Double trouble? Impact of frozen embryo transfer on the monozygotic twinning rate: a retrospective cohort study from 8459 cycles

Purpose To compare monozygotic twinning (MZT) rates in patients undergoing fresh embryo transfer (ET) and frozen embryo transfer.

All clinical pregnancies after single ET carried out in our IVF center between 2014 and 2018 (n = 8459) were retrospectively analyzed for the incidence of MZT. MZT rate was compared in women who underwent fresh ET (n = 3876) and frozen ET (n = 4583).

There was a total of 120 MZT identified in the fresh ET group (3.10%) and 103 MZT in the frozen ET group (2.25%), which was significant (p = 0.015). In the univariate analysis, the risk of MZT was decreased with frozen embryo transfer (OR 0.72; 95% CI, 0.55–0.94, p = 0.016) and increased with mild stimulation protocol in the fresh cycle (OR 1.90; 95% CI, 1.04–3.45, p = 0.036). Multivariable logistic regression revealed that frozen embryo transfer was associated with a significant decrease risk of MZT (adjusted OR 0.66; 95% CI, 0.46–0.90, p = 0.011).

Frozen ET is associated with a lower risk of MZT.

Fresh embryo transfer, Frozen embryo transfer, Monozygotic twinning, In vitro fertilization

Monozygotic twinning (MZT) occurs when a single zygote divides early in embryonic development. MZT is classified according to their chorionicity and amniocity. If the embryo splitting occurs between day 0 and day 4 after fertilization, the resulting twins are dichorionic-diamniotic. If it occurs between day 4 and day 8, the resulting twins are monochorionic-diamniotic. If it occurs after day 8, the twins will be monochorionic-monoamniotic [1]. MZT occurs rarely in spontaneous conception, with an estimated incidence of 0.4% [2, 3]. Interestingly, the use of assisted reproductive techniques (ART) has been suggested to be associated with an increased risk of MZT, which is approximately 0.7–13.2% [4–6]. MZT carries a significantly higher risk for both fetuses and mothers compared with singleton and dichorionic twin pregnancies, such as fetal growth restriction, preterm delivery, birth weight discordance, and perinatal mortality [7, 8]. The true incidence of MZT after IVF and in natural conception is likely underestimated, as dichorionic, diamniotic twins occurring after the double-embryo transfer is often assumed to be dizygous twins. The mechanisms producing MZT have remained unclear. Several factors may contribute to the increased rate of MZT observed after IVF, including ovarian hyperstimulation, assisted hatching (AH), maternal age, extended culture, and genetic factors. Theoretically, the increased MZT rate after IVF could be due to one or more of the following: firstly, damaged or prematurely breached zona could lead to blastomeres of the early cleavage embryo separate to form two fetuses. Secondly, ovarian hyperstimulation may increase the number of splitting-prone oocytes and increase the MZT rate. Thirdly, the absence of growth factors or cytokines and the presence of high-level free radicals can induce apoptosis leading to disruption of the inner cell mass, thus resulting in zygotic splitting [9]. Besides, not all multiples born after the single-embryo transfer are sex concordant, which could be explained by concurrent spontaneous conception during IVF. We aimed to compare the MZT rate between fresh embryo transfer and frozen embryo transfer by adjusting for covariates using a large, electronic database.

