Incidence Of Monozygotic Twinning Research Articles

O-213 Analysis of risk factors for monozygotic twins following in vitro fertilization-embryo transfer

Abstract Study question To investigate the risk factors associated with the incidence of monozygotic twinning（MZT）following in vitro fertilization and embryo transfer (IVF-ET). Summary answer MZT following IVF-ET may be associated with a reduced endometrial thickness on the day of embryo transfer and a decrease in estradiol levels post-transfer. What is known already MZT pregnancy arises from the division of a single fertilized ovum, resulting in offspring with identical genetic material. These twins exhibit markedly higher rates of complications compared to dizygotic counterparts, including increased incidences of miscarriage, congenital anomalies, intrauterine fetal demise, growth restriction, and preterm delivery. The underlying biological processes leading to MZT remain elusive. Presently, scholarly discourse centers around two predominant models and one hypothesis: the zygote division model, the embryonic membrane fusion model, and the over-mature gametes hypothesis, respectively. IVF-ET has been implicated in the heightened occurrence of MZT, suggesting a potential iatrogenic element to this phenomenon. Study design, size, duration A retrospective cohort study has been conducted. We analyzed 582 IVF-ET cycles at a large teaching hospital from January 2007 to December 2022, including 291 clinically confirmed MZTs, and 291 matched non-MZTs. Participants/materials, setting, methods A total of 582 participants, 291 in both MZT group and non-MZT group. There were 146 fresh embryo transfer cycles and 145 frozen-thawed cycles in the MZT group. Each cycle type was matched on a 1:1 basis by age and embryo transfer date with a control group that did not result in MZT following IVF-ET. We examined the disparities between the MZT group and the non-MZT group, and performed the Logistics regression analysis. Main results and the role of chance Significant differences were observed in the MZT group compared to the non-MZT group in the fresh embryo transfer cycle, particularly regarding estradiol (E2) and progesterone (P) levels on day 7 post-transfer, E2/P ratios on day 14 post-transfer, and endometrial thickness on the day of transfer (p<0.05). Logistic regression analysis indicated a correlation between reduced endometrial thickness on the transfer day and the occurrence of MZT. During the frozen-thawed cycle, significant differences were noted in E2/P ratios, E2 levels on day 7 post-transfer, and endometrial thickness on embryo transfer day (p<0.05). Logistic regression analysis suggested that lower E2 levels on day 7 post-transfer and reduced endometrial thickness on embryo transfer day were predictive of MZT. Additional potential influencing factors included the duration of the participants’ menstrual cycle, the protocol of ovulation induction, and the duration of embryo culture in vitro. Limitations, reasons for caution Primarily, the retrospective design may introduce bias, notably due to the potential absence of comprehensive data. Secondly, the study is constrained by the sample size, with 146 fresh cycles and 145 frozen cycles, which may not provide a fully representative statistical power. Wider implications of the findings The incidence of MZT may implicate signaling pathways at the maternal-fetal interface, with early embryonic split potentially serving as a compensatory mechanism in response to suboptimal estrogen levels or diminished endometrial thickness, thus enhancing embryonic viability. Trial registration number not applicable

Risk factors associated with monozygotic twinning in offspring conceived by assisted reproductive technology.

