T ﬁ ed/warmedsingleblastocysttransfercycles Blastocoeleexpansiondegreepredictslivebirthaftersingleblastocysttransferforfreshandvitri

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Blastocoele expansion degree predicts live birth after single blastocyst transfer for fresh and vitriﬁed/warmed single blastocyst transfer cycles

Q6 Qing-Yun Du, M., En-Yin Wang, Ph.D., Yan Huang, M., Xiao-Yi Guo, M., Yu-Jing Xiong, M., Yi-Ping Yu, M., Gui-Dong Yao, Ph.D., Sen-Lin Shi, Ph.D., and Ying-Pu Sun, M.D.

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Reproductive Medical Center, the First Afﬁliated Hospital of Zhengzhou University, Zhengzhou, People's Republic of China

Objective: To evaluate the independent effects of the degree of blastocoele expansion and the inner cell mass (ICM) and trophectoderm (TE) grades on predicting live birth after fresh and vitriﬁed/warmed single blastocyst transfer.

Design: Retrospective study.

Setting: Reproductive medical center.

Patient(s): Women undergoing 844 fresh and 370 vitriﬁed/warmed single blastocyst transfer cycles.

Intervention(s): None.

Main Outcome Measure(s): Live-birth rate correlated with blastocyst morphology parameters by logistic regression analysis and Spearman correlations analysis.

Result(s): The degree of blastocoele expansion was the only blastocyst morphology parameter that exhibited a significant ability to predict live birth in both fresh and vitrified/warmed single blastocyst transfer cycles by multivariate logistic regression and Spearman correlations analysis. Although the ICM grade was significantly related to live birth in fresh cycles according to the univariate model, its effect was not maintained in the multivariate logistic analysis. In vitrified/warmed cycles, neither ICM nor TE grade was correlated with live birth by logistic regression analysis.

Conclusion(s): This study is the first to confirm that the degree of blastocoele expansion is a better predictor of live birth after both fresh and vitrified/warmed single blastocyst transfer cycles than ICM or TE grade. (Fertil Steril^Ò

2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.)

Key Words: Blastocyst morphology, blastocoele expansion degree, live birth, single blastocyst transfer, ART

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T

he goals of assisted reproductive technology are to attain a high live-birth rate of healthy chil- dren and to minimize the risk of multiple pregnancies(1–4). New generations

of culture media (5, 6)have increased the rate of two pronuclei reaching the blastocyst stage (7, 8). Vitriﬁcation is a successful method of blastocyst cryopreservation (9–11). Artiﬁcial

shrinkage (12) of the blastocoele before vitrification improves the survival rates of vitrified/warmed blastocysts (13, 14). With the application of vitrification and artificial shrinkage, vitrified/warmed blastocyst transfer results in a live- birth rate that is similar to that of fresh blastocyst transfer (11, 15). Studies demonstrate that single blastocyst transfer results in clinical pregnancy and live birth at rates that are similar to those with double ET and higher than those with single cleavage-stage ET. Furthermore, for fresh and

Received June 9, 2015; revised November 24, 2015; accepted December 8, 2015.

Qing-Yun Du and En-Yin Wang should be considered similar in author order.

Supported by the National Natural Science Foundation of China (no.U1304314) and by grants from the Henan Province Education Department (no.13A320461) and the First Hospital of Zhengzhou University Youth Foundation (2014, to E.-Y.W.).

Reprint requests: Ying-Pu Sun, M.D., the First Afﬁliated Hospital of Zhengzhou University, Jianshe Dong Road, Erqi District, Zhengzhou City, Henan Province, People's Republic of China (E-mail:

[email protected]).

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vitriﬁed/warmed blastocysts, single blastocyst transfer gener- ates a reduced incidence of multiple pregnancies than double ET(4,15–17).

Several classiﬁcation and grading systems have been pro- posed to evaluate blastocysts. The blastocyst grading system by Gardner and Schoolcraft (18)is based on morphological parameters and remains largely unchallenged. However, studies continue to debate which parameter is the most important predictor of

Q2 live birth. Several studies have reported that the inner cell mass (ICM) grade is positively correlated with the clinical pregnancy rate (19, 20). However, other recent studies have demonstrated that the trophectoderm (TE) grade correlates with clinical pregnancy and live-birth rates in both fresh and vitriﬁed/warmed blastocyst cycles (21–24). In addition, evidence suggests that the degree of blastocoele expansion is a predictor of clinical outcomes after single blastocyst transfer(23, 25, 26).

Therefore, this study aimed to estimate the independent effects of ICM, TE, and degree of blastocoele expansion on the live-birth rate after fresh and vitriﬁed/warmed single blastocyst transfer cycles.

