In this study, all selected cycles met the requirement of 2 cleavage embryos for fresh transfer or freezing. Additionally, there was at least 1 extended culture embryo(s) for blastocysts. Finally, a total of 7026 OPU cycles with at least 1 spare embryo were performed in blastocyst culture in addition to two top cleavages transfers or cryopreservation. In these cycles, adverse blastocyst culture results contributed to 17.62%. Specifically, 12.43% (n = 873) of all cycles showed no blastocyst (CNB), and 5.19% (n = 365) of the included cycles yielded blastocysts but were not available (NAB). In addition, 5788 cycles (CHB, 82.38%) had two cleavage embryos and at least 1 valuable blastocyst (Fig. 1).
The number of day 3 embryos was obviously related to blastocyst culture outcome. As the number of cultured embryos was elevated, the possibility of blastocyst formation failure gradually decreased. The blastocyst failure rate reached a plateau when the number of cultured embryos was more than 6. In these cycles with a large amount of spare cleavage embryos, there were about approximately 3% cycles without blastocysts (Fig. 2). To investigate possible factors of blastocyst formation, binary logistic regression analysis was operated. As Table 1 shows, crude and adjusted for confounding factors (BMI, basal reproductive endocrine, doses of gonadotropin), advanced aged women had more difficulty obtaining blastocysts (aOR = 0.97, [95% CI 0.94–0.99] P < 0.05), for every year of growth, the odds of blastocyst formation decreased by 3%. The short agonist stimulation protocol showed a higher chance of blastocyst failure (aOR = 0.18, [95% CI 0.04–0.88] P < 0.05), but the number of embryos for blastocyst culture was a positive factor (aOR = 1.71, [95% CI 1.63–1.78] P < 0.01). These were independent variables for blastocyst failure. Furthermore, basal LH instead of the ovary stimulation protocol had a positive effect on blastocyst quality (aOR = 1.05, [95% CI 1.00-1.10] P < 0.05). Female age (aOR = 0.98, [95% CI 0.92–0.99] P < 0.05) and the numbers of embryos for blastocyst culture (aOR = 1.36, [95% CI 1.29–1.44] P < 0.01) were still related to the results of whether there were available blastocysts, while both female age and numbers of extending embryos had a weaker effect on blastocyst quality than blastocyst formation (Table 2).
Table 1
Association between blastocyst formation and patient's characteristic
Characteristic
|
OR (95% CI)
|
aOR (95% CI)
|
Female age
|
0.92(0.91, 0.93)
|
0.97(0.94, 0.99)
|
Male age
|
0.95(0.93, 0.96)
|
0.99(0.96, 1.03)
|
Infertility duration
|
0.93(0.92, 1.08)
|
0.93(0.81, 1.01)
|
Infertility type(ref: primary infertility)
|
0.88(0.66, 1.18)
|
1.04(0.88, 1.25)
|
Tubal factor
|
1.01(0.96, 1.06)
|
0.94(0.78, 1.14)
|
PCOS factor
|
0.39(0.27, 0.56)
|
0.71(0.47, 1.08)
|
Male factor
|
1.19(1.02, 1.41)
|
1.02(0.86, 1.23)
|
BMI
|
0.90(0.96, 1.04)
|
0.89(0.84, 1.02)
|
Basal FSH
|
0.94(0.93, 1.02)
|
0.92(0.91, 1.05)
|
Basal E2
|
1.01(0.99, 1.02)
|
0.90(0.69, 2.14)
|
Basal LH
|
1.04(0.81, 1.32)
|
0.82(0.74, 1.10)
|
Dose of gonadotrophin
|
0.80(0.70, 1.02)
|
0.97(0.92, 1.00)
|
Stimulation protocol (ref: mild stimulation)
|
|
|
Long protocol
|
2.86(2.08, 3.95)
|
1.01(0.70, 1.45)
|
Short protocol
|
0.09(0.02, 0.36)
|
0.18(0.04, 0.88)
|
Ultra-long protocol
|
3.19(1.84, 5.56)
|
1.63(0.90, 2.97)
|
Modified ultra-long protocol
|
1.98(1.28, 3.07)
|
0.81(0.49, 1.32)
|
GnRHA protocol
|
1.84(1.31, 2.60)
|
1.06(0.73, 1.55)
|
Luteal phase
|
1.58(1.09, 2.29)
|
0.87(0.58, 1.32)
|
Insemination method (ref: IVF)
|
1.30(1.10, 1.54)
|
1.22(0.65, 1.49)
|
Number of cultured embryos
|
1.73(1.66, 1.81)
|
1.71(1.63, 1.78)
|
Note: aOR = adjusted odds ratio; CI = confidence interval; OR = odds ratio, PCOS = polycystic ovary syndrome; BMI = body mass index; E2 = estradiol; FSH = follicle stimulating hormone; GnRHA = gonadotropin releasing hormone antagonist; IVF = fertilization in vitro; ref = reference |
Binary logistic regression considered reporting patient and treatment characteristics. Final models included female and male age (years), infertility duration (years), infertility type (primary infertility yes, no), infertility factors (tubal, PCOS, male yes, no), BMI, basal endocrine (FSH, E2, LH), dose of gonadotrophin, ovary stimulation protocol (reference = mild stimulation), insemination method (IVF yes, no), and number of cultured embryos.
