Maternal age is associated with embryo splitting after single embryo transfer: a retrospective cohort study.

Purpose To determine whether maternal age has an impact on monozygotic twinning (MZT) rates in women undergoing single embryo transfer (SET).

This is a retrospective cohort study analyzed for the incidence of MZT of all clinical pregnancies after a single embryo transfer was carried out between 2014 and 2018. The effect of different assisted reproductive technology (ART) parameters on the incidence of MZT was evaluated.

There were a total of 8459 cycles resulting in pregnancy during the study period. Of these pregnancies, 8236 were singletons and 223 were MZT. The preterm birth rate, miscarriage rate, and cesarean section rate were higher in MZT. Birth weight and gestational age at delivery were lower and smaller. In the univariate analysis, the risk of MZT was decreased with frozen embryo transfer (ET). A nonlinear relationship was observed between maternal age and MZT. A negative relationship between maternal age and MZT was observed in the patients’ age ≥ 36 years.

Advanced maternal age was associated with a lower rate of MZT. A threshold female age of 36 years existed for lower MZT.

IVF, Monozygotic twins, Maternal age

In vitro fertilization (IVF) treatment is complicated by a high rate of multiple-gestation pregnancies [1]. Elective single embryo transfer (eSET) has been proposed as a way of obtaining singleton pregnancies, which has been advocated in many IVF centers worldwide. Although eSET is capable of reducing multiple pregnancy rates, it can still arise due to the early splitting of the embryo resulting in two identical twins [2]. Monozygotic twinning (MZT) is known to carry a significantly higher risk of perinatal morbidity and mortality than singleton and dizygotic twins [3], such as twin-to-twin transfusion syndrome, which occurs in 10–20% of MZT [4]. The occurrence of monozygotic twinning in spontaneous conceptions is estimated to be approximately 0.4–0.45%. In contrast, the incidence of MZT after ART is higher (2–12-fold) [5–8]. MZT could have a significant effect on clinical outcomes and needs particular attention. Although the risk factors and mechanisms responsible for embryo splitting are still under discussion, it has been argued that IVF procedure and patient characteristics may play an important role in the development of MZT. Previous studies have pointed out that blastocyst transfer [9], assisted hatching (AH) [10], and oocyte age [11] may affect the risk of MZT pregnancies, while other studies contradict these findings. Therefore, our aim was to determine whether maternal age has an impact on MZT rates in women undergoing single embryo transfer.

Ethical approval This study was approved by the ethics committee of the Northwest Women’s and Children’s Hospital (number 2018002). Because of the nature of the retrospective cohort study, informed consent was therefore waived. Study design and patients This is a retrospective cohort study of 8459 cycles undergoing single embryo transfer between 2014 and 2018 in the Center for Assisted Reproductive Technology of Northwest Women’s and Children’s Hospital, China. We confirm that all protocols were performed in accordance with relevant guidelines and regulations. Because the purpose of our study was to determine whether maternal age has an impact on MZT, we chose to define an MZT as the presence of a gestational sac. This allowed us to capture as many MZT as possible, as some MZT may appear spontaneous reduction or miscarriage. Pregnant women underwent transvaginal ultrasound 4 to 5 weeks after embryo transfer, and the number of gestational sacs was recorded. MZT pregnancy was defined as those in which more than one fetal heart beat was seen in the gestational sacs on ultrasonography in the cycle with a single embryo transfer. The exclusion criteria were donor oocyte cycles, history of recurrent pregnancy loss, uterine pathology, and gender discordance twins. Statistical analysis Data are presented as mean ± SD or median (range) for continuous variables and as n (%) for categoric variables. The Student t test or Kruskal-Wallis rank test was used for parametric and nonparametric data, respectively. The chi-square test or Fisher exact test for categoric variables was used for each group. Univariate analysis was used to identify factors associated with MZT. We then applied a generalized additive model to estimate the independent relationship between maternal age and MZT, with adjustment for potential confounders. The adjusted variables included infertility duration, body mass index, no. of oocyte retrieved, fertilization type, infertility type, cause of infertility, type of ET, and embryo stage. We further applied a two-piecewise linear regression model to examine the threshold effect of maternal age on MZT using a spline smoothing function. The turning point was determined with the use of trial and error, including the selection of turning points along with a predefined interval and then choosing the turning point that provided the maximum model likelihood. Crude odds ratios (OR) and adjusted ORs (aOR) with 95% confidence interval (CI) were calculated. A P value was considered significant if < 0.05. Data were analyzed with the use of the statistical packages R (The R Foundation; http://www.r-project.org; version 3.4.3) and Empower (R) (www.empowerstates.com, X&Y Solutions, Inc., Boston, MA).