MATERIALS AND METHODS Study Design

This project was a retrospective study of single blastocyst transfer, including fresh cycles and vitrified/warmed cycles, performed from August 2009 to September 2014 at the Repro- ductive Medical Center of the First Affiliated Hospital of Zhengzhou University in China. The inclusion criteria included patients undergoing elective or nonelective single blastocyst transfer on day 5 in fresh or vitrified/warmed cycles. The main exclusion criteria included the oocyte donation cycle, fresh cycles undergoing assisted hatching, and cycles undergoing preimplantation genetic diagnosis. The First Affiliated Hospital of Zhengzhou University Ethics Commit- tee approved this retrospective study.

Ovarian Stimulation Protocol

Patients underwent pituitary suppression with SC administration of triptorelin acetate (0.1 mg Decapeptyl, Ferring; or 3.75 mg Diphereline, Ipsen). When the patient achieved the criteria for pituitary suppression, ovarian stimulation was initiated with gonadotropin (Gonal-F, Merck Serono; Pure- gon, Organon; or Fostimon, ISBA). When the lead follicle was R20 mm and more than half the follicles were R16 mm, hCG (Ovitrelle, Merck Serono) was injected to trigger oocyte maturation. Follicle aspiration guided by trans- vaginal ultrasound was conducted 36–38 hours after hCG administration.

Blastocyst Culture and Grading

Oocytes were identiﬁed and isolated from the follicular aspi- rate and rinsed in culture medium (G-MOPS, Vitrolife).

Insemination was achieved by IVF for 4 hours of incubation (27)or via the intracytoplasmic sperm injection (ICSI) tech- nique in fresh preequilibrated culture medium (G-IVF, Vitro- life). The inseminated oocytes were then placed in cleavage

medium (G1, Vitrolife), and fertilization was conﬁrmed based on the formation of two pronuclei after 16–18 hours. On the third day, cleavage-stage embryos were graded according to the following criteria(12): number and regularity of blasto- meres, percentage of fragmentation, and presence of granula- tion. On day 3, the embryos were regrouped, and selected embryos were cultured for 48 hours in a speciﬁc medium to form blastocysts (G2, Vitrolife).

On day 5, the blastocysts were evaluated by at least two local embryologists according to Gardner and School- craft's grading system(18). Brieﬂy, blastocysts were mainly evaluated based on three morphological parameters: the degree of blastocoele expansion and the grades of the ICM and TE. Expansion was categorized based on the following degrees: 1, an early blastocyst with its blastocoele less than half its size; 2, an early blastocyst with a blastocoele over half its size; 3, a full blastocyst with a blastocoele ﬁlling the space; 4, an expanded blastocyst with a blastocoele larger than its size; 5, a hatching blastocyst escaping from the zona pellucida; and 6, a hatched blastocyst that has completely escaped the zona pellucida.

ICM included the following grades: A, numerous tightly packed cells; B, a few loosely grouped cells; and C, very few cells. The TE was evaluated based on the following cat- egories: A, many cells forming a cohesive epithelium; B, several cells forming a loose epithelium; and C, very few cells. High-quality blastocysts were transplanted with guidance from ultrasound within 2 hours of grading, and the remaining blastocysts were vitriﬁed on day 5 or 6 according to the procedure described below.

Vitriﬁcation and Warming

The blastocoele was artificially shrunken using a laser pulse (OCTAX laser shot, MTG) before vitrification. Vitrification and warming protocols were conducted following traditional methods(28)according to the instructions of the Vit Kit (Ki- tazato Biopharma). Each blastocyst was transferred to equili- bration solution (7.5% [v/v] DMSO þ 7.5% [v/v] ethylene glycol) for 8–10 minutes. The blastocyst was then transferred into vitrification solution (15% [v/v] DMSO þ 15% [v/v]

ethylene glycol þ 0.5 M sucrose) for 30 seconds. Subse- quently, the blastocyst was placed on the Cryotop, and excess vitriﬁcation medium was removed by aspiration. The blastocyst was stored under liquid nitrogen.

During warming, the Cryotop was removed under liquid nitrogen, and the blastocyst was immersed in thawing solution (1.0 M sucrose) for 1 minute. The blastocyst was then transferred to diluent solution (0.5 M sucrose) at room temperature for 3 minutes. The blastocyst was washed twice in washing solution at room temperature for 10 minutes and cultured in blastocyst medium for 3 hours. A blastocyst was regarded as surviving if more than half the cells were intact and the blastocoele expanded again. Vitriﬁed/warmed blastocysts were graded as described above according to Gardner and Schoolcraft's grading system(18)2 hours after warming of blastocysts(28).

Laser-assisted hatching was subsequently conducted as described elsewhere (29, 30). Two-thirds of the zona

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pellucida thickness was thinned with laser treatment (OC- TAX laser shot, MTG). The power of the laser was set at 100%, and the pulse lasted for 0.5 ms. Irradiation was initiated at one point and continued until one-quarter of the zona pellucida was thinned. Laser-assisted hatching was applied to vitriﬁed/warmed blastocysts except for those blastocysts with blastocoele re-expansion degrees of 5 and 6. Each surviving blastocyst was transferred within 3 hours of assisted hatching.

