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Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates
ORIGINAL ARTICLE: ASSISTED REPRODUCTION
Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates Mohamad Irani, M.D.,a David Reichman, M.D.,a Alex Robles, M.D.,b Alexis Melnick, M.D.,a Owen Davis, M.D.,a Nikica Zaninovic, Ph.D.,a Kangpu Xu, Ph.D.,a and Zev Rosenwaks, M.D.a a The Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine; and b Department of Obstetrics and Gynecology, Weill Cornell Medicine, New York, New York
Objective: To determine whether blastocyst grading can predict pregnancy outcomes in the frozen-thawed embryo transfer (FET) of euploid blastocysts. Design: Retrospective cohort study. Setting: Academic medical center. Patient(s): Women who underwent FET of euploid embryo(s) between January 2013 and December 2015, with blastocysts were divided into four groups based on their morphologic grading before cryopreservation: excellent (R3AA), good (3–6AB, 3–6BA, 1–2AA), average (3–6BB, 3–6AC, 3–6CA, 1–2AB, 1–2BA), and poor (1–6BC, 1–6CB, 1–6CC, 1–2BB). Intervention(s): FET. Main Outcomes Measure(s): Ongoing pregnancy rate (OPR). Result(s): A total of 417 FET cycles (477 embryos) were included. Excellent-quality embryos (n ¼ 38) yielded a statistically significantly higher OPR than poor-quality embryos (n ¼ 106) (84.2% vs. 35.8%; adjusted odds ratio 11.0; 95% confidence interval, 3.8–32.1) and average-quality embryos (n ¼ 197) (84.2% vs. 55.8%; adjusted odds ratio 4.8; 95% confidence interval, 1.7–13.3). Good-quality embryos (n ¼ 76) were associated with a statistically significantly higher OPR than poor-quality embryos (61.8% vs. 35.8%). These odds ratios were adjusted for patient's age, body mass index, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of trophectoderm biopsy (5 or 6), and total number of euploid embryos for each patient. An inner cell mass grade of A yielded a statistically significantly higher OPR than ICM grade C (76.2% vs. 13.5%) or grade B (76.2% vs. 53.6%) after controlling for all confounders. Conclusion(s): Contrary to prior published studies, the current data suggest that blastocyst morphologic grading and particularly inner cell mass grade is a useful predictor of OPR per euploid embryo. Morphologic grading should be used to help in the selection among euploid blastocysts. (Fertil SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) Key Words: Blastocyst morphologic grading, euploid embryo, inner cell mass, IVF outcome, PGS, preimplantation genetic screening Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/ 16110-fertility-and-sterility/posts/13330-23094
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dvances in clinical and laboratory techniques over the last few decades have substantially improved pregnancy rates after in vitro fertilization (IVF) but have also increased multifetal pregnancy rates. These outcomes underscore the importance of having the ability to select and transfer the single best embryo that has
the highest potential of achieving a live birth. Preimplantation genetic screening (PGS) and blastocyst morphologic grading have been used to select the best embryo(s) in a given cohort (1–6). Embryo aneuploidy is one of the main factors influencing IVF success rates (7, 8). Screening strategies to select euploid embryos have been a focus of
Received September 17, 2016; revised November 2, 2016; accepted November 14, 2016. M.I. has nothing to disclose. D.R. has nothing to disclose. A.R. has nothing to disclose. A.M. has nothing to disclose. O.D. has nothing to disclose. N.Z. has nothing to disclose. K.X. has nothing to disclose. Z.R. has nothing to disclose. M.I. and D.R. should be considered similar in author order. Reprint requests: David Reichman, M.D., 40 Worth Street Suite 401, New York, New York 10013 (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2016.11.012 VOL. - NO. - / - 2016
the field for the last two decades. Over the last several years, blastocyst biopsy with analysis of all chromosomes has supplanted day 3 analysis via fluorescence in situ hybridization. Three randomized controlled trials have shown a beneficial role of PGS using comprehensive chromosome screening technology on trophectoderm (TE) cells (2–4). In general, PGS allows for a decrease in embryo transfer number, higher implantation rates per transfer, and a lower rate of miscarriage once implantation occurs for older patients undergoing IVF. Conventional blastocyst grading systems include the following three
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The Weill Cornell Medicine institutional review board approved this study. All FET cycles in which only PGS euploid embryos were transferred at the Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine from January 2013 through December 2015 were reviewed for inclusion. Only the first FET cycle for each patient was included to avoid repeated measures bias. Cycles in which patients had transfer of two embryos of different grading or patients with a history of uterine factor infertility were excluded from the study.
mean diameter of R2 follicles attained R17 mm. The following slide scale was used to adjust the dose of hCG according to serum E2 levels on the day of trigger: E2 <1,500 pg/mL: hCG 10,000 IU; E2 1,501–2,500 pg/mL: hCG 5,000 IU; E2 2,501– 3,000 pg/mL: hCG 4,000 IU; E2 >3,000 pg/mL: hCG 3,300 IU, dual trigger (GnRH-agonist 2 mg, and hCG 1,500 IU) or GnRH agonist 4 mg. Patients underwent ultrasound-guided oocyte retrieval under conscious sedation 35 to 37 hours after the trigger. Confirmed euploid embryos were transferred in subsequent FET cycles. The embryos selected for transfer were the best morphologically graded euploid embryo available for any individual patient. Patients who underwent transfer of excellent, good, or average morphologically graded blastocysts had also poorer graded embryos for selection. Those who underwent transfer of poor morphologically graded blastocysts did not have better graded euploid embryos for selection. Patients with regular menstrual cycles typically underwent ‘‘natural FET,’’ in which they were monitored with serial serum E2 and luteinizing hormone (LH) measurements. Embryo transfer was performed 5 days after detection of the LH surge. Based on physician preference, some patients started vaginal progesterone supplementation (Endometrin, Ferring Pharmaceuticals) 1 day after embryo transfer. Alternatively, ‘‘programmed FET’’ cycles were performed in which E2 patches were serially escalated typically up to a dose of 0.4 mg and attainment of an endometrial thickness R7 mm. Then daily intramuscular progesterone supplementation was started, and the E2 dose was decreased to 0.2 mg. Progesterone and estrogen levels were measured after transfer to confirm the adequacy of the supplementation. Embryos transfers were performed 5 days after starting progesterone using Wallace catheters (Marlow/Cooper Surgical).