What are the factors influencing the occurrence of monozygotic (MZ) twins in offspring conceived by assisted reproductive technology (ART)? Parental ages, the transfer of fresh versus frozen embryos, and the grade of blastocysts are all related to MZ twinning in ART offspring. Offspring conceived by ART have significantly increased risk of MZ twins, which may be due to the characteristics of the infertile population. The objective of this study was to explore the incidence of monozygotic (MZ) twins after ART and to clarify the risk factors for MZ twinning. A total of 255 monozygotic twins were enrolled in this cohort study, and then matched with singletons at a ratio of 1:4 randomly (with 1020 in the control group). All offspring were conceived by single embryo transfer. The collected data were divided into the following three aspects for analysis: characteristics of the infertile population, gamete or embryo manipulations, and factors related to embryo development. The incidence of MZ twins was 1.638% (255 out of 15 567 pregnancies after single embryo transfers). Compared to singleton births, a significantly lower rate of frozen embryo transfers (FET; 78.0% vs 86.1% P = 0.002) was seen amongst the MZ twins. Amongst fresh ETs, the rate of blastocyst transfers in the MZ twins group was higher compared to that in the control group (92.9% vs 75.4%, P = 0.005). We also found that certain grades of blastocysts in terms of trophectoderm (TE) development, inner cell mass + TE development and the classification of 'top-quality' embryos were associated with the incidence of MZ twinning (P = 0.025, P = 0.012, P = 0.020, respectively). Logistic regression analysis revealed that higher paternal age (odds ratio (OR) = 0.94, 95% CI = 0.89-1.00, P = 0.029) and FET (OR = 0.48, 95% CI = 0.33-0.68, P = 0.001) may be protective factors against MZ twinning. However, higher maternal age (OR = 1.07, 95% CI = 1.01-1.13, P = 0.027) and the transfer of blastocysts (OR = 4.31, 95% CI = 1.46-12.73, P = 0.008) appeared to be associated with an increased risk of MZ twinning. Amongst blastocyst transfers, a C grade TE may be protective factor against MZ twinning (B: OR = 1.90, 95% CI = 1.18-3.07, P = 0.009; A: OR = 1.58, 95% CI = 0.93-2.67, P = 0.089). First, our definition of MZ twins was based on twins' birth after single embryo transfers (SET), rather than ultrasound examination during early pregnancy. Second, the parental characteristics of the two groups were homogenous, so it was difficult to find any associations between infertility factors and the incidence of MZ twins. This multifaceted analysis of the risk factors for MZ twinning provides some information for clinical interventions in high-risk populations. This study was supported by the National Key Technology Research and Developmental Program of China (2022YFC2704404), CAMS Innovation Fund for Medical Sciences (2021-I2M-5-001), Taishan Scholars Program for Young Experts of Shandong Province (tsqn201909195), the Basic Science Center Program (31988101), and the Shandong Provincial Key Research and Development Program (2020ZLYS02). All authors have no conflicts of interest to declare. N/A.

The Impact of Assisted Hatching on Monozygotic Twinning is Related to Female Age and Insemination Method: A New Perspective.

Whether assisted hatching (AH) is associated with a higher incidence of monozygotic twinning (MZT) in women undergoing assisted reproductive technology remains controversial; the aim of the study was to demonstrate the relationship between AH and MZT. A total of 8900 clinical pregnancies were selected among embryo transfer cycles from January 2011 to October 2019. Women receiving day (D) 3 embryos were divided into groups A-C: group A (n = 1651) and group B (n = 1045) included women aged ≤37 or ≥38 years, respectively, with zona pellucida (ZP) thinning; group C (n = 3865) included women aged ≤37 years without AH. Women aged ≤37 years who underwent blastocyst transfer and/or blastocyst ZP breaching were included in group D (n = 2339). The incidence of MZT was compared among groups A, B and C, and between groups C and D. The incidence of MZT in group B (2.2%) was significantly higher than in group A (1.0%), especially following intracytoplasmic sperm injection (ICSI), while the incidence of MZT in group A (1.0%) was significantly lower than in group C (2.2%). The MZT rate with invitro fertilization was higher in group D (2.8%) than in group C (2.2%), but the MZT rate following ICSI was not significantly different between the two groups. ZP thinning of D3 embryos may increase the risk of MZT in older women (≥38 years), but decrease it in younger women (≤37 years). ZP breaching may be useful to reduce the incidence of MZT in ICSI-generated blastocysts.