Endometrial Preparation

For natural cycles, blastocyst transfer was performed 6 days after ovulation. For hormone-supplemented cycles, patients were administered 3 mg of E2 valerate (Progynon, Bayer) daily for 5 days starting on cycle day 3. Then the E₂valerate dose was adjusted according to endometrial thickness. The date to perform the vitriﬁed/warmed blastocyst transfer was determined according to endometrial thickness and the results of a serum hormone test. P supplementation was continued until a pregnancy test was performed. If the test was positive, P supplementation was continued for another 5 weeks.

Clinical Outcome

The primary outcome was live birth, deﬁned as the birth of a healthy newborn.

Statistical Analysis

Statistical analyses were performed using SPSS version 19.0 (IBM). Quantitative variables are presented as means andQ3

SD, and categorical variables are presented as n and %. Logis- tic regression analysis was performed to detect significant confounders that affected the live-birth rate and identify significant predictors of live birth among the blastocyst morphological parameters that were examined. Chi-square test and Spearman's rank correlation test were performed to identify potential relationships between blastocyst morphological parameters and live birth. P< .05 was considered statistically significant.

RESULTS

This study included 844 fresh and 370 vitriﬁed/warmed single blastocyst transfer cycles. The blastocysts were transferred on day 5.

TABLE 1

Patients and morphology characteristics versus live birth outcomes for fresh and vitriﬁed/warmed cycles.

Variable

Fresh cycles Vitriﬁed/warmed cycles Live birth

(n [ 321)

No live birth

(n [ 523) P value

Live birth (n [ 81)

No live birth

(n [ 289) P value

Age at transfer 28.4 3.5 29.2 4.2 .001 30.2 4.6 30.5 5.1 .55

Age at oocyte retrieval – – – 29.7 4.5 30.0 5.0 .53

Body mass index, kg/m² 22.4 3.0 22.5 3.3 .65 21.9 3.1 22.5 3.1 .17

Endometrium thickness, mm 12.5 2.6 12.0 2.6 .01 9.9 2.3 9.8 2.0 .70

Days of gonadotropin 11.8 1.8 10.9 1.7 .13 – – –

Dosage of gonadotropin, IU 1,692 602 1,751 653 .19 – – –

Aspirated oocytes 18.1 3.8 18.1 4.8 .98 17.0 6.8 17.3 7.5 .72

No. of good-quality cleavage-stage embryos3 8.6 3.8 8.4 3.8 .53 5.9 3.3 6.4 4.1 .30

Fertilization protocol, n (%) .66 .01

IVF 194 (60.4) 286 (54.7) 69 (85.2) 140 (48.4)

ICSI 127 (39.6) 237 (45.3) 12 (14.8) 149 (51.6)

Endometrial preparation protocol, n (%) .30

Programmed cycles – – – 63 (77.8) 208 (72.0)

Natural cycles – – – 18 (22.2) 81 (28.0)

Type of infertility, n (%) .10 .26

Primary 194 (60.4) 286 (54.7) 45 (55.6) 140 (48.4)

Secondary 127 (39.6) 237 (45.3) 36 (44.4) 149 (51.6)

Survival rate, % (n) – – – 96.4 (81/84) 91.5 (289/316) .12

Expansion degree, n (%) .01 .02

1 28 (8.7) 82 (15.7) 8 (9.9) 68 (23.5)

2 84 (26.2) 160 (30.6) 27 (33.3) 111 (38.4)

3 146 (45.5) 189 (36.1) 33 (40.7) 87 (30.1)

4 54 (16.8) 82 (15.7) 12 (14.8) 20 (6.9)

5 9 (2.8) 9 (1.7) 1 (1.2) 2 (0.7)

6 0 1 (0.2) 0 1 (0.3)

ICM, n (%) .04 .33

A 222 (69.2) 326 (62.3) 31 (38.3) 92 (31.8)

B 99 (30.8) 192 (36.7) 49 (60.5) 196 (67.8)

C 0 5 (1.0) 1 (1.2) 1 (0.3)

TE, n (%) .54 .97

A 178 (55.5) 271 (51.8) 20 (24.7) 68 (23.5)

B 122 (38.0) 219 (41.9) 45 (55.6) 162 (56.1)

C 21 (6.5) 33 (6.3) 16 (19.8) 59 (20.4)

Note: For categorical variables n (%) is presented, and for continuous variables mean SD is presented.

Du. Blastocoele expansion degree predicts birth. Fertil Steril 2016.

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Patient and Cycle Characteristics for Fresh and Vitriﬁed/Warmed Cycles

Table 1presents the patient and cycle characteristics according to live-birth outcome for fresh and vitriﬁed/warmed cycles.