Clinical Protocols
Laboratory Procedures and Blastocyst Grading
Controlled ovarian hyperstimulation, human chorionic gonadotropin (hCG) and/or leuprolide acetate trigger, oocyte retrieval, embryo culture, and embryo transfer were conducted per our standard protocols (14). The majority of cycles used gonadotropin-releasing hormone (GnRH) antagonist protocols; treatment with gonadotropins (Follistim, Merck; Gonal-F, EMD-Serono; and/or Menopur, Ferring) was followed by pituitary suppression using GnRH antagonists (Ganirelix acetate, 0.25 mg, Organon; Cetrotide, 0.25 mg, EMD-Serono) when either estradiol (E2) level surpassed 300 pg/mL or the lead follicle reached 13 mm. Alternatively, patients were downregulated with a GnRH agonist (Lupron, Abbott Pharmaceuticals). Women with diminished ovarian reserve were pretreated with E2 patches or oral contraceptive pills for follicular synchronization. Gonadotropin doses were formulated according to patient's age, weight, antral follicular count, antim€ ullerian hormone, and previous response to stimulation. Serial E2 levels and transvaginal ultrasounds were performed to monitor response to stimulation; the gonadotropin dose was adjusted accordingly. Final oocyte maturation was triggered with hCG (Pregnyl, Schering-Plough; Novarel, Ferring Pharmaceuticals; Profasi, EMD-Serono) and/or GnRH-agonist (Lupron, Abbott Pharmaceuticals) when the
Embryos were cultured using the EmbryoScope (Vitrolife) time-lapse system. Blastocysts were graded immediately before TE biopsy according to the degree of expansion and ICM and TE morphology (1). The degree of expansion and hatching status were as follows: [1] the blastocoel filling <50% of the nonexpanded embryo; [2] the blastocoel filling >50% of the embryo; [3] the blastocoel filling 100 % of the full blastocyst; [4] an expanded blastocyst with a thin zona pellucida; [5] a hatching blastocyst; [6] a blasctocyst that has completely hatched out of the zona pellucida. The ICM grading was as follows: [A] tightly packed cells; [B] loosely grouped cells; [C] cells that are not identifiable. Similarly TE grading was [A] many cells creating cohesive epithelial layer; [B] uneven size cells; [C] few large cells squeezed to the side (1). The embryologists were blinded to results of PGS, as the embryos were graded before TE biopsy. For the purpose of this study, and to compare our results to the findings of the published study by Capalbo et al. (13), blastocysts were divided into four groups based on their morphologic grading assessed immediately before TE biopsy: excellent (R3AA), good (3AB, 4AB, 5AB, 6AB, 3BA, 4BA, 5BA, 6BA, 1AA, and 2AA), average (3BB, 4BB, 5BB, 6BB, 3AC, 4AC, 5AC, 6AC, 3CA, 4CA, 5CA, 6CA, 1AB, 2AB, 1BA,
morphologic parameters: degree of blastocoel expansion, inner cell mass (ICM), and TE cells. These parameters are good predictors of live-birth rate (LBR) after fresh and frozenthawed embryo transfer (FET) cycles (1, 9–11). Moreover, morphologic grading has been used to predict the ploidy status of blastocysts (12). However, this association is not perfect as 48% of top-quality blastocysts were aneuploid and 37% of poor-quality blastocysts were euploid in a representative study (12). Capalbo et al. (13) recently reported that blastocyst grading does not predict FET outcome of euploid embryos (using the same four-tier rubric presented here). However, they only included 13 poor-quality embryos. On the contrary, our anecdotal experience suggested that blastocyst grading provides additional valuable information that can be used as an adjunct to PGS to select the best embryo(s) for transfer. Thus, the current retrospective cohort study was developed to evaluate the hypothesis that blastocyst grading allows for further optimization of pregnancy rates in transfers of known euploid embryos.
MATERIALS AND METHODS Cycle Selection
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Fertility and Sterility® and 2BA), and poor (1BC, 2BC, 3BC, 4BC, 5BC, 6BC, 1CB, 2CB, 3CB, 4CB, 5CB, 6CB, 1CC, 2CC, 3CC, 4CC, 5CC, 6CC, 1BB, and 2BB). Embryos were biopsied either on day 5 or day 6, depending on when blastulation occurred. To perform the TE biopsy, the embryos were immobilized with a holding pipette, and the zona pellucida was perforated via laser pulses (ZILOS-tk Laser). Three to seven cells were aspirated using a biopsy pipette with a 20-mm internal diameter. After exposing the biopsied specimens to wash buffer, the cells were loaded into 0.2 mL polymerase chain reaction (PCR) tubes with 2 mL of lysis buffer. Specimens were analyzed at the Weill Cornell PGS laboratory using the Illumina (BlueGnome) 24SureV3 chip (array comparative genomic hybridization) (15). All embryos were vitrified within 2 hours of biopsy using the Kitazato-based method.
Outcome Variables Assessed Our primary outcome was ongoing pregnancy rate. The secondary outcomes included the implantation and spontaneous abortion rates. The ongoing pregnancy rate was defined as the proportion of transfers resulting in viable pregnancies beyond 24 weeks' gestation. Implantation rate was defined as the total number of gestational sacs seen on transvaginal ultrasound divided by the total number of transferred embryos. The spontaneous abortion (SAB) rate was defined as the proportion of clinical pregnancies (identified intrauterine gestational sac on pelvic ultrasound) that resulted in first-trimester spontaneous abortion. The biochemical pregnancy rate was defined as the proportion of transfers resulting in a transient elevation in serum b-hCG levels without sonographic evidence of a gestational sac. The patients were divided into four groups according to the morphologic grading of their transferred blastocysts. Outcomes and baseline demographic characteristics were compared among the four groups. The extracted demographic characteristics included age, body mass index (BMI), gravidity, parity, type of frozen cycle, and peak endometrial thickness.
Statistical Analysis Chi-square and Fisher's exact tests were used to compare the categorical variables. The continuous variables were
expressed as mean standard error of the mean (SEM). They were tested for normality; Student's t test was used for parametric data. Odds ratios (OR) with 95% confidence intervals (CI) were calculated and adjusted for patient's age, BMI, number of transferred embryos, type of frozen cycle (natural versus programmed), peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. P< .05 was considered statistically significant. All data analyses were performed with STATA statistical software version 14 (StataCorp LP).
RESULTS A total of 417 FET cycles in which 477 embryos were transferred were included. Cycles were divided into four groups based on the morphologic grading of transferred embryos: excellent-quality embryos (n ¼ 38), good-quality embryos (n ¼ 76), average-quality embryos (n ¼ 197), and poorquality embryos (n ¼ 106). The demographic characteristics of patients in the four groups are summarized in Table 1. There were no differences in age, BMI, gravidity, parity, type of frozen cycle, or number of transferred embryos among the four groups. The likelihood of ongoing pregnancy rate as well as SAB rate were not affected by patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, or day of TE biopsy (5 or 6). A higher number of available euploid embryos was associated with a higher ongoing pregnancy rate (P¼ .04). However, after adjusting for blastocyst grading of transferred embryos, there was no statistically significant difference between patients with different numbers of available euploid embryos (adjusted OR 1.0; 95% CI, 0.9–1.1). Excellent-quality embryos yielded a statistically significantly higher ongoing pregnancy rate than good-quality embryos (84.2% vs. 61.8%; P¼ .008; adjusted OR 4.2; 95% CI, 1.4–12.3), average-quality embryos (84.2% vs. 55.8%; P¼ .002; adjusted OR 4.8; 95% CI, 1.7–13.3), and poorquality embryos (84.2% vs. 35.8%; P< .001; adjusted OR 11.0; 95% CI, 3.8–32.1) (Fig. 1). These odds ratios were adjusted for patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day
TABLE 1 The demographic characteristics of excellent, good, average, and poor euploid blastocyst FET cycles. P value
Grade Characteristic Age (y) BMI (kg/m2) Gravidity Parity Peak endometrial thickness (mm) No. of transferred embryos % Natural FET
Excellent
Good
Average
Poor
P1
P2
P3
P4
P5
P6
36.1 0.8 22.8 0.7 1.9 0.7 0.5 0.1 9.4 0.4 1.0 0.0 71.0
35.7 0.6 23.2 0.7 1.8 0.2 0.4 0.1 9.2 0.2 1.0 0.0 64.4
36.5 0.3 23.0 0.3 2.1 0.1 0.5 0.1 9.4 0.1 1.1 0.0 68.5
36.7 0.3 23.7 0.3 1.9 0.2 0.6 0.0 9.2 0.1 1.1 0.0 60.3
.58 .85 .75 .68 .66 .82 .53
.50 .84 .16 .72 .72 .43 .84
.64 .29 .73 .19 .61 .34 .32
.13 .94 .12 .32 .19 .18 .56
.17 .33 .51 .07 .87 .14 .64
.72 .12 .14 .13 .07 .82 .16
Note: Values were expressed as mean standard error of the mean (SEM). P1 ¼ P value comparing excellent versus good groups; P2 ¼ P value comparing excellent versus average groups; P3 ¼ P value comparing excellent versus poor groups; P4 ¼ P value comparing good versus average groups; P5 ¼ P value comparing good versus poor groups; P6 ¼ P value comparing average versus poor groups; BMI ¼ body mass index; FET ¼ frozen embryo transfer. Irani. Euploid blastocyst morphology. Fertil Steril 2016.