Table 2
Association between blastocyst quality and patient's characteristic
Characteristic
|
OR (95% CI)
|
aOR (95% CI)
|
Female age
|
0.94(0.92, 0.96)
|
0.98(0.92, 0.99)
|
Male age
|
0.96(0.94, 0.97)
|
0.97(0.97, 1.03)
|
Infertility duration
|
0.88(0.73, 1.13)
|
1.01(0.96, 1.05)
|
Infertility type(ref: primary infertility)
|
1.02(0.79, 1.18)
|
0.84(0.66, 1.08)
|
Tubal factor
|
0.69(0.44, 1.37)
|
0.88(0.68, 1.14)
|
PCOS factor
|
0.36(0.21, 0.64)
|
0.57(0.31, 1.03)
|
Male factor
|
0.93(0.74, 1.17)
|
0.80(0.63, 1.01)
|
BMI
|
0.65(0.54, 1.17)
|
0.99(0.96, 1.02)
|
Basal FSH
|
0.68(0.94, 1.01)
|
1.04(0.98, 1.09)
|
Basal E2
|
0.92(0.70, 1.32)
|
0.83(0.69, 1.21)
|
Basal LH
|
1.13(1.02, 1.25)
|
1.05(1.00, 1.10)
|
Dose of Gonadotrophin
|
0.76(0.65, 1.01)
|
0.91(0.80, 1.17)
|
Stimulation protocol (ref: mild stimulation)
|
|
|
Long protocol
|
2.53(1.58, 4.09)
|
1.29(0.72, 2.05)
|
Short protocol
|
0.63(0.27, 3.38)
|
0.31(0.02, 4.22)
|
Ultra-long protocol
|
1.35(0.70, 2.61)
|
0.93(0.46, 1.85)
|
Modified ultra-long protocol
|
2.97(1.45, 6.11)
|
1.71(0.81, 3.62)
|
GnRHA protocol
|
2.40(1.41, 4.07)
|
1.58(0.91, 2.74)
|
Luteral phase
|
2.07(1.17, 3.69)
|
1.32(0.73, 2.40)
|
Insemination method (ref: IVF)
|
0.84(0.68, 2.14)
|
1.04(0.79, 1.38)
|
Number of cultured embryos
|
1.37(1.31, 1.43)
|
1.36(1.29, 1.44)
|
Note: aOR = adjusted odds ratio; CI = confidence interval; OR = odds ratio, PCOS = polycystic ovary syndrome; BMI = body mass index; E2 = estradiol; FSH = follicle stimulating hormone; GnRHA = gonadotropin releasing hormone antagonist; IVF = fertilization in vitro; ref = reference |
Binary logistic regression considered reporting patient and treatment characteristics. Final models included female and male age (years), infertility duration (years), infertility type (primary infertility yes, no), infertility factors (tubal, PCOS, male; yes, no), BMI, basal endocrine (FSH, E2, LH), dose of gonadotrophin, ovary stimulation protocol (reference = mild stimulation), insemination method(IVF yes, no), and number of cultured embryos.
There were 2997 cycles that underwent cleavage-stage fresh embryo transfer; 453 cycles without any blastocysts, 203 cycles without available blastocysts, and 2341 cycles with at least one blastocyst. Compared with the number of cycles of frozen blastocysts, the pregnancy rate was significantly decreased in the CNB (aOR = 0.57; 95% CI 0.46–0.70; P < 0.001, reference = the NHB group) and NAB groups (aOR = 0.64; 95% CI 0.47–0.86; P < 0.001). Female age was a risk factor for clinical pregnancy (aOR = 0.94; 95% CI 0.93–0.96; P < 0.001). In the study, the female age distribution was different in the three cycles (Table 3). Patients in the CNB groups age were older than those in the other two groups. The protocols of stimulation resulted in differences in the pregnancy rate. The long protocol (aOR = 1.62; 95% CI 1.05–2.49; P < 0.05) and modified ultralong protocol (aOR = 2.77; 95% CI 1.63–4.71; P < 0.001) were beneficial for improving clinical pregnancy in fresh cleavage-stage transfer (Table 4).