For the fresh cycles, the live-birth rate was 38.0% (321/

844). Female age (28.4 3.5 years) was reduced for patients who experienced live birth compared with those who did not, whereas endometrium thickness (12.5 2.6 mm) was higher for patients who experienced live birth than for those

who did not. For those who achieved live birth, the key component was a blastocoele expansion degree of 2 (26.2%), 3 (45.5%), or 4 (16.8%). A larger proportion of patients who achieved live birth had ICM of grade A (69.2%) rather than grade B (30.8%).

For vitriﬁed/warmed cycles, the live-birth rate was 21.9%

(81/370). For vitrified/warmed blastocysts, the fertilization protocol in fresh cycles was a potentially significant factor that influenced live birth (P<.05). Large proportions of transferred blastocysts were of blastocoele re-expansion degrees 2 (33.3%) and 3 (40.7%), ICM grade B (60.5%), and TE grade B

FIGURE 1

print&web4C=FPO

(A) Examples of blastocyst grading in fresh cycles. (FB1)1AA blastocyst; (FB2)2AA blastocyst; (FB3)3AA blastocyst; (FB4)5AA blastocyst; (FB5)3AB blastocyst; (FB6)4AB blastocyst; (FB7)3BB blastocyst; (FB8)3BC blastocyst. (B) Examples of blastocyst grading in vitriﬁed/warmed cycles. (VB1)1BB blastocyst; (VB2)2BB blastocyst; (VB3)3BB blastocyst; (VB4)3AB blastocyst; (VB5)3AA blastocyst; (VB6)3BC blastocyst; (VB7)4BB blastocyst; (VB8)

5AB blastocyst. Bars ¼ 50 mm. ^Q4

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(55.6%). Among the three blastocyst morphology parameters, the blastocoele re-expansion degree was a signiﬁcant factor related to live birth on c²-test.

Blastocyst Morphology and Live Birth by Logistic Regression Analysis

Table 2presents the correlations between live birth and both blastocyst morphology parameters and cycle characteristics by logistic regression analysis for fresh and vitriﬁed/warmed cycles.

For fresh cycles, the live-birth rate was lower for patients over 35 years old (odds ratio [OR], 0.28; 95% confidence inter- val [CI], 0.13–0.60; P<.01) according to the univariate logistic regression analysis. The other characteristics examined, such as body mass index, number of retrieved oocytes, fertilization protocol, and number of good-quality embryos on day 3, were not significantly correlated with the live-birth rate in this sample. With regard to blastocyst morphology, the degree of blastocoele expansion and ICM grade were significant predictors of live birth in the univariate logistic regression model.

Although ICM grade B (34.0%) yielded a lower live-birth rate than ICM grade A (40.5%), the univariate logistic regression failed to detect a significant difference between ICM A and ICM B. No significant difference was found among TE grades A, B, and C. The live-birth rates for blastocoele expansion degrees 1, 2, 3, 4, and 5 were 25.5%, 34.4%, 43.6%, 39.7%, and 50.0%, respectively, and these values differed significantly (P<.05) based on the c²-tests. In the univariate logistic regression model, the live-birth rate was higher for blastocysts with a blastocoele expansion degree of 3 than for those with expansion degree 2 (OR, 1.47; 95% CI, 1.05–2.07; P¼.03) or degree 1 (OR, 2.26; 95% CI, 1.40–3.66; P¼.001). The number of blastocysts with ICM grade C, TE grade C, and blastocoele expansion degrees 5 and 6 was too small to perform any meaningful comparisons. After adjusting for the confounder (female age) in the multivariate analysis, only the blastocoele expansion degree remained significantly correlated with live birth (P<.05). The live-birth rate was higher for blastocysts with blastocoele expansion degree 3 than for those with expansion degree 2 (OR, 1.52; 95% CI, 1.08–2.15; P¼ .02) or degree 1 (OR, 2.20; 95% CI, 1.36–3.58;

P¼ .001). Compared with blastocysts with a blastocoele expansion degree of 1, the live-birth rate was higher for blastocysts with a blastocoele expansion degree of 4 (OR, 1.93;

95% CI, 1.11–3.34; P¼ .02; adjusted OR, 1.80; 95% CI, 1.03–3.13; P¼.04) or 5 (OR, 2.93; 95% CI, 1.06–8.11;

P¼ .04; adjusted OR, 3.24; 95% CI, 1.14–9.19; P¼ .03).