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FIGURE 1
Ongoing pregnancy rate of different morphologic grading of euploid blastocyst frozen embryo transfer (FET) cycles. *P<.01. **P<.001. Irani. Euploid blastocyst morphology. Fertil Steril 2016.
of TE biopsy (5 or 6), and total number of euploid embryos for each patient. Good-quality embryos were associated with a statistically significantly higher ongoing pregnancy rate than poorquality embryos (61.8% vs. 35.8%; P¼ .004; adjusted OR 2.6; 95% CI, 1.3–5.0) but comparable ongoing pregnancy rate with average-quality embryos (61.8% vs. 55.8%; P¼ .6; adjusted OR 1.1; 95% CI, 0.6–2.0). Average-quality embryos were also associated with a statistically significantly higher ongoing pregnancy rate than poor-quality embryos (55.8% vs. 35.8%; P¼ .002; adjusted OR 2.2; 95% CI, 1.3–3.8) (Table 2). There was an overall increase in ongoing pregnancy rate when transferring a higher-grade blastocyst (P< .001 by chi-squared test for trend). Excellent-quality embryos (n ¼ 41) yielded a statistically significantly higher implantation rate compared with goodquality embryos (n ¼ 85) (80.4% vs. 63.5%; P¼ .02; adjusted OR 2.8; 95%CI, 1.1–7.3), average-quality embryos (n ¼ 228) (80.4% vs. 57.8%; P¼ .02; adjusted OR 2.7; 95% CI, 1.1– 6.5), and poor-quality embryos (n ¼ 123) (80.4% vs. 44.7%; P¼ .001; adjusted OR 4.6; 95% CI, 1.8–11.7). Averagequality embryos yielded a statistically significantly higher implantation rate than poor-quality embryos (57.8% vs. 44.7%; P¼ .02; adjusted OR 1.7; 95% CI, 1.0–2.7). These odds ratios were adjusted for patient's age, BMI, type of frozen cycle, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. There was no statistically significant difference in implantation rate between good-quality and average-quality embryos (63.5% vs. 57.8%; P¼ .8; adjusted OR 0.9; 95% CI, 0.5–1.6) or between good-quality and poor-quality embryos (63.5% vs. 44.7%; P¼ .1; adjusted OR 1.6; 95% CI, 0.8–3.0). There was an overall improvement in implantation rate after transferring a higher-grade blastocyst (P< .001 by chi-square test for trend). 4
An ICM grade of A yielded a statistically significantly higher ongoing pregnancy rate than ICM grade C (76.2% vs. 13.5%; P< .001; adjusted OR 10.6; 95% CI, 2.9–38.9) and grade B (76.2% vs. 53.6%; P¼ .01; adjusted OR 2.1; 95% CI, 1.1–4.2). An ICM grade B was also associated with a higher ongoing pregnancy rate than grade C (53.6% vs. 13.5%; P¼ .004; adjusted OR 4.8; 95% CI, 1.6–14.1). These odds ratio were adjusted for TE grade, blastocoel expansion grade, patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. Furthermore, TE grade A was associated with a statistically significantly higher ongoing pregnancy rate (82.3%) than grade B (56.3%; P¼ .007) and grade C (27.5%; P< .001) after adjusting for patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. Additionally, TE grade B was associated with a statistically significantly higher ongoing pregnancy rate than grade C (56.3% vs. 27.5%; P¼ .001; adjusted OR 3.1; 95% CI, 1.6–6.1) after adjusting for the same confounding factors. However, after adjusting for ICM grade and all other confounding factors, the differences in ongoing pregnancy rates between TE grades A and C (P¼ .07), between TE grades A and B (P¼ .2), and between TE grades B and C (P¼ .07) were not statistically significant. The degree of blastocoel expansion was not correlated with ongoing pregnancy rate (P¼ .09). Poor-quality euploid embryos (n ¼ 51 cycles) were associated with a statistically significantly higher rate of SAB
TABLE 2 Ongoing pregnancy rate and spontaneous abortion rate of different morphologic grading of euploid blastocyst graded immediately before trophectoderm biopsy.
Grade Blastocyst Excellent Good Average Poor ICM A B C TE A B C Expansion 2 3 4 5
Ongoing pregnancy No. of cycles Rate (%) P value
Spontaneous abortion Rate (%)
P value
38 76 197 106
84.2 61.8 55.8 35.8
vs. Excellent .008 .002 < .001
0 2.7 11.3 18.9
.001 .009 .01 vs. Poor
80 300 37
76.2 53.6 13.5
vs. A .01 < .001
1.6 11.5 58.3
< .001 < .001 vs. C
34 325 58
82.3 56.3 27.5
vs. A .2 .07
0 12.4 15.7
.06 .3 vs. C
54 111 249 3
44.4 52.2 57.8 33.3
vs. 2 .6 .7 .4
20 13.4 8.8 0
vs. 2 .4 .3 1
Note: P values are adjusted for patient's age, body mass index, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of trophectoderm biopsy (5 or 6), and total number of euploid embryos for each patient. The P value of ICM, TE, and expansion groups were also adjusted for the other morphologic grading characteristics (ICM, TE, and expansion). ICM ¼ inner cell mass; TE ¼ trophectoderm. Irani. Euploid blastocyst morphology. Fertil Steril 2016.
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FIGURE 2
Spontaneous abortion rate of different morphologic grading of euploid blastocyst frozen embryo transfer (FET) cycles. *P<.05. **P<.01. Irani. Euploid blastocyst morphology. Fertil Steril 2016.
(25.4%) than average-quality embryos (n ¼ 123 cycles) (10.5%; P¼ .01; adjusted OR 2.9; 95% CI, 1.2–7.4), goodquality embryos (n ¼ 50 cycles) (6%; P¼ .009; adjusted OR 6.4; 95% CI, 1.5–26.0), and excellent-quality embryos (n ¼ 32 cycles) (0; P¼ .001) (Fig. 2). These odds ratios were adjusted for patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. The degree of blastocoel expansion and TE grade were not correlated with SAB rate (P¼ .07; P¼ .1, respectively). An ICM grade C yielded a statistically significantly higher SAB rate than an ICM grade A (58.3% vs. 1.6%; P< .001; adjusted OR 304.8; 95% CI, 14.4–6455.4) or grade B (58.3% vs. 11.5%; P< .001; adjusted OR 23.4; 95% CI, 4.2–130.8). An ICM grade B yielded a statistically significantly higher SAB rate than an ICM grade A (11.5% vs. 1.6%; P¼ .02; adjusted OR 12.9; 95% CI, 1.3–123.8). These odds ratios were adjusted for patient's age, BMI, number of transferred embryos, type of frozen cycle, TE grade, blastocoel expansion grade, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient.