P-139 The incidence of monozygotic twins after the usage of calcium ionophore or after trophectoderm biopsy in ART treatment

Abstract Study question To investigate whether the usage of calcium ionophore or trophectoderm biopsy affect the occurrence of monozygotic twins (MZT) in assisted reproductive treatment (ART) treatment. Summary answer Despite the increase was not statistically significant, higher percentage of MZT were observed in both group with calcium ionophore usage and trophectoderm biopsy. What is known already The incidence of monozygotic twins (MZT) in natural conception is approximately 0.4% worldwide but it is estimated that there is at least a 2-fold rise in the incidence of MZT after assisted reproductive technology compared with natural conception, although the reason is still under debate. The effectiveness of calcium ionophore in assisting oocytes activation and fertilisation has been widely studied. Most studies reported no impact upon embryo viability and reproductive potential after trophectoderm biopsy. However, the impact of calcium ionophore or trophectoderm biopsy on subsequent in vivo development of the blatocyst remains unclear. Study design, size, duration This is a retrospective observational cohort study of frozen embryos transfers (FET) that have achieved clinical pregnancy between January 2019 and October 2021. In this study, 709 FET cycles were included and analysed based on i) with or without calcium ionophore treatment after ICSI/IVF on oocytes retrieval day; ii) with or without trophectoderm biopsy on day 5 or 6. The results were analysed to identify possible risk factors affecting the prevalence of monozygotic twins. Participants/materials, setting, methods The cases with positive beta-hCG test were followed-up and analysed for the occurrence of MZT. The diagnosis of MZT was established by transvaginal ultrasound performed at 4-6 weeks after the transfer. The visualization of uteri containing more than one fetus, either in one sac (monochorionic monoamniotic) or in two sacs separated by a septum (monochorionic diamniotic) is suggestive of MZT. Data were analysed by Fisher’s exact test and Odd Ratio. Main results and the role of chance Data comprising a total of 709 FET cycles in a single clinical setting were analysed. Among 449 cases of clinical pregnancies, 8 cases of MZT occurred. A higher percentage of MZT (1.78%) after blastocyst transfer in ART treatment was observed as compared to natural conception (0.04%). There was a higher incidence of MZT in trophectoderm biopsy group as compared with non-biopsy group (2.3% vs 1.45% respectively, odds ratio [OR]= 1.59, 95% CI 0.39–6.46). However, there was no statistically significant (P-value=0.556, CI = 95%). The incidence of MZT following the calcium ionophore usage was higher compared with the group without calcium ionophore usage (2.33% versus 1.72%, OR = 1.36, 95% CI 0.16–11.3). There was no significantly higher risk of MZT with calcium ionophore versus non-calcium ionophore cycles (P-value= 0.493, CI = 95%). Limitations, reasons for caution The sample examined in this study is limited in size thus more studies are needed to achieve satisfactory statistical power. MZT diagnosed with ultrasound is difficult to differentiate from dichorionic monozygotic twins. The definitive diagnosis of monozygosity is only possible by DNA analysis of all the twins born. Wider implications of the findings These findings can draw attention to study the effect of calcium ionophore on embryo development and the mechanism behind MZT. Based on the findings, the patients undergoing ART treatment should be informed with the risk associated with higher incidence of MZT. Trial registration number NA

P-760 Monozygotic twin rate among IVF centers: A multicenter analysis of data from 18 Italian units