For the vitriﬁed/warmed cycles, although the live-birth rate for cycles with ICM grade B blastocysts (20.0%) was lower than that for cycles with ICM grade A blastocysts (25.2%), this difference was not signiﬁcant (P>.05). Blastocysts of TE grades A, B, and C resulted in live-birth rates of 22.7% (20/

88), 21.7% (45/207), and 21.3% (16/75), respectively. TE, ICM, and the degree of blastocyst re-expansion were examined by univariate logistic regression analysis, but only blastocoele re-expansion degree was a signiﬁcant predictor of live birth. In the univariate logistic regression model, age of oocyte retrieval, fertilization protocol, and the degree of blas-

tocoele re-expansion were correlated with live-birth rate. Ac- cording to the analysis, the live-birth rate was lower for patients 35 years or older when oocytes were retrieved (OR, 0.38; 95% CI, 0.17–0.86; P¼.02) and for patients who underwent ICSI (OR, 0.42; 95% CI, 0.22–0.82; P¼ .01). The live-birth rate was higher for blastocysts with a blastocoele re- expansion degree of 4 than for those with a re-expansion degree of 2 (OR, 2.67; 95% CI, 1.18–6.04; P¼.02) or 1 (OR, 5.10;

95% CI, 1.83–14.20; P¼.002) in the univariate logistic regression model. Compared with cycles with a blastocoele re- expansion degree of 1, the live-birth rate was higher for those with a blastocoele re-expansion degree of 3 (OR, 3.22; 95% CI, 1.40–7.43; P¼.006). In the multivariate logistic regression analysis, age when oocytes were retrieved, fertilization protocol, and the degree of blastocyst re-expansion remained sig- niﬁcant factors with regard to the live-birth rate in this population. After adjusting for confounders (i.e., age when oocytes were retrieved and fertilization protocol) in the logistic regression model, live-birth rate was higher for blastocysts with a blastocoele re-expansion degree of 4 than for those with a re-expansion degree of 2 (OR, 2.37; 95% CI, 1.03–

5.42; P¼.04) or with a re-expansion degree of 1 (OR, 3.97;

95% CI, 1.40–11.28; P¼.01). The other characteristics examined, such as age at transfer, endometrial thickness, endometrial preparation protocol, type of infertility, number of retrieved oocytes, and number of good-quality embryos, were not signiﬁcant factors with regard to the live-birth rate in this sample.

Live Birth by Combined Blastocyst Morphology for Fresh and Vitriﬁed/Warmed Cycles

Table 3shows the live-birth rate associated with different degrees of blastocoele expansion after adjusting for confounders for fresh and vitriﬁed/warmed cycles with the same ICM and TE grades. In this analysis, for fresh and vitri- ﬁed/warmed cycles, the composite score of ICM and TE grades for live births consisted mainly of AA, AB, BB, and BC. For blastocysts with the same ICM and TE grades, the sequence of live-birth rates is 1, 2, 3, and 4 of blastocoele expansion degree from low to high.

For fresh cycles in patients younger than 35 years old, the live-birth rate for AA increased with expansion from degree 1 (24.1%) to 2 (40.8%) and 3 (42.0%). The live-birth rate was higher for cycles with a blastocoele expansion degree of 5 (OR, 5.66; 95% CI, 1.42–22.60; P¼.01) compared with a degree of 1. The live-birth rates for BB were 27.6%, 27.0%, 46.9%, and 33.3% for expansion degrees of 1, 2, 3, and 4, respectively, and this difference was signiﬁcant (P<.05) based on the c²-test. For patients younger than 35 years old with a BB score, the live-birth rate was higher in cycles with a blastocoele expansion degree of 3 versus 1 (OR, 2.34;

95% CI, 1.03–5.28; P¼.04) or 2 (OR, 2.47; 95% CI, 1.22–

5.01; P¼.01). When the patients were younger than 35 years old and had BC stage blastocysts, the live-birth rates were 35.7%, 45.5%, and 75% for blastocoele expansion degrees of 1, 2, and 3, respectively.

For the vitriﬁed/warmed cycles, the fertilization protocol and female age of oocyte retrieval were adjusted. For patients

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TABLE 2

Logistic model for predicting live birth for fresh and vitriﬁed/warmed cycles.

Variable

Fresh cycles Vitriﬁed/warmed cycles

Live birth, % (n) OR (95% CI) P value Adjusted OR (95% CI) P value Live birth, % (n) OR (95% CI) P value Adjusted OR (95% CI) P value Age at transfer

