Study design and patients
This retrospective study involved women who had undergone FET during the period from January 2010 to December 2017, which was performed at the Department of Assisted Reproduction of the Ninth People’s Hospital of Shanghai Jiao Tong University School of Medicine. Women who met the following inclusion criteria were involved in the study: BMI <30 kg/m2, the transfer of blastocyst resulting in a live singleton birth.
The exclusion criteria were vanishing twin syndrome, congenital uterine malformations, and other diseases as determined by ultrasound or hysteroscopy. And exclude women with gestational diabetes, pregnancy-induced hypertension and preeclampsia, as these pregnancy-related factors may have bad effect on intrauterine fetal growth. Further, only the first pregnancy was retained for the women wo had more than one delivery during the study period. This study was approved by the Institutional Review Board of the hospital.
Laboratory protocols
The procedures of ovarian stimulation, oocyte retrieval and IVF/ICSI have been described in previous studies [18, 19]. In brief, IVF or ICSI was conventionally carried out based on semen parameters and previous fertilization histories. For IVF, oocytes were inseminated in human tubal fluid (HTF; Irvine Scientific) with approximately 300 000 progressively motile spermatozoa, and was supplemented with 10%(v/v) serum substitute supplement (SSS; Irvine Scientific). For ICSI, oocytes were transferred into dishes immediately after microinjection with HTF+10% SSS. The assessment of fertilization was performed 16-18 h post insemination/injection. A dish containing preequilibrated culture medium was then prepared for the transfer of zygotes. In this study, blastocysts were cultured in early cleavage medium (Irvine Scientific) before Day 3 and then in multiblast medium (Irvine Scientific) before 2013. However, a continuous single culture medium (Irvine Scientific) was introduced after January 2013.
All embryos were cultured under mineral oil and grew in the incubator at 37 ℃, under 5% O2 and 6% CO2 concentration (the balance gas was nitrogen). The assessement of embryo development was performed on Day 3, and only high-quality cleavage-stage embryos were selected for cryopreservation, which was at least six blastomeres with ≤20% fragmentation. Except for the change of culture media types, no change was made for the other laboratory conditions and IVF protocols throughout the study period.
Endometrial preparation and vitrification
Protocols of endometrial priming for FET have been previously described [20]. Briefly, a natural cycle FET was suitable for women having regular menstrual cycles with the use of hCG for triggering ovulation. Artificial cycles were offered for women with irregular cycles according to the discretion of treating physicians. The procedure of vitrification and thawing were previously described [19]. In short, Cryotop carrier system with dimethylsulfoxide–ethylene glycol–sucrose was used as cryoprotectants for embryo vitrification. Dilution solution in a sequential manner (1 mol/L to 0.5 mol/L to 0 mol/L sucrose) was used for thawing of embryos. All embryos were thawed on the day of transfer.
Maternal age
Maternal age at the birth of the child was the key explanatory variable, which was divided into the following categories: <30, 30–34, 35-39, and ≥40 years old. The age group 30–34 years old was set as the reference category in our analyses, as most of the ART births were to women in this age group [13].
Outcome measures
The main neonatal outcomes was live singleton birthweight. The definition for live singleton birth was a delivery of a singleton viable infant after the 20th gestational week. Gestational age (GA) in FET cycles was calculated from the day of embryo transfer (Day 17 for cleavagestage embryo transfer) [21]. The definition for PTB and very PTB were delivery before 37 and 32 completed gestational weeks, respectively. Z-scores were calculated according to gestational age and newborn gender on birthweight based on the national birthweight reference as previously described [22,23]. Birthweight of infant was divided into the following categories: LBW (<2500 g), very LBW (<1500 g), high birthweight (HBW) (>4500 g) and normal birthweight. The neonatal parameters included birthweight percentiles also based on the national birthweight reference [23]: SGA defined as birthweight <10th percentile, very SGA defined as birthweight <3rd percentile, large for gestational age (LGA) defined as birthweight >90th percentile and very LGA defined as birthweight >97th percentile. The information of pregnancy and neonatal outcomes were obtained from electronic medical records. The missing data was perfected by contact with midwives or treating obstetricians.
Statistical analysis
One-way analysis of variance was performed for continuous data, while Pearson’s chi-squared test or Fisher’s exact test were applied for categorical data. A post hoc Bonferroni correction was performed for multiple comparisons. The association between maternal age and neonatal outcomes was detected by multivariable logistic regression analysis, while the independent effect of maternal age on neonatal outcomes was analyzed by a multiple linear regression.