DISCUSSION The present study examined the role of blastocyst morphologic grading in selecting the best euploid embryo(s). Our data show that euploid embryos graded as excellent are associated with statistically significantly higher implantation and ongoing pregnancy rates than euploid embryos graded as good, average, or poor. Furthermore, embryos graded as poor are associated with a statistically significantly higher SAB rate than embryos graded as average, good, or excellent. Among the three criteria used to grade embryos, ICM grade conveyed the greatest impact towards ongoing pregnancy and SAB. The initial methods to screen embryos for aneuploidy was performed using fluorescence in situ hybridization on biopsy of cleavage-stage embryos (16–18). Although subsequent VOL. - NO. - / - 2016
studies revealed the pitfalls of this practice, more recent randomized controlled trials investigating PGS via comprehensive chromosome screening technology on TE cells demonstrated benefit (2–4, 16–18). Yang et al. (4) recruited patients with good prognosis (no prior miscarriage and age <35 years) who were attempting their first IVF cycle. Patients who underwent single blastocyst transfer on day 6 after PGS had a statistically significantly higher clinical pregnancy rate and ongoing pregnancy rate compared with patients whose embryos were selected for transfer solely based on their morphology (4). Forman et al. (3) included infertile patients younger than 43 years old with antim€ ullerian hormone concentration of R1.2 ng/mL and day 3 follicle-stimulating hormone concentration of <12 IU/L. Patients who had transfer of a single euploid blastocyst had similar ongoing pregnancy rates (R24 weeks) but statistically significantly lower rates of multiple gestation (0 versus 53.4%) compared with patients who had transfer of two untested best-quality blastocysts (3). Subsequently, Scott et al. (2) enrolled patients aged 21 to 42 years (female partner or oocyte donor) with %1 prior IVF failures. Patients in the PGS group had statistically significantly higher sustained implantation rates (R20 weeks) and delivery rates compared with patients transferring untested blastocysts (2). Capalbo et al. (13) recently reported that top-quality and lower-quality euploid blastocysts (using the same classification schema presented here) have similar pregnancy outcomes. The investigators reported a 53.8% ongoing implantation rate of poor-quality embryos, which was not statistically significantly different than the ongoing implantation rate of average (43.3%), good (59.4%), or excellent (49.1%) quality embryos (13). However, Capalbo et al. (10) only included 13 embryos of poor quality. Additionally, this was a multicenter study encompassing patients who underwent controlled ovarian stimulation, oocyte retrieval, blastocyst grading, PGS, and embryo transfer in two different centers, which could affect the consistency of their results. Moreover, blastocyst morphologic parameters have been shown to correlate with the implantation rate of embryos that are not tested with PGS (1, 10). Our data confirm that blastocyst grading is statistically significantly correlated with the developmental potential of euploid embryos. The morphology of non-PGS blastocysts has been suggested to correlate with SAB rate in some studies, but not in others (11, 19). Kovacic et al. (19) classified 1,396 embryos into categories according to their morphology and found that the worst-quality embryos had a statistically significantly higher SAB rate (83.3%) compared with top-quality embryos (12.8%). Conversely, Hill et al. (11) reviewed 694 single-blastocyst transfer cycles and showed no statistically significant correlation between blastocyst grade and miscarriage rate. Our study is the first to correlate the morphology of euploid blastocyst to miscarriage rate showing that poor blastocyst morphologic grading conveys a statistically significantly higher SAB rate. These findings demonstrate that abnormal early embryologic development reflected by poor blastocyst grading could be associated with an increased SAB rate of euploid embryos and could potentially be an 5
ORIGINAL ARTICLE: ASSISTED REPRODUCTION etiology for unexplained SAB. Abnormal gene expression in the developing embryo or inappropriate oocyte cytoplasmic maturation may explain the early embryonic developmental delay of some euploid embryos leading to this higher rate of SAB (20–22). Our data are consistent with previously reported data by Kovacic et al. (19) who stated that, among suboptimal blastocysts, those with normal ICM were associated with the highest implantation rate. Similarly, Richter et al. (23) showed that quantitative measurements of ICM, but not TE cell number or blastocoel expansion, were highly indicative of blastocyst implantation rate. In contrast, several studies have indicated that TE morphology statistically significantly predicted LBR (9, 11, 24–26). Thompson et al. (24) analyzed 3,151 fresh single-blastocyst transfer cycles and noted that TE morphology and blastocoel expansion, but not ICM morphology, were statistically significant predictors of clinical pregnancy rate and LBR. Likewise, Ahlstrom et al. (25) reviewed 1,117 fresh blastocyst cycles and found that TE was a stronger predictor of LBR than blastocoel expansion or ICM morphology. Similarly, Honnma et al. (26) examined 1,087 frozen-thawed single-blastocyst transfer cycles and reported that TE morphology was a statistically significant predictor of ongoing pregnancy rate and SAB rate after adjusting for all confounders including ICM morphology. Logistic regression analysis showed that ICM morphology and blastocyst expansion were not correlated with ongoing pregnancy rate or SAB rate (26). Hill et al. (11) also found that TE morphology was correlated with LBR in fresh autologous IVF, whereas ICM morphology was not. Additionally, Ahlstr€ om et al. (9) reviewed 1,089 single-blastocyst FET cycles and found that the prefreezing blastocoel expansion and TE grade were statistically significant predictors of LBR. It is important to note that all these mentioned studies included blastocysts that were not biopsied to evaluate their ploidy status (9, 11, 24–26). The TE grade is inversely correlated with the aneuploidy rate; blastocysts with a TE grade C have a 2.5-fold increase in the rate of aneuploidy compared with embryos with a TE grade A (12). This correlation may explain the statistically significant role of TE grade in predicting the pregnancy rate of blastocysts of unknown ploidy; poor TE grade is associated with a higher aneuploidy rate, which results in a higher rate of SAB and lower rate of ongoing pregnancy. The role of TE grade of known euploid blastocysts may not be as important in predicting cycle outcomes given that ploidy has already been determined in cases involving PGS. There are conflicting data regarding the predictive value of blastocoel expansion on cycle outcome. Some studies have shown that it is a strong predictor of LBR in frozen-thawed blastocyst transfer (9, 27). However, others showed that blastocyst expansion is not related to LBR after adjusting for confounders (25, 26). All these studies included blastocysts of unknown ploidy. It is possible that blastocoel expansion was not a good predictor of FET outcomes in the current study because euploid embryos with high ICM grade might continue expanding 6
after TE biopsy regardless of the blastocoel expansion before biopsy. This study may potentially be limited by the small sample size of euploid blastocysts with TE grade A or blastocoel expansion grades 5 and 6. A larger sample size might show that TE grade and/or blastocoel expansion are also statistically significant predictors of ongoing pregnancy rate and/ or SAB rate after controlling for all confounders including ICM grade. The emerging goal of assisted reproductive technology is to select and transfer the single best embryo, leading to a healthy singleton pregnancy. We conclude from the current study that blastocyst morphologic grade can be used as an adjunct to PGS (in those cases in which PGS is indicated) to select the best blastocyst associated with the highest probability of live birth. It is also important to note that patients who have only poor-quality euploid embryos (1–6BC; 1–6CB; 1– 6CC; 1–2BB) should still consider undergoing embryo transfer as they yield a lower but acceptable ongoing pregnancy rate (35.8%). Moreover, when selecting among poor-quality or average-quality embryos, priority should be given to those with better ICM morphology because it is a better predictor of pregnancy outcomes than TE morphology or blastocoel expansion.