<35 39.5 (313) 1 1 23.8 (70) 1

R35 15.4 (8) 0.28 (0.13–0.60) .001 0.27 (0.12–0.59) .001 14.5 (11) 0.54 (0.20–1.08) .08

Age at oocyte retrieval

<35 – 24.3 (74) 1

R35 – 10.8 (7) 0.38 (0.17–0.86) .02 0.40 (0.17–0.92) .03

Body mass index, kg/m²

<25 37.9 (251) 1 22.0 (67) 1

R25 38.5 (70) 1.02 (0.73–1.43) .89 21.2 (14) 0.95 (0.56–1.51) .88

Endometrium thickness, mm

<8 20.2 (2) 1 25.2 (69) 1 1

R8 38.2 (319) 2.48 (0.52–11.74) .25 12.5 (12) 0.42 (0.22–0.82) .01 0.46 (0.23–0.90) .02

Fertilization protocol

IVF 37.6 (230) 1 25.2 (69) 1 1

ICSI 39.2 (91) 1.07 (0.79–1.46) .66 12.5 (12) 0.42 (0.22–0.82) .01 0.46 (0.23–0.90) .02

Aspirated oocytes

<17 36.8 (127) 1 22.6 (40) 1

R17 38.9 (194) 1.09 (0.82–1.45) .54 21.2 (41) 0.92 (0.56–1.51) .88

No. of good-quality cleavage-stage embryos

<3 25.0 (5) 1 20.0 (12) 1

R3 38.3 (316) 1.87 (0.67–5.19) .23 22.3 (69) 1.15 (0.58–2.28) .70

Expansion degree Re-expansion degree ^Q5

1 25.5 (28) 0.65 (0.39–1.08) .09 0.69 (0.42–1.15) .16 10.5 (8) 0.48 (0.21–1.13) .09 0.54 (0.23–1.27) .16

2 34.4 (84) 1 1 19.6 (27) 1

3 43.6 (146) 1.47 (1.05–2.07)^a .03 1.52 (1.08–2.15) .02 27.5 (33) 1.56 (0.87–2.79)^d .13 1.52 (0.84–2.73) .17

4 39.7 (54) 1.25 (0.81–1.94)^b .31 1.24 (0.80–1.92) .33 37.5 (12) 2.67 (1.18–6.04)^e .02 2.37 (1.03–5.42) .04

5 50.0 (9) 1.91 (0.73–4.98)^c .19 2.24 (0.83–6.01) .11 33.3 (1) – –

ICM

A 40.5 (222) 1 25.2 (31) 1

B 34.0 (99) 0.76 (0.56–1.02) .07 20.0 (49) 0.74 (0.44–1.24) .26

C – – 50.0 (1) –

TE

A 39.7 (178) 1 22.7 (20) 1

B 35.8 (122) 0.85 (0.63–1.14) .27 21.7 (45) 0.94 (0.52–1.72) .85

C 38.9 (21) 0.97 (0.54–1.72) .92 21.3 (16) 0.92 (0.44–1.94) .83

Note: Live-birth rate (%) is stated for patients with the variable value. The total number of patients for each variable value can be calculated by dividing 100% by the live-birth rate stated (%) and then multiplying the result by n. For descriptive purposes, continuous variables have been transformed into categorical variables. Univariate logistic regression has been used, and those variables signiﬁcant in the univariate model have been adopted and analyzed in a multivariate logistic regression model.

aExpansion degree of 3 versus 1, OR (95% CI), 2.26 (1.40–3.66), P¼ .001; adjusted OR (95% CI), 2.20 (1.36–3.58), P¼ .001.

bExpansion degree of 4 versus 1, OR (95% CI), 1.93 (1.11–3.34), P¼ .02; adjusted OR (95% CI), 1.80 (1.03–3.13), P¼ .04.

cExpansion degree of 5 versus 1, OR (95% CI), 2.93 (1.06–8.11), P¼ .04; adjusted OR (95% CI), 2.93 (1.06–8.11), P¼ .04.

dExpansion degree of 3 versus 1, OR (95% CI), 3.22 (1.40–7.43), P¼ .01; adjusted OR (95% CI), 2.82 (1.21–6.57), P¼ .02.

eExpansion degree of 4 versus 1, OR (95% CI), 5.10 (1.83–14.20), P¼ .002; adjusted OR (95% CI), 3.97 (1.40–11.28), P¼ .01.

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younger than 35 years old with AA grade blastocysts that were fertilized by ICSI, the live-birth rates were 0%, 25.0%, 28.5%, and 50.0% for blastocoele re-expansion degrees of 1, 2, 3, and 4, respectively. For patients younger than 35 years old with BB grade blastocysts that were fertilized by IVF, the live-birth rates based on the degree of blastocoele re- expansion were 15.8% (degree 1), 14.6% (degree 2), 37.8%

(degree 3), and 50.0% (degree 2). For BB grade blastocysts in females who were younger than 35 years old and fertilized by IVF, the live-birth rate increased from 15.8% for those with blastocoele re-expansion degrees of 1 to 37.8% for those with blastocoele re-expansion degrees of 3 (OR, 4.31; 95% CI, 1.12–16.63; P¼.03). When the patients were younger than 35 years old and fertilized by IVF, yielding blastocysts with BC grade, the live-birth rates with respect to the degree of blastocoele re-expansion were 23.1% and 38.5% for degrees of 1 and 2, respectively. The live-birth rates for AA, AB, BB, and BC blastocysts were 20.5%, 35.9%, 18.0%, and 21.9%, respectively, and these differences were not signiﬁcant based on c²-tests.