Abstract Study question Could factors inherent to individual centers, related to local clinical or laboratory variables, play a role in the increased incidence of monozygotic twins in IVF? Summary answer The rate of MZT did not significantly vary among IVF centers What is known already The risk of monozygotic twins (MZT) is increased in couples undergoing in vitro fertilization (IVF). Several systematic reviews have investigated the possible determinants linked to IVF, but results obtained have been inconclusive. Associations have been identified with the prolonged culture at blastocyst stage and young female age. Conventional IVF compared to ICSI, and assisted hatching emerged as possible additional risk factors, but evidence is controversial. Meta-analyses, however, cannot provide robust evidence because of the difficulty in performing multivariate analyses. Moreover, available investigations were retrospective and involved relatively small sample sizes hampering the quality of the collected data. Study design, size, duration This is a multicenter retrospective cohort study using the Italian ART National Registry database and involving the Centers reporting data from individual IVF cycles from 2015 to 2019. Eighteen IVF centers were included with a total of 87,076 IVF cycles reported during the study period. The primary aim of the study was to investigate whether the incidence of MZT differed among centers. Participants/materials, setting, methods Only single embryo transfer cycles were considered. Women who had sex-discordant twin deliveries were excluded. MZT rate was calculated as the number of multiple pregnancies out of the total number of clinical pregnancies. A binomial distribution model was used to determine the 95% CI of the frequency of MZT. The rates among centers were deemed heterogeneous if the 95% CI in at least one center did not include the mean MZT rate. Main results and the role of chance Ten centers reported data from 2015 to 2019, eight from 2017 to 2019. An analysis of the main characteristics of the centers revealed considerable variability. The total number of clinical pregnancies fulfilling our inclusion criteria were 10,440. Of these, 7 were found to be twin pregnancies with discordant gender and were excluded, leaving 10,433 for data analyses. The number of cases included per center varied between 11 and 2,823. Overall, 162 pregnancies were found to be multiple: 48 ended in a miscarriage, 28 spontaneously reduced to singletons and 86 women delivered two or more newborns. The vast majority were twins (n = 160), one was triplet, and one was quadruplet. Considering the denominator of 10,433 pregnancies, the rate of MZT was thus 1.5% (95%CI: 1.3 - 1.8%). The MZT rate among centers varied between 0% (95%CI: 0.0 - 25.9%) and 3.2% (95%CI: 1.3 - 8.1%). All the 95%CIs of the rates of MZT include the 1.5% common rate, thus rejecting the hypothesis that this incidence significantly differed among centers. Spearman correlations between the rate of MZT and variables associated with individual center policies could not identify any significant association. Even when restricting the analyses only to the largest centers, no association emerged. Limitations, reasons for caution Our study was limited by its retrospective nature. Furthermore, some centers provided less than 100 cases for data analysis and were therefore poorly informative. External confirmation from larger registries is therefore required. Wider implications of the findings This study provides evidences that the rate of MZT did not differ among IVF centers. Furthermore, no clinical and laboratory variables inherent to individual centers were identified in the determinism of IVF-associated MZT. Further studies are needed to identify the causes of increased risk of MZT in IVF practice. Trial registration number not applicable

Blastocyst morphology is associated with the incidence of monozygotic twinning in assisted reproductive technology

An increased incidence of monozygotic twinning after a blastocyst transfer has been previously reported in assisted reproductive technology treatment. It is uncertain whether this phenomenon is due to the extended culture time, culture medium, or inherent blastocyst parameters. This study aimed to investigate the association between blastocyst parameters (invitro culture time, blastocyst stage, and inner cell mass and trophectoderm grading) and the incidence of monozygotic twinning after assisted reproductive technology. This was a retrospective cohort study employing data from a multicenter, large, electronic database from 4 academic hospitals. All clinical pregnancies after a single blastocyst transfer between January 2014 and February 2020 were included. Blastocyst morphology was evaluated based on the Gardner grading system, considering the blastocyst stage, and inner cell mass and trophectoderm grading (grades A, B, and C). Monozygotic twinning was defined as ≥2 fetal heartbeats in a single gestational sac or 2 gestational sacs with sex concordance at birth. The multivariable predicted marginal proportions from logistic regression models were used to compute adjusted relative risks for the association between blastocyst parameters and the incidence of monozygotic twinning. The overall monozygotic twinning rate was 1.53% (402 of 26,254 cases). The monozygotic twinning was not associated with the culture time invitro (day 5 vs day 6) or blastocyst stage (early, blastocyst, expanded, hatching, and hatched). Alternatively, monozygotic twinning was associated with lower inner cell mass grading (B vs A: adjusted relative risk, 1.67 [95 % confidence interval, 1.28-2.25]; C vs A: adjusted relative risk, 1.98 [95% confidence interval, 1.18-3.11]) and higher trophectoderm grading (B vs C: adjusted relative risk, 1.38 [95% confidence interval, 1.03-1.92]; A vs C: adjusted relative risk, 2.14 [95% confidence interval, 1.45-3.20]). The incidence of monozygotic twinning was the lowest in blastocysts with grade A inner cell mass and grade B or C trophectoderm (0.82%, as the reference) and the highest in blastocysts with grade B or C inner cell mass and grade A trophectoderm (2.40%; adjusted relative risk, 2.62; 95% confidence interval, 1.60-4.43). The incidence of monozygotic twinning in blastocysts with consistent inner cell mass or trophectoderm grading was somewhere in between (both A: 1.58%; adjusted relative risk, 1.86 [95% confidence interval, 1.23-3.04]; both B or C: 1.59%; adjusted relative risk, 1.84 [95% confidence interval, 1.29-2.90]). Higher risk of monozygotic twinning was associated with blastocyst morphology specific to those blastocysts with loosely arranged inner cell mass cells combined with tightly packed trophectoderm cells.