A total of 8459 patients resulting in pregnancy were identified during the study period. Of these pregnancies, 223 monozygotic twin pregnancies were identified (2.64%). As expected, MZT was more likely to occur in fresh transfers and have a higher rate of preterm birth, miscarriage, and cesarean section, while birth weight and gestational age at delivery were smaller (Table 1). Table 1. | Variable | Singletons (n = 8236) | MZT (n = 223) | P value | |---|---|---|---| | Female age (year) | 30.27 ± 3.93 | 30.11 ± 3.65 | 0.556a | | Infertility duration (year) | 3.34 ± 2.45 | 3.32 ± 2.42 | 0.888 a | | BMI (kg/m2) | 22.52 ± 3.29 | 22.72 ± 3.70 | 0.368 a | | Infertility type (n, %) | 0.515 b | || | Primary infertility | 4221 (52.14%) | 118 (54.38%) | | | Secondary infertility | 3874 (47.86%) | 99 (45.62%) | | | Cause of infertility (n, %) | 0.657 b | || | Tubal | 3304 (40.85%) | 99 (45.62%) | | | Endometriosis | 89 (1.10%) | 1 (0.46%) | | | Ovulation disorder | 601 (7.43%) | 12 (5.53%) | | | Male factor | 1616 (19.98%) | 36 (16.59%) | | | Mixed factor | 1936 (23.93%) | 55 (25.35%) | | | Unexplained | 278 (3.44%) | 8 (3.69%) | | | PGT | 245 (3.03%) | 6 (2.76%) | | | Other | 20 (0.25%) | 0 (0.00%) | | | No. of oocyte retrieved (n) | 12.42 ± 6.26 | 11.99 ± 6.26 | 0.315 a | | Fertilization type (n, %) | 0.664 b | || | IVF | 6082 (73.93%) | 167 (75.23%) | | | ICSI | 2145 (26.07%) | 55 (24.77%) | | | Type of ET (n, %) | 0.015 b | || | Fresh ET | 3756 (45.60%) | 120 (53.81%) | | | Frozen ET | 4480 (54.40%) | 103 (46.19%) | | | Embryo stage (n, %) | 0.050 b | || | Cleavage | 1536 (18.79%) | 30 (13.57%) | | | Blastocyst | 6639 (81.21%) | 191 (86.43%) | | | Assisted hatching (n, %) | 397(4.82%) | 16 (7.17%) | 0.263 b | | Preterm birth rate (n, %) | 442 (5.37%) | 91 (40.81%) | < 0.001 b | | Miscarriage rate (n, %) | 1153 (14.00%) | 47 (21.08%) | 0.003 b | | Birth weight (kg) | 3.31 ± 0.51 | 2.47 ± 0.59 | < 0.001 a | | Gestational age at delivery (weeks) | 38.79 ± 1.72 | 36.90 ± 2.72 | < 0.001 a | | Cesarean section rate (n, %) | 3686 (60.92%) | 121 (68.75%) | 0.036 b | BMI, body mass index; AFC, antral follicle count; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; PGT, preimplantation genetic testing; ET, embryo transfer; MZT, monozygotic twinning Data presented as n (percent) or mean ± standard deviation aStudent’s t test bFisher’s exact test Univariate analysis was performed to evaluate each variable’s effect on MZT (Table 2). Frozen embryo transfer was negatively associated with MZT. Table 2. | Covariate | Statistics | OR (95% CI) | P