Spearman Correlations between Blastocyst Morphology and Live Birth

Supplemental Table 1demonstrates the correlation between blastocyst morphology parameters and live birth for fresh and vitriﬁed/warmed cycles by rank sum test and Spearman

correlation test. In this analysis, for fresh cycles, both the degree of blastocoele expansion (r ¼ 0.106, P¼ .002) and the ICM (r ¼ 0.072, P¼.036) were correlated with live birth. For vitriﬁed/warmed cycles, only the degree of blastocoele re- expansion was correlated with live birth (r ¼ 0.183, P¼.000).

DISCUSSION

Our study found that the degree of blastocoele expansion was the only blastocyst morphology parameter that was signiﬁ- cantly related to the live-birth rate for fresh single blastocyst transfer cycles and that this re-expansion degree was the most important morphological predictor of live birth for vitriﬁed/

warmed single blastocyst transfer. For fresh cycles, only the degree of blastocoele expansion was significantly associated with the live-birth rate after adjusting for confounders for fresh single blastocyst transfer cycles. For fresh cycles, the ICM grade was significantly concerned with the live-birth rate in the univariate model but not in the multivariate logistic analysis. For vitrified/warmed cycles, the degree of blastocoele re-expansion, but not TE or ICM grade, was significantly correlated with the live-birth rate.

The importance of blastocoele expansion for clinical outcomes has been previously documented. A study by Goto et al.

(19)examined 1,488 single frozen blastocyst transfer cycles and found that blastocyst grade, which is mainly deﬁned by the grade of expansion, signiﬁcantly affected pregnancy TABLE 3

Live-birth rate by combined blastocyst morphology for fresh and vitriﬁed/warmed cycles.

Variable Age

Expansion degree

Fresh cycles Vitriﬁed/warmed cycles

Live birth, % (n) OR (95% CI) P value Live birth of IVF, % (n)

Live birth of ICSI, % (n)

AA <35 1 24.1 (7/29) 1 0/5 0/3

2 40.8 (29/71) 2.17 (0.82–5.74) .12 18.2 (2/11) 25.0 (1/4)

3 42.0 (79/188) 2.28 (0.93–5.59) .07 17.4 (4/23) 28.6 (2/7)

4 37.7 (40/106) 1.91 (0.75–4.86) .18 40.0 (6/15) 50.0 (1/2)

5 64.3 (9/14) 5.66 (1.42–22.60) .01 — —

AA R35 1 0/3 0/6 0/3

2 0/1 0/3 —

3 35.3 (6/17) 20.0 (1/5) —

4 33.3 (1/3) 0/1 0/1

5 0/3 — —

AB <35 2 51.9 (14/27) 1 75.0 (3/4) 33.3 (1/3)

3 43.8 (28/64) 0.72 (0.29–1.78) .48 28.6 (4/14) 37.5 (3/8)

4 53.3 (8/15) 1.06 (0.30–3.76) .93 25.0 (1/4) 100.0 (1/1)

R35 2 0/3

3 33.3 (1/3)

4 0/1

BB <35 1 29.1 (16/55) 1 15.8 (3/19) 0/9

2 27.9 (31/111) 0.95 (0.46–1.93) .82 14.6 (6/41) 0/18

3 48.9 (23/47) 2.34 (1.03–5.28)^a .04 37.8 (14/37)^b 14.3 (1/7)

4 33.3 (1/3) 1.22 (0.10–14.41) .88 50.0 (2/4) 0/1

5 0/1 — 0/1 —

6 0/1 — — 0/1

BC <35 1 35.7 (5/14) 1 23.1 (3/13) 12.5 (1/8)

2 45.5 (10/22) 1.50 (0.38–5.95) .56 38.5 (10/26) 0/6

3 75.0 (6/8) 5.40 (0.78–37.51) .09 0/1 50.0 (1/2)

Note: AA, AB, BA, BB, and BC are combined grades of ICM and TE.

aFresh cycles, expansion degree of 3 versus 2, OR (95% CI), 2.47 (1.22–5.01), P¼ .012.

bVitriﬁed/warmed cycles, expansion degree of 3 versus 1, OR (95% CI), 4.31 (1.12–16.63), P¼ .03.

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outcomes. Moreover, neither ICM nor TE affected the pregnancy outcomes for blastocysts of the same expansion degree. Thompson et al.(23)and Ahlstrom et al.(21)reported that the blastocyst expansion stage signiﬁcantly and inde- pendently predicted live-birth rates in a multivariate logistic model for fresh single blastocyst transfer cycles. In a study of the correlations between post-thaw morphological characteristics and live births after vitriﬁed/warmed blastocyst transfer cycles, Ahlstrom et al.(26)found that the degree of re-expansion was the best predictor of the live-birth rate.

Our results strongly support the hypothesis that the degree of blastocoele expansion is correlated with live birth in fresh single blastocyst transfer cycles and vitriﬁed/warmed single blastocyst transfer cycles.