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

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

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

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.

Double trouble? Clinic-specific risk factors for monozygotic twinning

To investigate clinic-specific risk factors for monozygotic twinning (MZT) using a large, electronic database. Retrospective case-control study. Infertility clinics. Using an electronic medical record system, viable clinical pregnancy (confirmation of a gestational sac(s) and presence of at least one fetal pole with a heartbeat on first trimester ultrasound), data were obtained from homologous invitro fertilization (IVF) cycles after single ET from June 1, 2004, to December 31, 2016. Monozygotic twinning was defined as a pregnancy with two fetal heartbeats on ultrasound with sex concordance at birth. None. Risk factors for MZT including cycle type, method of insemination, and method of cryopreservation. Of the 28,265 IVF cycles that met inclusion criteria over the study period, 8,749 (31.0%) resulted in a viable intrauterine clinical pregnancy. There were 102 (2.7%) MZT in the fresh cycle cohort and 133 (2.7%) in the frozen cycle cohort. Neither cryopreservation nor the method of cryopreservation was a significant risk factor for MZT. However, the use of sequential media was an independent risk factor for MZT in fresh, but not frozen, ETs (odds ratio = 1.72, 95% confidence interval, 1.10-2.68). Significant differences were seen in the incidence of MZT between clinics, and this difference persisted after controlling for known risk factors (clinic 0, reference; clinic 2, odds ratio = 2.22; 95% confidence interval, 1.48-3.32; clinic 3, odds ratio = 1.93; 95% confidence interval, 1.30-2.87). Differences in MZT rates exist between individual IVF clinics, suggesting that variations in practice patterns may contribute to this event. The present study noted the use of sequential media was an independent risk factor for fresh but not frozen cycles.

Monozygotic twinning following embryo biopsy at the blastocyst stage.

Objective:Monozygotic twinning incidence following preimplantation genetic testing in embryos at cleavage-stage does not appear to increase; however, data regarding the possible impact of the blastocyst-stage preimplantation genetic testing is lacking. We compared the incidence of monozygotic twinning in preimplantation genetic testing cycles performed at the blastocyst-stage, versus cycles without PGT, following single embryo transfer.Methods:In this retrospective cohort study, we analyzed the incidence of twin pregnancies in patients undergoing intracytoplasmic sperm injection and blastocyst-preimplantation genetic testing (253 cycles), versus a period-matched control population of patients undergoing intracytoplasmic sperm injection and single embryo transfer without preimplantation genetic testing (606 cycles).Results:The overall monozygotic twinning rate was 14/859 (1.6%) per clinical pregnancy. The incidence of zygotic splitting following intracytoplasmic sperm injection and preimplantation genetic testing was 3.5% (95% Confidence interval 1.8%-6.6%) versus 0.8% (95% Confidence interval 0.3%-1.9%) following intracytoplasmic sperm injection without preimplantation sperm injection. After adjusting for potential confounders, preimplantation genetic testing cycles were associated with an increase in the incidence of monozygotic twinning when compared to cycles without embryo biopsy (Odd ratio 3.44, 95% Confidence interval 1.05-11.27, p=0.041).Conclusions:Our findings indicate that embryo biopsy for preimplantation genetic testing performed at the blastocyst stage is associated to an increase in the incidence of monozygotic twinning. Further validation in larger sample size studies is warranted. Patients undergoing preimplantation genetic testing must receive proper counselling about the potential risks of the technique.

Factors associated with embryo splitting and clinical outcome of monozygotic twins in pregnancies after IVF and ICSI.