Study design and patients All fresh and frozen IVF/ICSI cycles with autologous oocytes from 2014 to 2018 at a single assisted reproductive center were retrospectively analyzed using data extracted from the electronic medical record database. This study was approved by the ethics committee of the Northwest Women’s and Children’s Hospital (number 2018002). Both consecutive fresh and frozen-thawed embryo transfer cycles were included in the study. Only SET cycles resulting in a clinical pregnancy confirmed by ultrasound were included in the final analysis. Cycles from donor oocyte cycles, history of recurrent pregnancy loss, uterine pathology, and gender discordance twins were excluded. Ovarian stimulation and luteal support Ovarian stimulation, oocyte retrieval, and fertilization were carried as previously described [10, 11]. Ultrasound guidance was used for embryo transfer. Frozen-thawed embryo transfer was proposed based on the following indications: high risk of OHSS, serum progesterone > 1.5 ng/ml on the day of hCG trigger, unfavorable endometrium at the time of oocyte retrieval, PGT cycles, and personal reason. Luteal support was administered in the form of vaginal progesterone (90 mg q.d.; Crinone, Serono, Hertfordshire, UK) or vaginal progesterone soft capsules (0.2 g t.i.d; Utrogestan, Besins, France), oral progesterone (10 mg t.i.d.; Dydrogesterone, Abbott Biologicals B.V., Netherlands) until the confirmation of biochemical pregnancy, and were maintained to 10 weeks of gestation. Clinical outcome The pregnancy test was performed 14 days after embryo transfer. Transvaginal ultrasound was performed 6–7 weeks after embryo transfer if the pregnancy test was positive. An MZT pregnancy was identified when more than one fetal heartbeat was visualized on the first-trimester ultrasound. The transvaginal ultrasound examination was repeated 1 week later to confirm the prior findings. All women were followed up until 1 year after embryo transfer. Data collection and statistical analysis The required information was retrieved from the electronic database of our IVF center. Statistical comparisons between fresh ET and frozen ET were performed with the use of the independent sample Student’s t test or Mann-Whitney U test for normally and nonnormally distributed variables, respectively. Continuous variables were reported as the mean ± standard error of the mean (SEM). Categorical variables were calculated with the use of the Chi-square or Fisher’s exact test. The crude odds ratio (OR) for the incidence of MZT with the corresponding 95% confidence intervals (CIs) was calculated through logistic regression analysis. A variance inflation factor (VIF) was calculated to test for collinearity among the predictors. To control for potential confounding effects, a multivariable logistic regression analysis was performed and an adjusted OR was calculated after accounting for the following potential confounders: female age at embryo transfer, female age at oocyte retrieval, infertility duration, fertilization type, body mass index, no. of oocyte retrieved, infertility type, cause of infertility, protocol in the fresh cycle, embryo stage and type of luteal support, and year of treatment. 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, Massachusetts). The level of significance was set at p < 0.05.