Table 3
A comparison of the baseline characteristic parameters in three group of blastocyst outcome
|
Characteristic
|
CNB groupa
|
NAB groupb
|
CHB groupc
|
n
|
453
|
203
|
2341
|
|
Age (mean ± SD)
|
32.64 ± 5.52
|
31.76 ± 5.58
|
31.01 ± 4.67
|
P < 0.01
|
Infertility diagnosis
|
|
|
|
|
Primary infertility (n, %)
|
221(48.79%)
|
100(49.26%)
|
1163(49.68%)
|
NS
|
Tubal factor (n, %)
|
171(37.75%)
|
78(38.42%)
|
928(39.64%)
|
NS
|
Endometriosis (n, %)
|
92(20.31%)
|
48(23.65%)
|
597(25.50%)
|
NS
|
Uterine factor (n, %)
|
20(4.42%)
|
10(4.93%)
|
110(4.70%)
|
NS
|
Ovulation dysfunction (n, %)
|
67(14.79%)
|
27(13.30%)
|
338(14.44%)
|
NS
|
Male factor (n, %)
|
188(41.50%)
|
78(38.42%)
|
958(40.92%)
|
NS
|
Unexplained (n, %)
|
49(10.82%)
|
25(12.32%)
|
261(11.15%)
|
NS
|
Duration (y) (mean ± SD)
|
3.77 ± 3.18
|
3.40 ± 2.96
|
3.25 ± 2.55
|
P < 0.01
|
BMI (kg/m2) (mean ± SD)
|
23.64 ± 4.74
|
23.47 ± 4.29
|
22.84 ± 4.41
|
NS
|
FSH (mIU/ml) (mean ± SD)
|
6.72 ± 3.75
|
7.04 ± 3.47
|
6.74 ± 3.81
|
NS
|
E2 (pg/ml) (mean ± SD)
|
44.75 ± 22.12
|
43.18 ± 30.8
|
41.51 ± 29.59
|
NS
|
LH (mIU/ml) (mean ± SD)
|
4.31 ± 2.43
|
4.02 ± 3.11
|
3.98 ± 3.24
|
NS
|
Oocytes collected (n)
|
12.82 ± 7.26
|
12.29 ± 6.91
|
13.05 ± 7.35
|
NS
|
Embryos (mean ± SD)
|
8.47 ± 5.25
|
8.63 ± 5.82
|
9.11 ± 6.79
|
NS
|
Good embryo rate of transferred (n, %)
|
698(77.04%)
|
318(78.33%)
|
3756(80.22%)
|
NS
|
Note: BMI = body mass index; E2 = estradiol; FSH = follicle stimulating hormone; LH = luteinizing hormone |
a CNB patients included 2 day-3 embryos were frozen or transferred, spare embryos were extended culturing, but there was no blastocyst formation. |
b NAB patients included 2 day-3 embryos were frozen or transferred, spare embryos were extended culturing, but there was no available blastocyst formation. |
c CHB patients included 2 day-3 embryos were frozen or transferred, spare embryos were extended culturing, but there was at least 1 available blastocyst formation. |
Table 4
Analysis of influencing factors of clinical pregnancy in cleavage stage transfer cycles
Characteristic
|
OR (95% CI)
|
aOR (95% CI)
|
Female age
|
0.94 (0.93, 0.96)
|
0.94 (0.93, 0.96)
|
Male age
|
0.96 (0.95, 0.97)
|
1.00 (0.99, 1.01)
|
Infertility duration
|
0.95 (0.93, 0.98)
|
0.98 (0.95, 1.01)
|
Infertility type(ref: primary infertility)
|
1.04 (0.90, 1.20)
|
0.87 (0.74, 1.02)
|
Tubal factor
|
0.95 (0.82, 1.10)
|
1.15 (0.97, 1.36)
|
PCOS factor
|
0.77 (0.64, 0.92)
|
0.78 (0.65, 0.95)
|
Male factor
|
0.80 (0.42, 1.53)
|
0.93 (0.48, 1.80)
|
BMI
|
0.97 (0.94, 0.99)
|
0.98 (0.96, 1.01)
|
Basal FSH
|
1.03 (0.99, 1.07)
|
1.03 (0.99, 1.07)
|
Basal E2
|
1.00 (0.99, 1.00)
|
0.91 (0.83, 1.04)
|
Basal LH
|
1.00 (0.96, 1.04)
|
0.99 (0.95, 1.03)
|
Stimulation protocol (ref: mild stimulation)
|
Long protocol
|
2.19 (1.45, 3.32)
|
1.62 (1.05, 2.49)
|
Short protocol
|
0.90 (0.16, 5.17)
|
0.98 (0.16, 6.10)
|
Ultra-long protocol
|
2.17 (1.27, 3.71)
|
1.59 (0.92, 2.76)
|
Modified ultra-long protocol
|
3.77 (2.25, 6.30)
|
2.77 (1.63, 4.71)
|
GnRHA protocol
|
1.34 (0.84, 2.14)
|
1.16 (0.71, 1.89)
|
Insemination method (ref: IVF)
|
0.95 (0.81, 1.12)
|
0.81 (0.68, 1.26)
|
Blastcyst formation result (ref: CHBa group)
|
CNBb group
|
0.51 (0.42, 0.63)
|
0.57 (0.46, 0.70)
|
NABc group
|
0.59 (0.44, 0.78)
|
0.64 (0.47, 0.86)
|
Note: aOR = adjusted odds ratio; CI = confidence interval; OR = odds ratio, PCOS = polycystic ovary syndrome; BMI = body mass index; E2 = estradiol; FSH = follicle stimulating hormone; GnRHA = gonadotropin releasing hormone antagonist; IVF = fertilization in vitro; ref = reference |