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Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000;73:1155–8. Scott RT Jr, Upham KM, Forman EJ, Hong KH, Scott KL, Taylor D, et al. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil Steril 2013;100: 697–703. Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, Levy B, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril 2013;100:100–7.e1. Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet 2012;5:24. Dahdouh EM, Balayla J, Garcia-Velasco JA. Comprehensive chromosome screening improves embryo selection: a meta-analysis. Fertil Steril 2015; 104:1503–12. Chen MSW, Junyan H, Song Q. Can comprehensive chromosome screening technology improve IVF/ICSI outcomes? A meta-analysis. PLoS One 2015; 10:e0140779. Werner M, Reh A, Grifo J, Perle MA. Characteristics of chromosomal abnormalities diagnosed after spontaneous abortions in an infertile population. J Assist Reprod Genet 2012;29:817–20. Scott RT Jr, Ferry K, Su J, Tao X, Scott K, Treff NR. Comprehensive chromosome screening is highly predictive of the reproductive potential of human embryos: a prospective, blinded, nonselection study. Fertil Steril 2012;97: 870–5. €m A, Westin C, Wikland M, Hardarson T. Prediction of live birth in Ahlstro frozen-thawed single blastocyst transfer cycles by pre-freeze and postthaw morphology. Hum Reprod 2013;28:1199–209. Van den Abbeel E, Balaban B, Ziebe S, Lundin K, Cuesta MJ, Klein BM, et al. Association between blastocyst morphology and outcome of singleblastocyst transfer. Reprod Biomed Online 2013;27:353–61. Hill MJ, Richter KS, Heitmann RJ, Graham JR, Tucker MJ, DeCherney AH, et al. Trophectoderm grade predicts outcomes of single-blastocyst transfers. Fertil Steril 2013;99:1283–9.e1. VOL. - NO. - / - 2016
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Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates Mohamad Irani, M.D.,a David Reichman, M.D.,a Alex Robles, M.D.,b Alexis Melnick, M.D.,a Owen Davis, M.D.,a Nikica Zaninovic, Ph.D.,a Kangpu Xu, Ph.D.,a and Zev Rosenwaks, M.D.a a The Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine; and b Department of Obstetrics and Gynecology, Weill Cornell Medicine, New York, New York
Objective: To determine whether blastocyst grading can predict pregnancy outcomes in the frozen-thawed embryo transfer (FET) of euploid blastocysts. Design: Retrospective cohort study. Setting: Academic medical center. Patient(s): Women who underwent FET of euploid embryo(s) between January 2013 and December 2015, with blastocysts were divided into four groups based on their morphologic grading before cryopreservation: excellent (R3AA), good (3–6AB, 3–6BA, 1–2AA), average (3–6BB, 3–6AC, 3–6CA, 1–2AB, 1–2BA), and poor (1–6BC, 1–6CB, 1–6CC, 1–2BB). Intervention(s): FET. Main Outcomes Measure(s): Ongoing pregnancy rate (OPR). Result(s): A total of 417 FET cycles (477 embryos) were included. Excellent-quality embryos (n ¼ 38) yielded a statistically significantly higher OPR than poor-quality embryos (n ¼ 106) (84.2% vs. 35.8%; adjusted odds ratio 11.0; 95% confidence interval, 3.8–32.1) and average-quality embryos (n ¼ 197) (84.2% vs. 55.8%; adjusted odds ratio 4.8; 95% confidence interval, 1.7–13.3). Good-quality embryos (n ¼ 76) were associated with a statistically significantly higher OPR than poor-quality embryos (61.8% vs. 35.8%). These odds ratios were adjusted for patient's age, body mass index, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of trophectoderm biopsy (5 or 6), and total number of euploid embryos for each patient. An inner cell mass grade of A yielded a statistically significantly higher OPR than ICM grade C (76.2% vs. 13.5%) or grade B (76.2% vs. 53.6%) after controlling for all confounders. Conclusion(s): Contrary to prior published studies, the current data suggest that blastocyst morphologic grading and particularly inner cell mass grade is a useful predictor of OPR per euploid embryo. Morphologic grading should be used to help in the selection among euploid blastocysts. (Fertil SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) Key Words: Blastocyst morphologic grading, euploid embryo, inner cell mass, IVF outcome, PGS, preimplantation genetic screening Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/ 16110-fertility-and-sterility/posts/13330-23094
A
dvances in clinical and laboratory techniques over the last few decades have substantially improved pregnancy rates after in vitro fertilization (IVF) but have also increased multifetal pregnancy rates. These outcomes underscore the importance of having the ability to select and transfer the single best embryo that has
the highest potential of achieving a live birth. Preimplantation genetic screening (PGS) and blastocyst morphologic grading have been used to select the best embryo(s) in a given cohort (1–6). Embryo aneuploidy is one of the main factors influencing IVF success rates (7, 8). Screening strategies to select euploid embryos have been a focus of
Received September 17, 2016; revised November 2, 2016; accepted November 14, 2016. M.I. has nothing to disclose. D.R. has nothing to disclose. A.R. has nothing to disclose. A.M. has nothing to disclose. O.D. has nothing to disclose. N.Z. has nothing to disclose. K.X. has nothing to disclose. Z.R. has nothing to disclose. M.I. and D.R. should be considered similar in author order. Reprint requests: David Reichman, M.D., 40 Worth Street Suite 401, New York, New York 10013 (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2016.11.012 VOL. - NO. - / - 2016
the field for the last two decades. Over the last several years, blastocyst biopsy with analysis of all chromosomes has supplanted day 3 analysis via fluorescence in situ hybridization. Three randomized controlled trials have shown a beneficial role of PGS using comprehensive chromosome screening technology on trophectoderm (TE) cells (2–4). In general, PGS allows for a decrease in embryo transfer number, higher implantation rates per transfer, and a lower rate of miscarriage once implantation occurs for older patients undergoing IVF. Conventional blastocyst grading systems include the following three
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The Weill Cornell Medicine institutional review board approved this study. All FET cycles in which only PGS euploid embryos were transferred at the Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine from January 2013 through December 2015 were reviewed for inclusion. Only the first FET cycle for each patient was included to avoid repeated measures bias. Cycles in which patients had transfer of two embryos of different grading or patients with a history of uterine factor infertility were excluded from the study.
mean diameter of R2 follicles attained R17 mm. The following slide scale was used to adjust the dose of hCG according to serum E2 levels on the day of trigger: E2 <1,500 pg/mL: hCG 10,000 IU; E2 1,501–2,500 pg/mL: hCG 5,000 IU; E2 2,501– 3,000 pg/mL: hCG 4,000 IU; E2 >3,000 pg/mL: hCG 3,300 IU, dual trigger (GnRH-agonist 2 mg, and hCG 1,500 IU) or GnRH agonist 4 mg. Patients underwent ultrasound-guided oocyte retrieval under conscious sedation 35 to 37 hours after the trigger. Confirmed euploid embryos were transferred in subsequent FET cycles. The embryos selected for transfer were the best morphologically graded euploid embryo available for any individual patient. Patients who underwent transfer of excellent, good, or average morphologically graded blastocysts had also poorer graded embryos for selection. Those who underwent transfer of poor morphologically graded blastocysts did not have better graded euploid embryos for selection. Patients with regular menstrual cycles typically underwent ‘‘natural FET,’’ in which they were monitored with serial serum E2 and luteinizing hormone (LH) measurements. Embryo transfer was performed 5 days after detection of the LH surge. Based on physician preference, some patients started vaginal progesterone supplementation (Endometrin, Ferring Pharmaceuticals) 1 day after embryo transfer. Alternatively, ‘‘programmed FET’’ cycles were performed in which E2 patches were serially escalated typically up to a dose of 0.4 mg and attainment of an endometrial thickness R7 mm. Then daily intramuscular progesterone supplementation was started, and the E2 dose was decreased to 0.2 mg. Progesterone and estrogen levels were measured after transfer to confirm the adequacy of the supplementation. Embryos transfers were performed 5 days after starting progesterone using Wallace catheters (Marlow/Cooper Surgical).
