morphology can also vary significantly based on culture systems. This may significantly affect the reproducibility of our model within other laboratories with alternate culture systems. Though our findings suggest better performance among euploid blastocysts of higher morphologic grade, the results were not statistically significant and were less pronounced than among PGT-A untested blastocysts. This lack of statistical significance could be attributable to a lack of power.
Clinicians and patients have conflicting evidence and no clear guide when selecting order of blastocyst transfer from a vitrified-warmed cohort. Among PGT-A untested blastocysts of unknown ploidy, assessment of blastocyst quality via morphological grading and day of cryopreservation can help select the blastocyst with the highest chance of live birth. However, when a blastocyst is deemed euploid by PGT-A, it appears to attenuate the value of these predictors.
Supplementary Material
FUNDING
This work was conducted with support from Harvard Catalyst | The Harvard Clinical and Translational Science Center (National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health Award UL1 TR001102) and financial contributions from Harvard University and its affiliated academic health care centers. The funding sources had no involvement in the study design, collection, analysis, or interpretation of data, the writing of the report, or the decision to submit the article for publication.
Footnotes
All authors have confirmed that there are no potential conflicts of interest or disclosures to report pertaining to this submission.
REFERENCES
- Ahlström A, Westin C, Reismer E, Wikland M, Hardarson T, 2011. Trophectoderm morphology: An important parameter for predicting live birth after single blastocyst transfer. Hum. Reprod 26, 3289–3296. 10.1093/humrep/der325 [DOI] [PubMed] [Google Scholar]
- Ahlström A, Westin C, Wikland M, Hardarson T, 2013. Prediction of live birth in frozen-thawed single blastocyst transfer cycles by pre-freeze and post-thaw morphology. Hum. Reprod 28, 1199–1209. 10.1093/humrep/det054 [DOI] [PubMed] [Google Scholar]
- Alfarawati S, S M, Fragouli E, Ph, D., Colls P, Ph, D., Stevens J, Wells D, Ph, D., Path FRC, 2011. The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender. Fertil. Steril 95, 520–524. 10.1016/j.fertnstert.2010.04.003 [DOI] [PubMed] [Google Scholar]
- Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011. The Istanbul consensus workshop on embryo assessment: Proceedings of an expert meeting. Reprod. Biomed. Online 22, 632–646. 10.1016/j.fertnstert.2018.05.024 [DOI] [PubMed] [Google Scholar]
- Basile N, Garcia-Velasco JA, 2016. The state of “freeze-for-all” in human ARTs. J. Assist. Reprod. Genet 33, 1543–1550. 10.1007/s10815-016-0799-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bosch E, Labarta E, Crespo J, Simón C, Remohí J, Jenkins J, Pellicer A, 2010. Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: Analysis of over 4000 cycles. Hum. Reprod 25, 2092–2100. 10.1093/humrep/deq125 [DOI] [PubMed] [Google Scholar]
- Campbell A, Fishel S, Bowman N, Duffy S, Sedler M, Thornton S, 2013. Retrospective analysis of outcomes after IVF using an aneuploidy risk model derived from time-lapse imaging without PGS. Reprod. Biomed. Online 27, 140–146. 10.1016/j.rbmo.2013.04.013 [DOI] [PubMed] [Google Scholar]