value | |---|---|---|---| | Female age (year) | 30.26 ± 3.92 | 0.99 (0.96, 1.02) | 0.556 | | Infertility duration | 3.34 ± 2.44 | 1.00 (0.94, 1.05) | 0.888 | | BMI (kg/m2) | 22.52 ± 3.30 | 1.02 (0.98, 1.06) | 0.368 | | No. of oocyte retrieved | 12.41 ± 6.26 | 0.99 (0.97, 1.01) | 0.315 | | Fertilization type | ||| | IVF | 6249 (73.96%) | Reference | | | ICSI | 2200 (26.04%) | 0.93 (0.69, 1.27) | 0.664 | | Infertility type (n, %) | ||| | Primary infertility | 4339 (52.20%) | Reference | | | Secondary infertility | 3973 (47.80%) | 0.91 (0.70, 1.20) | 0.516 | | Cause of infertility (n, %) | ||| | Tubal | 3403 (40.97%) | Reference | | | Endometriosis | 90 (1.08%) | 0.37 (0.05, 2.72) | 0.332 | | Ovulation disorder | 613 (7.38%) | 0.67 (0.36, 1.22) | 0.189 | | Male | 1652 (19.89%) | 0.74 (0.51, 1.09) | 0.132 | | Mixed | 1991 (23.97%) | 0.95 (0.68, 1.32) | 0.755 | | Unexplained | 286 (3.44%) | 0.96 (0.46, 1.99) | 0.914 | | PGT | 251 (3.02%) | 0.82 (0.35, 1.88) | 0.636 | | Other | 20 (0.24%) | 0.00 (0.00, inf.) | 0.970 | | Type of ET (n, %) | ||| | Fresh ET | 3876 (45.82%) | Reference | | | Frozen ET | 4583 (54.18%) | 0.72 (0.55, 0.94) | 0.016 | | Embryo stage (n, %) | ||| | Cleavage stage | 1566 (18.65%) | Reference | | | Blastocyst stage | 6830 (81.35%) | 1.47 (1.00, 2.17) | 0.051 | MZT, monozygotic twinning; OR, odds ratio; CI, confidence interval; BMI, body mass index; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; PGT, preimplantation genetic testing; ET, embryo transfer The nonlinear relationship between maternal age and MZT was observed, and a threshold maternal age of 36 years existed for lower MZT adjusting for infertility duration, body mass index, no. of oocyte retrieved, fertilization type, infertility type, and cause of infertility (Fig. 1). The regression coefficient was 1.01 (95% CI 0.94, 1.08) for female age < 36 years while 0.73 (95% CI 0.52, 0.91) for female age ≥ 36 years after adjusted for potential covariates (Table 3). Table 3. | Crude model a | Adjusted model b | ||| |---|---|---|---|---| | OR (95%CI) | P value | aOR (95%CI) | P value | | | Maternal age | |||| | < 36 years | 1.02 (0.96, 1.07) | 0.502 | 1.01 (0.94, 1.08) | 0.349 | | ≥ 36 years | 0.74 (0.53, 0.95) | 0.043 | 0.73 (0.52, 0.91) | 0.042 | OR, odds ratio; CI, confidence interval; aOR, adjusted odds ratio; ET, embryo transfer aWe did not adjust other covariates bAdjusted for infertility duration, body mass index, no. of oocyte retrieved, fertilization type, infertility type, cause of infertility, type of ET, embryo stage, and assisted hatching