A total of 39,673 IVF cycles, where embryo transfer had been performed, were identified from the years 2014 to 2018, and 8459 cycles resulted in clinical pregnancy and met the inclusion criteria in the final analysis (Fig. 1). Of these, 3876 were in the fresh cycle and 4583 in the frozen cycle. Among our total cohort, we identified 223 MZT pregnancies. The incidence of MZT in fresh ET and frozen ET were 120 (3.10%) and 103 (2.25%), respectively (Fig. 2). Baseline characteristics are shown in Table 1. Female age at oocyte retrieval, infertility duration, and infertility type were similar between the two groups. Compared with fresh ET, women in frozen ET were older at embryo transfer (30.61 ± 4.01 vs 29.85 ± 3.76; p < 0.001), have lighter BMI (22.45 ± 3.33 vs 22.61 ± 3.27; p = 0.023), have more oocyte retrieved (13.42 ± 7.18 vs 11.25 ± 4.75; p < 0.001), performed more ICSI (27.92% vs 23.81; p < 0.001), have higher birth weight (3.32 ± 0.54 vs 3.26 ± 0.51; p < 0.001), and more cesarean section rate (78.99% vs 50.31%; p < 0.001). However, women in frozen ET have less tubal factor of infertility (38.83% vs 43.44%; p < 0.001) and smaller gestational age at delivery (38.68 ± 1.85 vs 38.79 ± 0.51; p < 0.001). Table 1. | Variable | Fresh ET (n = 3876) | Frozen ET (n = 4583) | P value | |---|---|---|---| | Female age at embryo transfer | 29.85 ± 3.76 | 30.61 ± 4.02 | < 0.001 | | Female age at oocyte retrieval | 29.85 ± 3.76 | 29.99 ± 4.06 | 0.114 | | Infertility duration | 3.40 ± 2.40 | 3.29 ± 2.48 | 0.056 | | BMI | 22.61 ± 3.27 | 22.45 ± 3.33 | 0.023 | | No. of oocyte retrieved | 11.25 ± 4.75 | 13.42 ± 7.18 | < 0.001 | | Fertilization type (n, %) | < 0.001 | || | IVF | 2953 (76.19%) | 3296 (72.08%) | | | ICSI | 923 (23.81%) | 1277 (27.92%) | | | Infertility type (n, %) | 0.367 | || | Primary infertility | 1995 (51.67%) | 2344 (52.66%) | | | Secondary infertility | 1866 (48.33%) | 2107 (47.34%) | | | Cause of infertility (n, %) | < 0.001 | || | Tubal | 1678 (43.44%) | 1725 (38.83%) | | | Endometriosis | 31 (0.80%) | 59 (1.33%) | | | Ovulation disorder | 258 (6.68%) | 355 (7.99%) | | | Male | 879 (22.75%) | 773 (17.40%) | | | Mixed | 856 (22.16%) | 1135 (25.55%) | | | Unexplained | 151 (3.91%) | 135 (3.04%) | | | PGT | 0 (0.00%) | 251 (5.65%) | | | Other | 10 (0.26%) | 10 (0.23%) | | | Protocol in the fresh cycle (n, %) | < 0.001 | || | Agonist | 3705 (95.66%) | 3337 (74.34%) | | | Antagonist | 148 (3.82%) | 923 (20.56%) | | | Mild stimulation | 20 (0.52%) | 229 (5.10%) | | | Embryo stage (n, %) | < 0.001 | || | Cleavage stage | 815 (21.03%) | 751 (16.62%) | | | Blastocyst stage | 3061 (78.97%) | 3769 (83.38%) | | | Type of pregnancy (n, %) | 0.015 | || | Singleton | 3756 (96.90%) | 4480 (97.75%) | | | Monozygotic twinning | 120 (3.10%) | 103 (2.25%) | | | Birth weight (kg) | 3.26 ± 0.51 | 3.32 ± 0.54 | < 0.001 | | Gestational age at delivery (weeks) | 38.79 ± 1.73 | 38.68 ± 1.85 | 0.023 | | Cesarean section rate (n, %) | 1950 (50.31%) | 1857 (78.99%) | < 0.001 | 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 are presented as % (n) or mean ± SD; significance for differences was measured using Chi-squared test, Kruskal-Wallis Univariate analysis was performed to evaluate each variable’s effect on the MZT rate (Table 2). The risk of MZT was decreased with frozen embryo transfer and increased with mild stimulation protocol in the fresh cycle. Table 2. | Covariate | Statistics | OR (95%CI) | P value | |---|---|---|---| | Type of ET | 0.54 ± 0.50 | 0.72 (0.55, 0.94) | 0.016 | | Female age at embryo transfer | 30.26 ± 3.92 | 0.99 (0.96, 1.02) | 0.556 | | Female age at oocyte retrieval | 29.92 ± 3.92 | 1.00 (0.96, 1.03) | 0.924 | | Infertility duration | 3.34 ± 2.44 | 1.00 (0.94, 1.05) | 0.888 | | Fertilization type | ||| | IVF | 6249 (73.96%) | Reference | | | ICSI | 2200 (26.04%) | 0.93 (0.69, 1.27) | 0.664 | | BMI | 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 | | Infertility type | ||| | Primary infertility | 4339 (52.20%) | Reference | | | Secondary infertility | 3973 (47.80%) | 0.91 (0.70, 1.20) | 0.516 | | Cause of infertility | ||| | 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) | 0.133 | | 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 | | Protocol in the fresh cycle | ||| | Agonist | 7042 (84.21%) | Reference | | | Antagonist | 1071 (12.81%) | 0.82 (0.53, 1.28) | 0.383 | | Mild stimulation | 249 (2.98%) | 1.90 (1.04, 3.45) | 0.036 | | Embryo stage | ||| | 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 Interaction and stratified analyses are shown in Table 3. Lower MZT was observed in patients with frozen ET in all subgroups of female age at embryo transfer, female age at oocyte retrieval, infertility duration, fertilization type, infertility type, protocol in the fresh cycle, and embryo stage. There were no significant interactions in any of the subgroups (p > 0.05 for all comparisons). Table 3. | Characteristic | No. of participants | OR (95%CI)a | P value | P for interaction | |---|---|---|---|---| | Female age at embryo transfer (year) | 0.071 | ||| | Tertile 1 (20–27) | 2095 | 0.77 (0.45, 1.33) | 0.347 | | | Tertile 2 (28–31) | 3506 | 0.92 (0.61, 1.38) | 0.683 | | | Tertile 3 (32–47) | 2858 | 0.50 (0.32, 0.80) | 0.004 | | | Female age at oocyte retrieval (year) | 0.140 | ||| | Tertile 1 (20–27) | 2340 | 0.78 (0.47, 1.31) | 0.351 | | | Tertile 2 (28–30) | 2682 | 0.73 (0.45, 1.17) | 0.188 | | | Tertile 3 (31–47) | 3349 | 0.64 (0.42, 0.98) | 0.040 | | | Infertility duration (year) | 0.788 | ||| | Tertile 1 (0–1.9) | 1953 | 0.62 (0.36, 1.09) | 0.098 | | | Tertile 2 (2.0–3.5) | 3344 | 0.67 (0.44, 1.03) | 0.065 | | | Tertile 3 (4–20) | 3162 | 0.85 (0.55, 1.30) | 0.448 | | | Fertilization type | 0.369 | ||| | IVF | 6249 | 0.80 (0.59, 1.09) | 0.154 | | | ICSI | 2200 | 0.51 (0.30, 0.88) | 0.015 | | | Infertility type | 0.756 | ||| | Primary infertility | 4339 | 0.69 (0.48, 0.99) | 0.045 | | | Secondary infertility | 3973 | 0.73 (0.49, 1.09) | 0.127 | | | Protocol in fresh cycle | 0.498 | ||| | Agonist | 7042 | 0.68 (0.50, 0.92) | 0.013 | | | Antagonist | 1071 | 0.44 (0.17, 1.15) | 0.093 | | | Mild stimulation | 249 | 0.96 (0.12, 7.83) | 0.969 | | | Embryo stage | 0.495 | ||| | Cleavage stage | 1566 | 1.09 (0.53, 2.24) | 0.821 | | | Blastocyst stage | 6830 | 0.66 (0.49, 0.88) | 0.005 | OR odds ratio, CI confidence interval, BMI body mass index, IVF in vitro fertilization, ICSI intracytoplasmic sperm injection aAdjusted for BMI, no. of oocyte retrieved, and cause of infertility except the subgroup variable A logistic regression model was then used to assess the association between type of ET and MZT while adjusting for potential confounders presented in Table 4. In the unadjusted model and adjusted model, frozen ET was associated with a decreased likelihood of MZT than fresh ET (OR 0.71; 95% CI, 0.54–0.94, p = 0.016; and adjusted OR 0.66; 95% CI, 0.46–0.90, p = 0.011, respectively). In addition, we separate blastocyst and cleavage stage in different models (Tables 5 and 6) and different protocols in different models (Tables 7 and 8) and found consistent results. Table 4. | Crude modela | Adjusted modelb | ||| |---|---|---|---|---| | OR (95%CI) | P value | OR (95%CI) | P value | | | Fresh ET | Reference | Reference | || | Frozen ET | 0.71 (0.54, 0.94) | 0.016 | 0.66 (0.46, 0.90) | 0.011 | OR odds ratio, ET embryo transfer, MZT monozygotic twinning, CI confidence interval aWe did not adjust other covariates bAdjusted for female age at embryo transfer, female age at oocyte retrieval, infertility duration, fertilization type, body mass index, no. of oocyte retrieved, infertility type, cause of infertility, protocol in the fresh cycle, embryo stage, type of luteal support, and year of treatment Table 5. | Crude model | Adjusted modelb | ||| |---|---|---|---|---| | OR (95%CI) | P value | OR (95%CI) | P value | | | Fresh ET | Reference | Reference | || | Frozen ET | 0.66 (0.49, 0.88) | 0.005 | 0.64 (0.45, 0.91) | 0.012 | OR odds ratio, ET embryo transfer, MZT monozygotic twinning, CI confidence interval aWe did not adjust other covariates bAdjusted for female age at embryo transfer, female age at oocyte retrieval, infertility duration, fertilization type, body mass index, no. of oocyte retrieved, infertility type, cause of infertility, protocol in fresh cycle, type of luteal support, and year of treatment Table 6. | Crude modela | Adjusted modelb | ||| |---|---|---|---|---| | OR (95%CI) | P value | OR (95%CI) | P value | | | Fresh ET | Reference | Reference | || | Frozen ET | 0.83 (0.62, 0.98) | 0.031 | 0.79 (0.61, 0.93) | 0.029 | OR odds ratio, ET embryo transfer, MZT monozygotic twinning, CI confidence interval aWe did not adjust other covariates bAdjusted for female age at embryo transfer, female age at oocyte retrieval, infertility duration, fertilization type, body mass index, no. of oocyte retrieved, infertility type, cause of infertility, protocol in fresh cycle, type of luteal support, and year of treatment Table 7. | Crude modela | Adjusted modelb | ||| |---|---|---|---|---| | OR (95%CI) | P value | OR (95%CI) | P value | | | Fresh ET | Reference | Reference | || | Frozen ET | 0.65 (0.49, 0.87) | 0.003 | 0.68 (0.50, 0.94) | 0.019 | OR odds ratio, ET embryo transfer, MZT monozygotic twinning, CI confidence interval aWe did not adjust other covariates bAdjusted for female age at embryo transfer, female age at oocyte retrieval, infertility duration, fertilization type, body mass index, no. of oocyte retrieved, infertility type, cause of infertility, embryo stage, type of luteal support, and year of treatment Table 8. | Crude modela | Adjusted modelb | ||| |---|---|---|---|---| | OR (95%CI) | P value | OR (95%CI) | P value | | | Fresh ET | Reference | Reference | || | Frozen ET | 0.73 (0.64, 0.91) | 0.026 | 0.69 (0.55, 0.84) | 0.031 | OR odds ratio, ET embryo transfer, MZT monozygotic twinning, CI confidence interval aWe did not adjust other covariates bAdjusted for female age at embryo transfer, female age at oocyte retrieval, infertility duration, fertilization type, body mass index, no. of oocyte retrieved, infertility type, cause of infertility, embryo stage, type of luteal support, and year of treatment