Clinical Protocols
Laboratory Procedures and Blastocyst Grading
Controlled ovarian hyperstimulation, human chorionic gonadotropin (hCG) and/or leuprolide acetate trigger, oocyte retrieval, embryo culture, and embryo transfer were conducted per our standard protocols (14). The majority of cycles used gonadotropin-releasing hormone (GnRH) antagonist protocols; treatment with gonadotropins (Follistim, Merck; Gonal-F, EMD-Serono; and/or Menopur, Ferring) was followed by pituitary suppression using GnRH antagonists (Ganirelix acetate, 0.25 mg, Organon; Cetrotide, 0.25 mg, EMD-Serono) when either estradiol (E2) level surpassed 300 pg/mL or the lead follicle reached 13 mm. Alternatively, patients were downregulated with a GnRH agonist (Lupron, Abbott Pharmaceuticals). Women with diminished ovarian reserve were pretreated with E2 patches or oral contraceptive pills for follicular synchronization. Gonadotropin doses were formulated according to patient's age, weight, antral follicular count, antim€ ullerian hormone, and previous response to stimulation. Serial E2 levels and transvaginal ultrasounds were performed to monitor response to stimulation; the gonadotropin dose was adjusted accordingly. Final oocyte maturation was triggered with hCG (Pregnyl, Schering-Plough; Novarel, Ferring Pharmaceuticals; Profasi, EMD-Serono) and/or GnRH-agonist (Lupron, Abbott Pharmaceuticals) when the
Embryos were cultured using the EmbryoScope (Vitrolife) time-lapse system. Blastocysts were graded immediately before TE biopsy according to the degree of expansion and ICM and TE morphology (1). The degree of expansion and hatching status were as follows: [1] the blastocoel filling <50% of the nonexpanded embryo; [2] the blastocoel filling >50% of the embryo; [3] the blastocoel filling 100 % of the full blastocyst; [4] an expanded blastocyst with a thin zona pellucida; [5] a hatching blastocyst; [6] a blasctocyst that has completely hatched out of the zona pellucida. The ICM grading was as follows: [A] tightly packed cells; [B] loosely grouped cells; [C] cells that are not identifiable. Similarly TE grading was [A] many cells creating cohesive epithelial layer; [B] uneven size cells; [C] few large cells squeezed to the side (1). The embryologists were blinded to results of PGS, as the embryos were graded before TE biopsy. For the purpose of this study, and to compare our results to the findings of the published study by Capalbo et al. (13), blastocysts were divided into four groups based on their morphologic grading assessed immediately before TE biopsy: excellent (R3AA), good (3AB, 4AB, 5AB, 6AB, 3BA, 4BA, 5BA, 6BA, 1AA, and 2AA), average (3BB, 4BB, 5BB, 6BB, 3AC, 4AC, 5AC, 6AC, 3CA, 4CA, 5CA, 6CA, 1AB, 2AB, 1BA,
morphologic parameters: degree of blastocoel expansion, inner cell mass (ICM), and TE cells. These parameters are good predictors of live-birth rate (LBR) after fresh and frozenthawed embryo transfer (FET) cycles (1, 9–11). Moreover, morphologic grading has been used to predict the ploidy status of blastocysts (12). However, this association is not perfect as 48% of top-quality blastocysts were aneuploid and 37% of poor-quality blastocysts were euploid in a representative study (12). Capalbo et al. (13) recently reported that blastocyst grading does not predict FET outcome of euploid embryos (using the same four-tier rubric presented here). However, they only included 13 poor-quality embryos. On the contrary, our anecdotal experience suggested that blastocyst grading provides additional valuable information that can be used as an adjunct to PGS to select the best embryo(s) for transfer. Thus, the current retrospective cohort study was developed to evaluate the hypothesis that blastocyst grading allows for further optimization of pregnancy rates in transfers of known euploid embryos.
MATERIALS AND METHODS Cycle Selection
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Fertility and Sterility® and 2BA), and poor (1BC, 2BC, 3BC, 4BC, 5BC, 6BC, 1CB, 2CB, 3CB, 4CB, 5CB, 6CB, 1CC, 2CC, 3CC, 4CC, 5CC, 6CC, 1BB, and 2BB). Embryos were biopsied either on day 5 or day 6, depending on when blastulation occurred. To perform the TE biopsy, the embryos were immobilized with a holding pipette, and the zona pellucida was perforated via laser pulses (ZILOS-tk Laser). Three to seven cells were aspirated using a biopsy pipette with a 20-mm internal diameter. After exposing the biopsied specimens to wash buffer, the cells were loaded into 0.2 mL polymerase chain reaction (PCR) tubes with 2 mL of lysis buffer. Specimens were analyzed at the Weill Cornell PGS laboratory using the Illumina (BlueGnome) 24SureV3 chip (array comparative genomic hybridization) (15). All embryos were vitrified within 2 hours of biopsy using the Kitazato-based method.
Outcome Variables Assessed Our primary outcome was ongoing pregnancy rate. The secondary outcomes included the implantation and spontaneous abortion rates. The ongoing pregnancy rate was defined as the proportion of transfers resulting in viable pregnancies beyond 24 weeks' gestation. Implantation rate was defined as the total number of gestational sacs seen on transvaginal ultrasound divided by the total number of transferred embryos. The spontaneous abortion (SAB) rate was defined as the proportion of clinical pregnancies (identified intrauterine gestational sac on pelvic ultrasound) that resulted in first-trimester spontaneous abortion. The biochemical pregnancy rate was defined as the proportion of transfers resulting in a transient elevation in serum b-hCG levels without sonographic evidence of a gestational sac. The patients were divided into four groups according to the morphologic grading of their transferred blastocysts. Outcomes and baseline demographic characteristics were compared among the four groups. The extracted demographic characteristics included age, body mass index (BMI), gravidity, parity, type of frozen cycle, and peak endometrial thickness.
Statistical Analysis Chi-square and Fisher's exact tests were used to compare the categorical variables. The continuous variables were
expressed as mean standard error of the mean (SEM). They were tested for normality; Student's t test was used for parametric data. Odds ratios (OR) with 95% confidence intervals (CI) were calculated and adjusted for patient's age, BMI, number of transferred embryos, type of frozen cycle (natural versus programmed), peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. P< .05 was considered statistically significant. All data analyses were performed with STATA statistical software version 14 (StataCorp LP).
RESULTS A total of 417 FET cycles in which 477 embryos were transferred were included. Cycles were divided into four groups based on the morphologic grading of transferred embryos: excellent-quality embryos (n ¼ 38), good-quality embryos (n ¼ 76), average-quality embryos (n ¼ 197), and poorquality embryos (n ¼ 106). The demographic characteristics of patients in the four groups are summarized in Table 1. There were no differences in age, BMI, gravidity, parity, type of frozen cycle, or number of transferred embryos among the four groups. The likelihood of ongoing pregnancy rate as well as SAB rate were not affected by patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, or day of TE biopsy (5 or 6). A higher number of available euploid embryos was associated with a higher ongoing pregnancy rate (P¼ .04). However, after adjusting for blastocyst grading of transferred embryos, there was no statistically significant difference between patients with different numbers of available euploid embryos (adjusted OR 1.0; 95% CI, 0.9–1.1). Excellent-quality embryos yielded a statistically significantly higher ongoing pregnancy rate than good-quality embryos (84.2% vs. 61.8%; P¼ .008; adjusted OR 4.2; 95% CI, 1.4–12.3), average-quality embryos (84.2% vs. 55.8%; P¼ .002; adjusted OR 4.8; 95% CI, 1.7–13.3), and poorquality embryos (84.2% vs. 35.8%; P< .001; adjusted OR 11.0; 95% CI, 3.8–32.1) (Fig. 1). These odds ratios were adjusted for patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day
TABLE 1 The demographic characteristics of excellent, good, average, and poor euploid blastocyst FET cycles. P value
Grade Characteristic Age (y) BMI (kg/m2) Gravidity Parity Peak endometrial thickness (mm) No. of transferred embryos % Natural FET
Excellent
Good
Average
Poor
P1
P2
P3
P4
P5
P6
36.1 0.8 22.8 0.7 1.9 0.7 0.5 0.1 9.4 0.4 1.0 0.0 71.0
35.7 0.6 23.2 0.7 1.8 0.2 0.4 0.1 9.2 0.2 1.0 0.0 64.4
36.5 0.3 23.0 0.3 2.1 0.1 0.5 0.1 9.4 0.1 1.1 0.0 68.5
36.7 0.3 23.7 0.3 1.9 0.2 0.6 0.0 9.2 0.1 1.1 0.0 60.3
.58 .85 .75 .68 .66 .82 .53
.50 .84 .16 .72 .72 .43 .84
.64 .29 .73 .19 .61 .34 .32
.13 .94 .12 .32 .19 .18 .56
.17 .33 .51 .07 .87 .14 .64
.72 .12 .14 .13 .07 .82 .16
Note: Values were expressed as mean standard error of the mean (SEM). P1 ¼ P value comparing excellent versus good groups; P2 ¼ P value comparing excellent versus average groups; P3 ¼ P value comparing excellent versus poor groups; P4 ¼ P value comparing good versus average groups; P5 ¼ P value comparing good versus poor groups; P6 ¼ P value comparing average versus poor groups; BMI ¼ body mass index; FET ¼ frozen embryo transfer. Irani. Euploid blastocyst morphology. Fertil Steril 2016.