What factors are associated with monozygotic twins (MZT) after autologous IVF/ICSI with fresh and frozen/thawed single embryo transfer (SET) and what is the outcome of MZT?Factors associated with increased MZT were blastocyst transfer and elective single embryo transfer (eSET), with MZT showing a lower gestational age at birth and neonatal weight but higher perinatal mortality only after fresh transfer.ART is associated with an increased incidence of MZT, which carries higher perinatal mortality. However, risk factors associated with MZT are still controversial.A population-based retrospective analysis of data extracted from ART cycles reported to the Latin American Registry of ART between January 2012 and December 2016 was used in order to study the frequency and outcome of MZT after SET.In total, 2925 clinical pregnancies obtained after autologous IVF/ICSI with fresh SET were used to study biomedical factors possibly associated with MZT, such as maternal age, type of insemination, use of assisted hatching, stage of embryo development at transfer, elective or non-elective SET and preimplantation genetic testing. Another group of 3085 clinical pregnancies obtained after SET of frozen-thawed embryo transfer (FET) was also used to study the possible association between embryo freezing and MZT. Only pregnancies with complete follow-up until birth were included in this analysis. The diagnosis of MZT was established by transvaginal ultrasound performed at 6–8 weeks of amenorrhea. The rate of MZT for each potential risk factor was obtained and a multivariable logistic regression was performed in order to account for the above-mentioned factors. Pregnancies were followed until birth and the early neonatal period in order to assess the rate of miscarriage and stillbirths, gestational age at birth, neonatal weight and early neonatal mortality.There were 76 MZT out of 2925 clinical pregnancies with fresh SET (2.6%) and 69 MZT out of 3085 clinical pregnancies after FET (2.2%) (odds ratio (OR) = 0.85, 95% CI 0.61–1.19). A statistically significantly increase in MZT rate was observed with blastocyst compared with cleavage stage ET (3.4 versus 2.0%, respectively; OR = 1.70, 95% CI 1.05–2.76). When confounding variables were considered, eSET was also significantly associated with an increase in the odds of MZT (OR = 1.74, 95% CI 1.04–2.92). Overall perinatal mortality was higher in MZT compared with singletons, but this rise was only significant after fresh ET.Limitations of the current study result from the fact that MZT were diagnosed with ultrasound performed at 6–8 weeks of amenorrhea; therefore, spontaneous embryo reductions taking place earlier were missed.Reproductive health providers must inform their patients that blastocyst transfer and eSET of fresh embryos are associated with a statistically significantly increase in the odds of MZT and that perinatal mortality after fresh ET is significantly higher in MZT than in singletons.The Latin American Registry of ART receives direct funding from Ferring Pharmaceuticals, but no specific funding was received to undertake this study. None of the authors declare conflict of interest.

The risk of secondary sex ratio imbalance and increased monozygotic twinning after blastocyst transfer: data from the Japan Environment and Children\u2019s Study

BackgroundSome studies have suggested that blastocyst transfer is associated with i) imbalance in the secondary sex ratio (SSR) (which favors male offspring), ii) increased incidence of monozygotic twins (MZT). In contrast, others have not found these changes. In this study, we evaluated the association between blastocyst transfer and SSR and MZT, considering potential parental confounders.MethodsThe Japan Environment and Children’s Study is a large, nationwide longitudinal birth cohort study funded by the Ministry of the Environment of Japan. We used this large dataset, including 103,099 pregnancies, to further investigate the association between blastocyst transfer, SSR and MZT, using spontaneously conceived pregnancies, non-assisted reproductive technology (non-ART) treatment (intrauterine insemination and ovulation induction with timed intercourse) and cleavage stage embryo transfer for comparison. We evaluated the association with each group, the SSR, and the frequency of MZT, calculating the adjusted odds ratio (AOR) using multivariable logistic regression analyses, adjusting for potential parental confounders such as basic health and socioeconomic status.ResultsFor each group (spontaneous conception vs. non-ART treatment vs. cleavage stage embryo transfer vs. blastocyst transfer), the percentages of males were 51.3% vs 50.7% vs 48.9% vs 53.4% and the monozygotic twinning rates per pregnancy were 0.27% vs 0.11% vs 0.27% vs 0.99% respectively. Multivariate logistic regression analyses indicated that blastocyst transfer was significantly associated with a higher SSR and higher incidence of MZT than the other three groups (SSR: AOR 1.095, 95% CI1.001–1.198; MZT: AOR 4.229, 95% CI 2.614–6.684).ConclusionsThere are significant relationships between blastocyst transfer and SSR imbalance and a higher occurrence of MZT.