However, certain factors remain unclear. Unfortunately, Goto et al.(19)did not evaluate the effect of each blastocyst parameter on ongoing pregnancy or delivery rates or the importance of the degree of blastocoele expansion on those dependent variables. In contrast to our study, several studies have reported that the ICM grade is important in predicting clinical outcome (20). Several recent studies also reported that the TE grade is a better predictor of clinical outcome after blastocyst transfer than the ICM grade. Ahlstrom et al.(21) and Hill et al. (22) found that the TE grade was the most important parameter when predicting the live-birth rates for fresh single blastocyst transfer cycles. Honnma et al. (31) and Chen et al.(24)reported that the TE grade, rather than the degree of expansion, is the most important factor in predicting clinical pregnancy after vitriﬁed/warmed blastocyst transfer cycles.

In our study, the degree of blastocoele re-expansion was almost the same as the degree of expansion before vitrification. Artificial shrinkage before vitrification improved the survival rate of expanded and hatched blastocysts(14) and decreased the average time for re-expansion of the surviving blastocysts compared with that of the non–artificial shrinkage group (13, 14, 32). This outcome may occur because the blastocyst with a faster re-expansion has faster developmental kinetics, and the Naþ/Kþ-ATPase (pump) is responsible for blastocoele re-expansion (33). The rate of development and the blastocoele stage are related to the suc- cess of blastocyst implantation (34, 35). A previous study found that expanded/hatched blastocysts have lower survival and live-birth rates than early blastocysts in the absence of artificial shrinkage before vitrification(32). How- ever, blastocysts with greater degrees of expansion have higher rates of survival and pregnancy with the application of artificial blastocoele shrinkage (14, 26). The degree of blastocoele expansion may be important because an increased number of cell divisions leads to a greater number of smaller cells with increased cell junction quality and a larger blastocoele. Blastocysts with a higher expansion degree have smaller cells. Owing to the greater surface- to-volume ratio of blastocysts with smaller cells, these blastocysts are more easily permeated by cryoprotectant and protected from osmotic stress and injury(36). Therefore, blastocysts with a higher expansion degree may better tolerate vitrification and cryoprotectant toxicity.

In both the fresh and vitrified/warmed cycles, the blastocoele expands owing to an influx of fluid, and fluid leakage is prevented by the tight junctions of the TE cells(37). Moreover, an expanded blastocyst is more likely to have cells with better contours (35). An increased degree of blastocoele re- expansion is related to a lower percentage of cell loss (26).

A strength of this study is that we evaluated both fresh and vitriﬁed/warmed single blastocyst transfer cycles during the same time period and found that the blastocoele expansion degree plays the most important role in predicting live birth.

Furthermore, to accurately represent the clinical situa- tion in China, at least three cleavage embryos of good quality were selected to form blastocysts, whereas other embryos were vitrified on day 3 when the patients were at risk of mild ovarian hyperstimulation syndrome (38) or multiple pregnancies. However, for those patients with fewer than three good-quality embryos, the whole embryos were cultured to the blastocyst stage on day 3. Moreover, our multivariate logistic regression analysis revealed that female age affected the live birth for fresh blastocyst transfer cycles, which is consistent with previous studies(21, 23). Regarding the vitrified/warmed cycles in our study, a significant correlation between age of oocyte retrieval and live birth was noted, similar to previous studies(39). The fertilization protocol during fresh cycles for vitrified/warmed blastocysts was related to the live-birth rate in vitrified/

warmed single blastocyst transfer cycles, contrary to the ﬁndings of Ahlstrom et al.(26).

For ethical reasons, we chose the best embryo for implantation based on Gardner and Schoolcraft's system and current conventions. This process introduces a bias of the data because most blastocysts were of ICM grade A/B and TE grade A/B. In our laboratory, ICM is preferred over TE and expansion degree in the selection of suitable blastocysts for transfer.

However, this study reveals that the degree of blastocoele expansion is a more important predictor of the live-birth rate than the ICM and TE grades. As shown inTable 3, the live-birth rates increased with the degree of blastocoele expansion or re-expansion for blastocysts with the same ICM and TE grades. This finding illustrates the overpowering effect of the degree of blastocoele expansion and suggests that adjustments to our blastocyst selection criteria may improve the live-birth rates. According to our results (Table 2), with ICM grade A/B and TE grade A/B, the live- birth rate in blastocysts with a blastocoele expansion degree of 3 in fresh cycles was significantly higher than in blastocysts with blastocoele expansion degrees of 1 and 2. More- over, with ICM grade A/B and TE grade A/B, the live-birth rate in blastocysts with a blastocoele re-expansion degree of 4 in vitrified/warmed cycles was significantly higher than in blastocysts with blastocoele re-expansion degrees of 1 and 2.

Our study was retrospective, so it is unknown whether the prospective application of the ﬁndings (if even feasible, since in many cases patients may not have multiple high-quality embryos available) would lead to better outcomes for the patients. In addition, the small sample size of our study limits the validity of our results.

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