- Capalbo A, Rienzi L, Cimadomo D, Maggiulli R, Elliott T, Wright G, Nagy ZP, Ubaldi FM, 2014. Correlation between standard blastocyst morphology, euploidy and implantation: An observational study in two centers involving 956 screened blastocysts. Hum. Reprod 29, 1173–1181. 10.1093/humrep/deu033 [DOI] [PubMed] [Google Scholar]
- Casper RF, Yanushpolsky EH, 2016. Optimal endometrial preparation for frozen embryo transfer cycles: Window of implantation and progesterone support. Fertil. Steril 105, 867–872. 10.1016/j.fertnstert.2016.01.006 [DOI] [PubMed] [Google Scholar]
- Chen M, Wei S, Hu J, Quan S, 2015. Can comprehensive chromosome screening technology improve IVF/ICSI outcomes? A meta-analysis. PLoS One 10, 1–21. 10.1371/journal.pone.0140779 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen Z-J, Shi Y, Sun Y, Zhang B, Liang X, Cao Y, Yang J, Liu J, Wei D, Weng N, Tian L, Hao C, Yang D, Zhou F, Shi J, Xu Y, Li J, Yan J, Qin Y, Zhao H, Zhang H, Legro RS, 2016. Fresh versus Frozen Embryos for Infertility in the Polycystic Ovary Syndrome. N. Engl. J. Med 10.1056/NEJMoa1513873 [DOI] [PubMed] [Google Scholar]
- Cimadomo D, Capalbo A, Levi-Setti PE, Soscia D, Orlando G, Albani E, Parini V, Stoppa M, Dovere L, Tacconi L, Ievoli E, Maggiulli R, Ubaldi FM, Rienzi L, 2018. Associations of blastocyst features, trophectoderm biopsy and other laboratory practice with post-warming behavior and implantation. Hum. Reprod 1–10. 10.1093/humrep/dey291 [DOI] [PubMed] [Google Scholar]
- Du QY, Wang EY, Huang Y, Guo XY, Xiong YJ, Yu YP, Yao GD, Shi SL, Sun YP, 2016. Blastocoele expansion degree predicts live birth after single blastocyst transfer for fresh and vitrified/warmed single blastocyst transfer cycles. Fertil. Steril 105, 910–919.e1. 10.1016/j.fertnstert.2015.12.014 [DOI] [PubMed] [Google Scholar]
- Du T, Wang Y, Fan Y, Zhang S, Yan Z, Yu W, Xi Q, Chen Q, Mol BW, Lyu Q, Kuang Y, 2018. Fertility and neonatal outcomes of embryos achieving blastulation on Day 7: are they of clinical value? Hum. Reprod 33, 1038–1051. 10.1093/humrep/dey092 [DOI] [PubMed] [Google Scholar]
- Ebner T, Tritscher K, Mayer RB, Oppelt P, Duba HC, Maurer M, Schappacher-Tilp G, Petek E, Shebl O, 2016. Quantitative and qualitative trophectoderm grading allows for prediction of live birth and gender. J. Assist. Reprod. Genet 33, 49–57. 10.1007/s10815-015-0609-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ferreux L, Bourdon M, Sallem A, Santulli P, Barraud-Lange V, Le Foll N, Maignien C, Chapron C, De Ziegler D, Wolf JP, Pocate-Cheriet K, 2018. Live birth rate following frozen-thawed blastocyst transfer is higher with blastocysts expanded on Day 5 than on Day 6. Hum. Reprod 10.1093/humrep/dey004 [DOI] [PubMed] [Google Scholar]
- Fragouli E, Alfarawati S, Spath K, Wells D, 2014. Morphological and cytogenetic assessment of cleavage and blastocyst stage embryos. Mol. Hum. Reprod 20, 117–126. 10.1093/molehr/gat073 [DOI] [PubMed] [Google Scholar]
- Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB, 2000. Blastocyst score affects implantation and pregnancy outcome: Towards a single blastocyst transfer. Fertil. Steril 73, 1155–1158. 10.1016/S0015-0282(00)00518-5 [DOI] [PubMed] [Google Scholar]
- Ghobara T, Gelbaya TA, Ayeleke RO, 2017. Cycle regimens for frozen-thawed embryo transfer. Cochrane Database Syst. Rev 10.1002/14651858.CD003414.pub3 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Honnma H, Baba T, Sasaki M, Hashiba Y, Ohno H, Fukunaga T, Endo T, Saito T, Asada Y, 2012. Trophectoderm morphology significantly affects the rates of ongoing pregnancy and miscarriage in frozen-thawed single-blastocyst transfer cycle in vitro fertilization. Fertil. Steril 98, 361–367. 10.1016/j.fertnstert.2012.05.014 [DOI] [PubMed] [Google Scholar]