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FIGURE 1
Ongoing pregnancy rate of different morphologic grading of euploid blastocyst frozen embryo transfer (FET) cycles. *P<.01. **P<.001. Irani. Euploid blastocyst morphology. Fertil Steril 2016.
of TE biopsy (5 or 6), and total number of euploid embryos for each patient. Good-quality embryos were associated with a statistically significantly higher ongoing pregnancy rate than poorquality embryos (61.8% vs. 35.8%; P¼ .004; adjusted OR 2.6; 95% CI, 1.3–5.0) but comparable ongoing pregnancy rate with average-quality embryos (61.8% vs. 55.8%; P¼ .6; adjusted OR 1.1; 95% CI, 0.6–2.0). Average-quality embryos were also associated with a statistically significantly higher ongoing pregnancy rate than poor-quality embryos (55.8% vs. 35.8%; P¼ .002; adjusted OR 2.2; 95% CI, 1.3–3.8) (Table 2). There was an overall increase in ongoing pregnancy rate when transferring a higher-grade blastocyst (P< .001 by chi-squared test for trend). Excellent-quality embryos (n ¼ 41) yielded a statistically significantly higher implantation rate compared with goodquality embryos (n ¼ 85) (80.4% vs. 63.5%; P¼ .02; adjusted OR 2.8; 95%CI, 1.1–7.3), average-quality embryos (n ¼ 228) (80.4% vs. 57.8%; P¼ .02; adjusted OR 2.7; 95% CI, 1.1– 6.5), and poor-quality embryos (n ¼ 123) (80.4% vs. 44.7%; P¼ .001; adjusted OR 4.6; 95% CI, 1.8–11.7). Averagequality embryos yielded a statistically significantly higher implantation rate than poor-quality embryos (57.8% vs. 44.7%; P¼ .02; adjusted OR 1.7; 95% CI, 1.0–2.7). These odds ratios were adjusted for patient's age, BMI, type of frozen cycle, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. There was no statistically significant difference in implantation rate between good-quality and average-quality embryos (63.5% vs. 57.8%; P¼ .8; adjusted OR 0.9; 95% CI, 0.5–1.6) or between good-quality and poor-quality embryos (63.5% vs. 44.7%; P¼ .1; adjusted OR 1.6; 95% CI, 0.8–3.0). There was an overall improvement in implantation rate after transferring a higher-grade blastocyst (P< .001 by chi-square test for trend). 4
An ICM grade of A yielded a statistically significantly higher ongoing pregnancy rate than ICM grade C (76.2% vs. 13.5%; P< .001; adjusted OR 10.6; 95% CI, 2.9–38.9) and grade B (76.2% vs. 53.6%; P¼ .01; adjusted OR 2.1; 95% CI, 1.1–4.2). An ICM grade B was also associated with a higher ongoing pregnancy rate than grade C (53.6% vs. 13.5%; P¼ .004; adjusted OR 4.8; 95% CI, 1.6–14.1). These odds ratio were adjusted for TE grade, blastocoel expansion grade, patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. Furthermore, TE grade A was associated with a statistically significantly higher ongoing pregnancy rate (82.3%) than grade B (56.3%; P¼ .007) and grade C (27.5%; P< .001) after adjusting for patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. Additionally, TE grade B was associated with a statistically significantly higher ongoing pregnancy rate than grade C (56.3% vs. 27.5%; P¼ .001; adjusted OR 3.1; 95% CI, 1.6–6.1) after adjusting for the same confounding factors. However, after adjusting for ICM grade and all other confounding factors, the differences in ongoing pregnancy rates between TE grades A and C (P¼ .07), between TE grades A and B (P¼ .2), and between TE grades B and C (P¼ .07) were not statistically significant. The degree of blastocoel expansion was not correlated with ongoing pregnancy rate (P¼ .09). Poor-quality euploid embryos (n ¼ 51 cycles) were associated with a statistically significantly higher rate of SAB
TABLE 2 Ongoing pregnancy rate and spontaneous abortion rate of different morphologic grading of euploid blastocyst graded immediately before trophectoderm biopsy.
Grade Blastocyst Excellent Good Average Poor ICM A B C TE A B C Expansion 2 3 4 5
Ongoing pregnancy No. of cycles Rate (%) P value
Spontaneous abortion Rate (%)
P value
38 76 197 106
84.2 61.8 55.8 35.8
vs. Excellent .008 .002 < .001
0 2.7 11.3 18.9
.001 .009 .01 vs. Poor
80 300 37
76.2 53.6 13.5
vs. A .01 < .001
1.6 11.5 58.3
< .001 < .001 vs. C
34 325 58
82.3 56.3 27.5
vs. A .2 .07
0 12.4 15.7
.06 .3 vs. C
54 111 249 3
44.4 52.2 57.8 33.3
vs. 2 .6 .7 .4
20 13.4 8.8 0
vs. 2 .4 .3 1
Note: P values are adjusted for patient's age, body mass index, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of trophectoderm biopsy (5 or 6), and total number of euploid embryos for each patient. The P value of ICM, TE, and expansion groups were also adjusted for the other morphologic grading characteristics (ICM, TE, and expansion). ICM ¼ inner cell mass; TE ¼ trophectoderm. Irani. Euploid blastocyst morphology. Fertil Steril 2016.
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FIGURE 2
Spontaneous abortion rate of different morphologic grading of euploid blastocyst frozen embryo transfer (FET) cycles. *P<.05. **P<.01. Irani. Euploid blastocyst morphology. Fertil Steril 2016.
(25.4%) than average-quality embryos (n ¼ 123 cycles) (10.5%; P¼ .01; adjusted OR 2.9; 95% CI, 1.2–7.4), goodquality embryos (n ¼ 50 cycles) (6%; P¼ .009; adjusted OR 6.4; 95% CI, 1.5–26.0), and excellent-quality embryos (n ¼ 32 cycles) (0; P¼ .001) (Fig. 2). These odds ratios were adjusted for patient's age, BMI, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient. The degree of blastocoel expansion and TE grade were not correlated with SAB rate (P¼ .07; P¼ .1, respectively). An ICM grade C yielded a statistically significantly higher SAB rate than an ICM grade A (58.3% vs. 1.6%; P< .001; adjusted OR 304.8; 95% CI, 14.4–6455.4) or grade B (58.3% vs. 11.5%; P< .001; adjusted OR 23.4; 95% CI, 4.2–130.8). An ICM grade B yielded a statistically significantly higher SAB rate than an ICM grade A (11.5% vs. 1.6%; P¼ .02; adjusted OR 12.9; 95% CI, 1.3–123.8). These odds ratios were adjusted for patient's age, BMI, number of transferred embryos, type of frozen cycle, TE grade, blastocoel expansion grade, peak endometrial thickness, day of TE biopsy (5 or 6), and total number of euploid embryos for each patient.