Identifying risk factors related to monozygotic twins after assisted reproductive technologies

ObjectiveThis secondary analysis aimed to identify the incidence and risk factors associated with monozygotic twins (MZTs) after assisted reproductive technology (ART). Study designIn this retrospective cohort observational study, the treatment cycles were compared between MZT and non-MZT pregnancies using logistic regression analyses. Of the 11,501 patients with 13,225 pregnancies, 166 MZTs were diagnosed (1.44% in ART pregnant patients; 1.25% in pregnant cycles). ResultsResults of the logistic analysis revealed that embryo stage at transfer and number of good-quality embryos were independent predictive factors for MZTs. The thresholds for two factors were 3.5 and 3.5. The efficacy of embryo stage at transfer and number of good-quality embryos for MZTs were evaluated using receiver-operating characteristic curves. The areas under the curve (AUCs) for these two parameters were 0.708 and 0.633, respectively. Adding number of good quality embryos increased the discriminative ability of the model (AUC = 0.745). ConclusionsThe incidence of MZTs, subsequent to ART, is quite high. Extended culture (≥3.5 days), together with the number of good-quality embryos (≥3.5), conferred the greatest risk of producing MZTs.

Neonatal outcomes among twins following assisted reproductive technology: an Australian population-based retrospective cohort study

BackgroundWhile their incidence is on the rise, twin pregnancies are associated with risks to the mothers and their babies. This study aims to investigate the likelihood of adverse neonatal outcomes of twins following assisted reproductive technology (ART) compared to non-ART twins.MethodsA retrospective population study using the Australian National Perinatal Data Collections (NPDC) which included 19,662 twins of ≥20 weeks gestational age or ≥ 400 g birthweight in Australia. Maternal outcomes and neonatal outcomes (preterm birth, low birth weight, resuscitation and neonatal death) were compared. Generalized Estimating Equations were used to assess the likelihood of any neonatal outcomes, with adjusted odds ratio (AOR) and 95% confidence intervals (CI) presented. Weinberg’s differential rule was used to estimate monozygotic twin rate.ResultsART mothers were 3.3 years older than non-ART mothers. The rates of pregnancy-induced hypertension and gestational diabetes were significantly higher for ART mothers than non-ART mothers (12.2% vs. 8.4%, p < 0.01) and (9.7% vs. 7.5%, p < 0.01) respectively. The incidence of monozygotic twins was 2.0% for ART twins and 1.1% for non-ART twins. Compared with non-ART twins, ART twins had higher rates of preterm birth (AOR 1.13, 95% CI: 1.05–1.22), low birth weight (AOR 1.13, 95% CI: 1.05–1.22), and resuscitation (AOR 1.26, 95% CI: 1.17–1.36). Liveborn ART twins had 28% (AOR 1.28, 95% CI 1.09–1.50) increased odds of having any adverse neonatal outcome compared to liveborn non-ART twins, especially for opposite-sex ART twins (AOR 1.42, 95% CI 1.11–1.82).ConclusionAs ART twins had higher rates of adverse outcome, special prenatal care is recommended. Couples accessing ART should be fully informed of the risk of adverse outcome of twin pregnancies.