In our retrospective study examining a large cohort of single-embryo transfer, we have found that frozen ET was associated with a lower risk of MZT. The overall incidence of MZT in this study was 3.10% and 2.25% after fresh and frozen ET. This is in general agreement with those of others in the field of IVF but represents a significant increase over MZT after natural conception (three to four per 1000 births) [12, 13]. The exact pathogenesis contributing to embryo splitting is still unknown, but evidence suggests that multiple factors might be responsible, including intracytoplasmic sperm injection, assisted hatching, and extended culture [9, 14, 15]. A previous study suggested that MZT decreased with higher FSH doses [16]. This result is similar to what we observed in our analysis. However, the association between the MZT rate and frozen ET remains controversial. A clear reason for this discrepancy is hard to provide. Differences in the uterine environment after hyper ovarian stimulation may explain the decreased incidence of MZT following frozen ET [17]. Data are still controversial regarding the influence of frozen embryo transfer on MZT. According to our data, frozen ET was associated with a lower MZT rate, which contradicts previous reports [18, 19]. Kawachiya S. reported that embryo freezing did not influence the MZT rate [18]. However, their study did not control the confounding factors of fresh and frozen ET on MZT. Nakasuji T et al. reported a similar MZT rate in fresh and frozen ET [19]. One limitation of that study was data regarding ovarian stimulation and fertilization methods were not available in patients undergoing frozen ET. Our study has several strengths to note. Firstly, the main strength of this study is that it included a large sample of SET cycles with adjustment for multiple potential confounders to obtain reliable clinical outcomes. Secondly, we chose single-embryo transfer to be assessed in the study, taking into consideration the possibility of concurrent natural conceptions. The limitations of our study include the retrospective nature of its design, which inherently lends itself to treatment bias. However, owing to the rarity of MZT, a prospective cohort study is not feasible. Using retrospective data allows us to look at large groups of certain patient subsets that we could not otherwise do prospectively. Ideally, a genetic test should be the gold standard of diagnosing MZT, but the high cost often eliminates this opportunity. The genetic background of MZT has not been collected, which may be a confounding factor [20]. A potential limitation of this study is that data is based on early ultrasound at 6–7 weeks after embryo transfer and cannot determine either whether one embryo loss occurred before this ultrasound, which is relevant for clinical outcomes and prognosis. Changes in clinical care and laboratory practice over 4 years of study period may affect the outcome. To reduce the effect of treatment changes, we added the year of treatment as a confounder. The average patient age was young; therefore, the results may not apply to most practices. Previous studies have suggested that blastocyst culture was associated with an increased MZT risk [18, 21], which could be a potential cofounder. In conclusion, we have used a database of 8459 pregnancies resulting from single-embryo transfer to compare the MZT rate between fresh ET and frozen ET. Our study demonstrated that frozen ET appears to be significantly associated with a decreased frequency of MZT compared with fresh ET. The risks associated with fresh ET should be considered when counseling infertility patients. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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