- Irani M, O’Neill C, Palermo GD, Xu K, Zhang C, Qin X, Zhan Q, Clarke RN, Ye Z, Zaninovic N, Rosenwaks Z, 2018. Blastocyst development rate influences implantation and live birth rates of similarly graded euploid blastocysts. Fertil. Steril 110, 95–102.e1. 10.1016/j.fertnstert.2018.03.032 [DOI] [PubMed] [Google Scholar]
- Irani M, Reichman D, Robles A, Melnick A, Davis O, Zaninovic N, Xu K, Rosenwaks Z, 2017. Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates. Fertil. Steril 107, 664–670. 10.1016/j.fertnstert.2016.11.012 [DOI] [PubMed] [Google Scholar]
- Irani M, Zaninovic N, Rosenwaks Z, Xu K, 2019. Does maternal age at retrieval influence the implantation potential of euploid blastocysts? Am. J. Obstet. Gynecol 220, 379.e1–379.e7. 10.1016/j.ajog.2018.11.1103 [DOI] [PubMed] [Google Scholar]
- Ishihara O, Kuwahara A, Saitoh H, 2011. Frozen-thawed blastocyst transfer reduces ectopic pregnancy risk: An analysis of single embryo transfer cycles in Japan. Fertil. Steril 95, 1966–1969. 10.1016/j.fertnstert.2011.02.015 [DOI] [PubMed] [Google Scholar]
- Kaing A, Kroener LL, Tassin R, Li M, Liu L, Buyalos R, Hubert G, Shamonki M, 2018. Earlier day of blastocyst development is predictive of embryonic euploidy across all ages: essential data for physician decision-making and counseling patients. J. Assist. Reprod. Genet 35, 119–125. 10.1007/s10815-017-1038-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kroener L, Ambartsumyan G, Briton-Jones C, Dumesic D, Surrey M, Munné S, Hill D, 2012. The effect of timing of embryonic progression on chromosomal abnormality. Fertil. Steril 98, 876–880. 10.1016/j.fertnstert.2012.06.014 [DOI] [PubMed] [Google Scholar]
- Levy B, Scott RT, Ferry KM, Forman EJ, Tao X, Hong KH, Treff NR, Taylor D, 2013. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil. Steril 100, 100–107.e1. 10.1016/j.fertnstert.2013.02.056 [DOI] [PubMed] [Google Scholar]
- Mackens S, Stubbe A, Landuyt L. Van, Racca A, Roelens C, Camus M, Vos M. De, Vijver A. Van De, Tournaye H, Blockeel C, 2020. To trigger or not to trigger ovulation in a natural cycle for frozen emgryo transfer: a randomized controlled trial. Hum. Reprod 00, 1–9. 10.1093/humrep/deaa026 [DOI] [PubMed] [Google Scholar]
- Maheshwari A, Pandey S, Raja EA, Shetty A, Hamilton M, Bhattacharya S, 2018. Is frozen embryo transfer better for mothers and babies? Can cumulative meta-analysis provide a definitive answer? Hum. Reprod. Update 10.1093/HUMUPD/DMX031 [DOI] [PubMed] [Google Scholar]
- Murphy LA, Seidler EA, Vaughan DA, Resetkova N, Penzias AS, Toth TL, Thornton KL, Sakkas D, 2019. To test or not to test? A framework for counselling patients on preimplantation genetic testing for aneuploidy (PGT-A). Hum. Reprod 34, 268–275. 10.1093/humrep/dey346 [DOI] [PubMed] [Google Scholar]
- Rienzi L, Cimadomo D, Delgado A, Minasi MG, Fabozzi G, Gallego R. del, Stoppa M, Bellver J, Giancani A, Esbert M, Capalbo A, Remohì J, Greco E, Ubaldi FM, Meseguer M, 2019. Time of morulation and trophectoderm quality are predictors of a live birth after euploid blastocyst transfer: a multicenter study. Fertil. Steril 112, 1080–1093.e1. 10.1016/j.fertnstert.2019.07.1322 [DOI] [PubMed] [Google Scholar]