DISCUSSION The present study examined the role of blastocyst morphologic grading in selecting the best euploid embryo(s). Our data show that euploid embryos graded as excellent are associated with statistically significantly higher implantation and ongoing pregnancy rates than euploid embryos graded as good, average, or poor. Furthermore, embryos graded as poor are associated with a statistically significantly higher SAB rate than embryos graded as average, good, or excellent. Among the three criteria used to grade embryos, ICM grade conveyed the greatest impact towards ongoing pregnancy and SAB. The initial methods to screen embryos for aneuploidy was performed using fluorescence in situ hybridization on biopsy of cleavage-stage embryos (16–18). Although subsequent VOL. - NO. - / - 2016
studies revealed the pitfalls of this practice, more recent randomized controlled trials investigating PGS via comprehensive chromosome screening technology on TE cells demonstrated benefit (2–4, 16–18). Yang et al. (4) recruited patients with good prognosis (no prior miscarriage and age <35 years) who were attempting their first IVF cycle. Patients who underwent single blastocyst transfer on day 6 after PGS had a statistically significantly higher clinical pregnancy rate and ongoing pregnancy rate compared with patients whose embryos were selected for transfer solely based on their morphology (4). Forman et al. (3) included infertile patients younger than 43 years old with antim€ ullerian hormone concentration of R1.2 ng/mL and day 3 follicle-stimulating hormone concentration of <12 IU/L. Patients who had transfer of a single euploid blastocyst had similar ongoing pregnancy rates (R24 weeks) but statistically significantly lower rates of multiple gestation (0 versus 53.4%) compared with patients who had transfer of two untested best-quality blastocysts (3). Subsequently, Scott et al. (2) enrolled patients aged 21 to 42 years (female partner or oocyte donor) with %1 prior IVF failures. Patients in the PGS group had statistically significantly higher sustained implantation rates (R20 weeks) and delivery rates compared with patients transferring untested blastocysts (2). Capalbo et al. (13) recently reported that top-quality and lower-quality euploid blastocysts (using the same classification schema presented here) have similar pregnancy outcomes. The investigators reported a 53.8% ongoing implantation rate of poor-quality embryos, which was not statistically significantly different than the ongoing implantation rate of average (43.3%), good (59.4%), or excellent (49.1%) quality embryos (13). However, Capalbo et al. (10) only included 13 embryos of poor quality. Additionally, this was a multicenter study encompassing patients who underwent controlled ovarian stimulation, oocyte retrieval, blastocyst grading, PGS, and embryo transfer in two different centers, which could affect the consistency of their results. Moreover, blastocyst morphologic parameters have been shown to correlate with the implantation rate of embryos that are not tested with PGS (1, 10). Our data confirm that blastocyst grading is statistically significantly correlated with the developmental potential of euploid embryos. The morphology of non-PGS blastocysts has been suggested to correlate with SAB rate in some studies, but not in others (11, 19). Kovacic et al. (19) classified 1,396 embryos into categories according to their morphology and found that the worst-quality embryos had a statistically significantly higher SAB rate (83.3%) compared with top-quality embryos (12.8%). Conversely, Hill et al. (11) reviewed 694 single-blastocyst transfer cycles and showed no statistically significant correlation between blastocyst grade and miscarriage rate. Our study is the first to correlate the morphology of euploid blastocyst to miscarriage rate showing that poor blastocyst morphologic grading conveys a statistically significantly higher SAB rate. These findings demonstrate that abnormal early embryologic development reflected by poor blastocyst grading could be associated with an increased SAB rate of euploid embryos and could potentially be an 5
ORIGINAL ARTICLE: ASSISTED REPRODUCTION etiology for unexplained SAB. Abnormal gene expression in the developing embryo or inappropriate oocyte cytoplasmic maturation may explain the early embryonic developmental delay of some euploid embryos leading to this higher rate of SAB (20–22). Our data are consistent with previously reported data by Kovacic et al. (19) who stated that, among suboptimal blastocysts, those with normal ICM were associated with the highest implantation rate. Similarly, Richter et al. (23) showed that quantitative measurements of ICM, but not TE cell number or blastocoel expansion, were highly indicative of blastocyst implantation rate. In contrast, several studies have indicated that TE morphology statistically significantly predicted LBR (9, 11, 24–26). Thompson et al. (24) analyzed 3,151 fresh single-blastocyst transfer cycles and noted that TE morphology and blastocoel expansion, but not ICM morphology, were statistically significant predictors of clinical pregnancy rate and LBR. Likewise, Ahlstrom et al. (25) reviewed 1,117 fresh blastocyst cycles and found that TE was a stronger predictor of LBR than blastocoel expansion or ICM morphology. Similarly, Honnma et al. (26) examined 1,087 frozen-thawed single-blastocyst transfer cycles and reported that TE morphology was a statistically significant predictor of ongoing pregnancy rate and SAB rate after adjusting for all confounders including ICM morphology. Logistic regression analysis showed that ICM morphology and blastocyst expansion were not correlated with ongoing pregnancy rate or SAB rate (26). Hill et al. (11) also found that TE morphology was correlated with LBR in fresh autologous IVF, whereas ICM morphology was not. Additionally, Ahlstr€ om et al. (9) reviewed 1,089 single-blastocyst FET cycles and found that the prefreezing blastocoel expansion and TE grade were statistically significant predictors of LBR. It is important to note that all these mentioned studies included blastocysts that were not biopsied to evaluate their ploidy status (9, 11, 24–26). The TE grade is inversely correlated with the aneuploidy rate; blastocysts with a TE grade C have a 2.5-fold increase in the rate of aneuploidy compared with embryos with a TE grade A (12). This correlation may explain the statistically significant role of TE grade in predicting the pregnancy rate of blastocysts of unknown ploidy; poor TE grade is associated with a higher aneuploidy rate, which results in a higher rate of SAB and lower rate of ongoing pregnancy. The role of TE grade of known euploid blastocysts may not be as important in predicting cycle outcomes given that ploidy has already been determined in cases involving PGS. There are conflicting data regarding the predictive value of blastocoel expansion on cycle outcome. Some studies have shown that it is a strong predictor of LBR in frozen-thawed blastocyst transfer (9, 27). However, others showed that blastocyst expansion is not related to LBR after adjusting for confounders (25, 26). All these studies included blastocysts of unknown ploidy. It is possible that blastocoel expansion was not a good predictor of FET outcomes in the current study because euploid embryos with high ICM grade might continue expanding 6
after TE biopsy regardless of the blastocoel expansion before biopsy. This study may potentially be limited by the small sample size of euploid blastocysts with TE grade A or blastocoel expansion grades 5 and 6. A larger sample size might show that TE grade and/or blastocoel expansion are also statistically significant predictors of ongoing pregnancy rate and/ or SAB rate after controlling for all confounders including ICM grade. The emerging goal of assisted reproductive technology is to select and transfer the single best embryo, leading to a healthy singleton pregnancy. We conclude from the current study that blastocyst morphologic grade can be used as an adjunct to PGS (in those cases in which PGS is indicated) to select the best blastocyst associated with the highest probability of live birth. It is also important to note that patients who have only poor-quality euploid embryos (1–6BC; 1–6CB; 1– 6CC; 1–2BB) should still consider undergoing embryo transfer as they yield a lower but acceptable ongoing pregnancy rate (35.8%). Moreover, when selecting among poor-quality or average-quality embryos, priority should be given to those with better ICM morphology because it is a better predictor of pregnancy outcomes than TE morphology or blastocoel expansion.
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