a CNB patients included 2 day-3 embryos were frozen or transferred, spare embryos were extended culturing, but there was no blastocyst formation. |
b NAB patients included 2 day-3 embryos were frozen or transferred, spare embryos were extended culturing, but there was no available blastocyst formation. |
c CHB patients included 2 day-3 embryos were frozen or transferred, spare embryos were extended culturing, but there was at least 1 available blastocyst formation. |
Binary logistic regression considered reporting patient and treatment characteristics. Final models included female and male age (years), infertility duration (years), infertility type (primary infertility yes, no), infertility factors (tubal, PCOS, male; yes, no), BMI, basal endocrine (FSH, E2,LH), ovary stimulation protocol (reference = mild stimulation), insemination method(IVF yes,no), and blastocyst formation result (reference: CHB group).
Available blastocysts were an indicator of sibling cleavage-stage embryo viability. The implantation rate was correlated with blastocyst culture outcome (Table 5). This rate was obviously higher in the CHB group (35.25%); only 23.40% of embryos were implanted in the CNB group, and 24.88% of embryo successfully implanted in the NAB group (P < 0.001). Women without available blastocysts were more likely to have a higher pregnancy loss rate (19.10%) than women in the other two groups (9.84% in the CNB group, and 10.70% in the CHB group, P < 0.05). A similar tendency appeared in the live birth rate (CHB 47.97%, NAB 34.48%, CNB 34.88%, P < 0.001). Regardless of whether there was a blastocyst until day 7 in the remaining embryo culture, there was no impact of blastocyst culture results on preterm birth (CNB 12.03%, NAB 18.21%, CHB 18.45%, respectively, P = 0.139). The incidence of very preterm birth before 32 weeks was significantly higher in only the poor-quality blastocyst group (NAB 7.04%, CNB 1.27%, CHB 1.77%, respectively, P < 0.01). Birth defects showed a similar occurrence rate in the three groups, 1.05% in the CNB group, 1.09% in the NAB group, and 0.89% in the CHB group (P = 0.966). Those cycles of transferring two top day 3 embryos had no difference in the sex ratio of the male proportion (CNB 0.57, NAB 0.55, CHB 0.53, respectively, P = 0.669).
Table 5
Reproductive outcomes after day-3 fresh embryo transfer in different blastocyst formation groups
|
CNB groupa
|
NAB groupb
|
CHB groupc
|
P value
|
transfer cylces
|
453
|
203
|
2341
|
|
implantation rate (n, %)
|
212(23.40)
|
101(24.88)
|
1650(35.24)
|
P < 0.001
|
clinical pregnancy rate (n, %)
|
183(40.40)
|
89(43.84)
|
1337(57.11)
|
P < 0.001
|
miscarriage rate (n, %)
|
18(9.84)
|
17(19.10)
|
143(10.70)
|
P < 0.05
|
live birth rate (n, %)
|
158 (34.88)
|
70(34.48)
|
1123(47.97)
|
P < 0.001
|
preterm rate (n, %)
|
21(12.03)
|
18(18.31)
|
229(18.45)
|
NS
|
birth defect rate (n, %)
|
2(1.05)
|
1(1.09)
|
6(0.89)
|
NS
|
sex ratio of male proportion
|
0.57
|
0.55
|
0.53
|
NS
|
a CNB patients included 2 day-3 embryos were frozen or transferred, spare embryos were extended culturing, but there was no blastocyst formation. |
b NAB patients included 2 day-3 embryos were frozen or transferred, spare embryos were extended culturing, but there was no available blastocyst formation. |
c CHB patients included 2 day-3 embryos were frozen or transferred, spare embryos were extended culturing, but there was at least 1 available blastocyst formation. |