The multivariable analyses included the following confounders: maternal BMI, parity, infertility cause and duration, insemination method, the type of endometrial preparation, endometrial thickness, the year of treatment and newborn gender. In multivariable models, continuous covariates (maternal BMI, infertility duration, endometrial thickness and the year of treatment) and categorical covariates were indicated in Table 1. Maternal age with 30-34 years was taken as a reference group in multivariable analyses. For the development of IVF techniques over time [24], a sensitivity analysis was performed on treating the year of treatment as a categorical variable. All analyses were conducted with SPSS Statistics (version 21.0) and P <0.05 was considered to be statistically significant.
Table 1 Patient treatment and demographic characteristics according to maternal age.
|
<30 y
n=3586
|
30-34 y
n=5461
|
35-39 y
n=2861
|
≥40 y
n=657
|
P value
|
Age
|
27.29±1.63
|
31.93±1.40
|
36.47±1.34
|
41.31±1.53
|
<0.001a
|
Maternal BMI (kg/m2)
|
21.21±2.75
|
21.45±2.66
|
21.69±2.61
|
21.95±2.37
|
<0.001a
|
Infertility cause
|
|
|
|
|
<0.001b
|
Female
|
2508 (69.9)
|
3821 (70.0)
|
2001 (69.9)
|
449 (68.3)
|
|
Male
|
504 (14.1)
|
653 (12.0)
|
284 (9.9)
|
64 (9.7)
|
|
Mixed
|
510 (14.2)
|
828 (15.2)
|
494 (17.3)
|
127 (19.3)
|
|
Unexplained
|
64 (1.8)
|
159 (2.9)
|
82 (2.9)
|
17 (2.6)
|
|
Parity
|
|
|
|
|
<0.001b
|
0
|
3489 (97.3)
|
5159 (94.5)
|
2478 (86.6)
|
471 (71.7)
|
|
>0
|
97 (2.7)
|
302 (5.5)
|
383 (13.4)
|
186 (28.3)
|
|
Infertility duration (years)
|
2.53±1.82
|
3.29±2.42
|
4.11±3.41
|
4.70±4.55
|
<0.001a
|
FET cycle rank
|
|
|
|
|
<0.001b
|
First
|
2332 (65.0)
|
3112 (57.0)
|
1444 (50.5)
|
298 (45.4)
|
|
High order
|
1254 (35.0)
|
2349 (43.0)
|
1417 (49.5)
|
359 (54.6)
|
|
Fertilization method
|
|
|
|
|
<0.001b
|
IVF
|
2210 (61.6)
|
3425 (62.7)
|
1871 (65.4)
|
414 (63.0)
|
|
ICSI
|
990 (27.6)
|
1407 (25.8)
|
762 (26.6)
|
225 (34.2)
|
|
IVF+ICSI
|
386 (10.8)
|
629 (11.5)
|
228 (8.0)
|
18 (2.7)
|
|
Number of embryos transferred
|
|
|
|
|
<0.001b
|
1
|
555 (15.5)
|
985 (18.0)
|
569 (19.9)
|
145 (22.1)
|
|
≥2
|
3031 (84.5)
|
4476 (82.0)
|
2292 (80.1)
|
512 (77.9)
|
|
Embryo developmental stage at transfer
|
|
|
|
|
0.464b
|
Day 3
|
3010 (83.9)
|
4573 (83.7)
|
2415 (84.4)
|
565 (86.0)
|
|
Day 5/6
|
576 (16.1)
|
888 (16.3)
|
446 (15.6)
|
92 (14.0)
|
|
FET endometrial preparation
|
|
|
|
|
<0.001b
|
Natural cycle
|
737 (20.6)
|
1338 (24.5)
|
750 (26.2)
|
176 (26.8)
|
|
Artificial cycle
|
2849 (79.4)
|
4123 (75.5)
|
2111 (73.8)
|
481 (73.2)
|
|
Endometrial thickness (mm)
|
|
|
|
|
<0.001b
|
<8
|
232 (6.5)
|
399 (7.3)
|
270 (9.4)
|
83 (12.6)
|
|
8-11
|
1958 (54.6)
|
3067 (56.2)
|
1610 (56.3)
|
369 (56.2)
|
|
>11
|
1396 (38.9)
|
1995 (36.5)
|
981 (34.3)
|
205 (31.2)
|
|
Year of treatment
|
|
|
|
|
<0.001b
|
2010–2012
|
276 (7.7)
|
465 (8.5)
|
218 (7.6)
|
33 (5.0)
|
|
2013–2015
|
1693 (47.2)
|
2561 (46.9)
|
1219 (42.6)
|
250 (38.1)
|
|
2016–2017
|
1617 (45.1)
|
2435 (44.6)
|
1424 (49.8)
|
375 (56.9)
|
|
Data are mean±SD or n (%).
aOne-way ANOVA.
bPearson chi-square test.