- Roque M, Lattes K, Serra S, Solà I, Geber S, Carreras R, Checa MA, 2013. Fresh embryo transfer versus frozen embryo transfer in in vitro fertilization cycles: A systematic review and meta-analysis. Fertil. Steril 99, 156–162. 10.1016/j.fertnstert.2012.09.003 [DOI] [PubMed] [Google Scholar]
- Sakkas D, Gardner D, 2012. Evaluation of embryo quality: Analysis of morphology and physiology, in: Gardner D, Weissman A, Howles C, Shoham Z (Ed.), Textbook of Assisted Reproductive Techniques; Boca Raton, FL, pp. 240–253. [Google Scholar]
- Sanchez T, Seidler EA, Gardner DK, Needleman D, Sakkas D, 2017. Will noninvasive methods surpass invasive for assessing gametes and embryos? Fertil. Steril 108, 730–737. 10.1016/j.fertnstert.2017.10.004 [DOI] [PubMed] [Google Scholar]
- Sanchez T, Venturas M, Aghvami SA, Yang X, Fraden S, Sakkas Denny. Needleman DJ, 2019. Combined noninvasive metabolic and spindle imaging as potential tools for embryo and oocyte assessment. Hum. Reprod 34, 2349–2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scott RT, Upham KM, Forman EJ, Zhao T, Treff NR, 2013. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: A randomized and paired clinical trial. Fertil. Steril 100, 624–630. 10.1016/j.fertnstert.2013.04.039 [DOI] [PubMed] [Google Scholar]
- Sha T, Yin X, Cheng W, Massey IY, 2018. Pregnancy-related complications and perinatal outcomes resulting from transfer of cryopreserved versus fresh embryos in vitro fertilization: a meta-analysis. Fertil. Steril 10.1016/j.fertnstert.2017.10.019 [DOI] [PubMed] [Google Scholar]
- Sunkara SK, Siozos A, Bolton VN, Khalaf Y, Braude PR, El-Toukhy T, 2010. The influence of delayed blastocyst formation on the outcome of frozen-thawed blastocyst transfer: A systematic review and meta-analysis. Hum. Reprod 25, 1906–1915. 10.1093/humrep/deq143 [DOI] [PubMed] [Google Scholar]
- Tiegs AW, Sun L, Patounakis G, Scott RT, 2019. Worth the wait Day 7 blastocysts have lower euploidy rates but similar sustained implantation rates as Day 5 and Day 6 blastocysts. Hum. Reprod 34, 1632–1639. 10.1093/humrep/dez138 [DOI] [PubMed] [Google Scholar]
- Tran D, Cooke S, Illingworth PJ, Gardner DK, 2019. Deep learning as a predictive tool for fetal heart pregnancy following time-lapse incubation and blastocyst transfer. Hum. Reprod 34, 1011–1018. 10.1093/humrep/dez042 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vaughan DA, Leung A, Resetkova N, Ruthazer R, Penzias AS, Sakkas D, Alper MM, 2017. How many oocytes are optimal to achieve multiple live births with one stimulation cycle? The one-and-done approach. Fertil. Steril 107, 397–404.e3. 10.1016/j.fertnstert.2016.10.037 [DOI] [PubMed] [Google Scholar]
- Venetis CA, Kolibianakis EM, Bosdou JK, Tarlatzis BC, 2013. Progesterone elevation and probability of pregnancy after IVF : a systematic review and meta-analysis of over 60 000 cycles 19, 433–457. 10.1093/humupd/dmt014 [DOI] [PubMed] [Google Scholar]
- Wang X, Zhen J, Sun Z, Yu Q, Deng C, Zhou Y, Wang H, He F, 2016. Effects of fifth day (D5) or sixth day (D6) frozen-thawed blastocysts on neonatal outcomes. Zygote. 10.1017/S0967199415000696 [DOI] [PubMed] [Google Scholar]
- Wong KM, van Wely M, Mol F, Repping S, Mastenbroek S, 2017. Fresh versus frozen embryo transfers in assisted reproduction. Cochrane database Syst. Rev 10.1002/14651858.CD011184.pub2 [DOI] [PMC free article] [PubMed] [Google Scholar]
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