In our retrospective study examining a large cohort of SET, we report a negative relationship between maternal age and MZT in patients over 36 years. With the growing popularity of ART, the incidence of MZT after ART is known to be significantly higher than the spontaneous incidence [12]. The exact pathogenesis leading to embryo splitting is not well demonstrated, but available evidence suggests that multiple factors might be responsible, including extended embryo culture, maternal age, cause of infertility and fertilization method, ovarian stimulation, and embryo morphology [13]. Factors may influence the twinning rates among older mothers. For example, increased parity, genetic history, higher body stature, or higher body mass index (BMI) [14]. One longitudinal study found parity and BMI can interact and contribute to age-related changes in twinning rates in natural fertility [15]. The authors conclude that the increase in twinning rates is substantially more influenced by mother’s age than by the studied time period. Several studies have reported a significant influence of maternal age on the MZT rate. Some studies showed an increase in the MZT rate in young maternal age [9, 11, 16]. Studies have demonstrated that blastocyst culture is associated with an increased incidence of MZT after SET. Although embryos derived from young females were more likely to be cultured to the blastocyst stage, the MZT rate increase remained significant when controlling for such factors. It can be speculated younger and healthier oocytes have superior reproductive potential. Young women with high-quality embryos may have a better chance of surviving MZT reduction than older women with relatively poor-quality embryos. As selective single blastocyst embryo transfer has been advocated worldwide, the MZT rate should be increased. However, studies did not detect an increase in MZT rate in recent years [17], which does not provide support for this possibility. On the contrary, some studies suggested advanced maternal age may increase the MZT rate [18]. The hypothesis is that the thickness of the zona pellucida (ZP) decreases with maternal age, which may change the procedure of normal hatching of blastocyst [19–21]. In vivo study suggested that zona remains present until embryo implantation. Premature disruption of ZP can interfere with signaling mechanisms in the embryos and allows for blastomere separation and in turn divisions [22]. Thus, advanced maternal age with thinner ZP may enhance the risk of MZT. However, if this is the case, the preimplantation genetic testing (PGT) with zona manipulation should also increase the MZT rate, which is not the case. In addition, the influence of growth factors such as insulin receptors may induce changes in signaling, cytoplasmic shifting, and polarity changes in the embryo, which may increase MZT [11, 16]. On the contrary, several studies suggested that maternal age did not influence the MZT rate [23–25]. Compared with these studies, we adjusted more covariates to make the results more reliable. MZT rate is contributed to various factors. Some low-quality embryos grow for a limited time and eventually vanish before clinical detection, making the true rate of MZT underestimated. Biological MZT reduction can be expected during implantation [26]. Our present work has some strengths of note. Firstly, the main strength of this study is that it included a large sample of SET cycles with adjustment for multiple potential confounders. Secondly, we ruled out the possibility of dizygotic births resulting from natural conception during an IVF cycle. This allows for a more accurate analysis of the true MZT rate. Thirdly, to the best of our knowledge, this is the first study to provide clear evidence of a nonlinear association between maternal age and MZT. Several limitations associated with the present study warrant mention. Firstly, the gold standard for the determination of monozygosity is DNA analysis, which is currently not routinely performed for twin pregnancies [27]. However, this method is costly, and obtaining consent from the patients is not always straightforward. The fact that monozygosity was not confirmed by genetic testing may have overestimated the prevalence of MZT. Secondly, we did not include patients with two or more embryos transferred since it may be difficult to detect zygocity of implanted embryos. In conclusion, advanced maternal age could be associated with a lower rate of MZT. A threshold female age of 36 years existed for lower MZT. According to the nonlinear effects of maternal age on MZT in our study, women < 36 years should be advocated SET to avoid multiple pregnancies. As MZT is a rare event, we could not adequately evaluate chorionicity by DNA analysis. Such evaluations will require multicenter studies in the future. Compliance with ethical standards This study was approved by the ethics committee of the Northwest Women’s and Children’s Hospital (number 2018002). Because of the nature of the retrospective cohort study, informed consent was therefore waived. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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