Elevated incidence of monozygotic twinning is associated with extended embryo culture, but not with zona pellucida manipulation or freeze-thaw procedure

To identify the incidence and risk factors associated with IVF-conceived monozygotic twinning (MZT). Retrospective study. Academic hospital. A total of 3,463 women with clinical pregnancies between January 2014 and February 2015 were analyzed. None. The measures were the incidence of MZT based on the number of embryos that were replaced, type of insemination method (conventional IVF or intracytoplasmic sperm injection [ICSI]), with or without the use of assisted hatching (AH), and day of embryo transferred in fresh and frozen cycles. Ninety-three women (2.69%) with MZT were observed. No statistically significant differences were observed in the cycle parameters of fresh or frozen cycles between MZT and other non-MZT pregnancies. Specific IVF procedures or techniques, such as the number of embryo replaced, zona pellucida manipulation (ICSI and AH), and freeze-thaw procedure, did not significantly increase the rate of MZT, except for the day of embryo transferred. Compared with day 3 transferred, day 4 and 5/6 transferred showed an increased probability of MZT (odds ratio [OR], 2.73; 95% confidence interval [CI], 1.16-6.42 for day 4 transferred and OR, 3.68; 95% CI, 2.29-5.93 for day 5/6 transferred). Extended culture (advanced embryo stage) in fresh and frozen cycles appeared to be associated with increased rates of MZT. The effect of the number of embryos transferred, ICSI and AH, and freeze-thaw procedures on the risk for MZT was not demonstrated.

Determinants of monozygotic twinning in ART: a systematic review and a meta-analysis.

The incidence of monozygotic twins (MZT) after ART appears to be higher than the incidence after spontaneous conceptions contradicting the aim of ART to avoid multiple pregnancies because of the associated risks. The aim was to study the frequency of MZT after IVF and ICSI and how it is influenced by the day of embryo transfer, maternal age, zona pellucida manipulation, controlled ovarian stimulation, stimulation protocol, culture media and embryo quality. Original studies and reviews were identified by searching the PubMed, Embase and Cochrane databases up to March 2017. The inclusion criterion was publications focusing on the five study questions related to MZT in our study. The exclusion criteria were articles that did not include blastocyst transfer, were on non-humans, were not published in peer-reviewed journals, and were based only on case studies. All of the articles were categorized according to the Oxford Centre for Evidence-based Medicine's 'Levels of Evidence', and quality and risk of bias assessment was performed with 'The Cochrane Collaboration's Risk of Bias Tools'. A meta-analysis was performed to study the impact of the day of embryo transfer on the MZT rate. The literature search resulted in a total of 42 articles, including 38 original studies, for analysis. The included original studies reported a MZT rate with blastocyst transfer from zero to 13.2%. Our meta-analysis found a higher frequency of MZT after blastocyst transfer compared with cleavage-stage embryos transfer: odds ratio = 2.18, 95% CI: 1.93-2.48 (fixed effect meta-analysis). A younger maternal age may increase the MZT rate, and recent studies regarding the use of zona pellucida manipulating techniques have disagreed with the previous suspicion of a higher MZT rate after the use of these methods. The extended culture to-blastocyst stage is a potential risk factor for MZT, but it is uncertain whether this phenomenon is due to the extended time, culture media or greater likelihood of younger oocytes to reach the blastocyst stage. An increased frequency of MZT following the GnRH-agonist suppression protocol has been suggested, as well as a decreased frequency of MZT with high gonadotrophin doses, which could reflect an age-related effect. Only limited literature has focused on the role of embryo morphology in the MZT rate, therefore, this issue remains unresolved. We found blastocyst transfer to be a risk factor for MZT. Hence, the results of this meta-analysis may weaken the previously proposed view that greater experience with blastocyst transfer and improved culture media could decrease the high rate of MZT after blastocyst transfer. To minimize the rate of MZT and the associated complications, the mechanisms underlying blastocyst transfer and MZT pregnancy must be elucidated.

Inner cell mass incarceration in 8-shaped blastocysts does not increase monozygotic twinning in preimplantation genetic diagnosis and screening patients.

BackgroundThe use of assisted reproductive technology (ART) has been reported to increase the incidence of monozygotic twinning (MZT) compared with the incidence following natural conception. It has been hypothesized that splitting of the inner cell mass (ICM) through a small zona hole may result in MZT. In this study, using a cohort of patients undergoing preimplantation genetic diagnosis/screening (PGD/PGS), we compared the clinical and neonatal outcomes of human 8-shaped blastocysts hatching with ICM incarceration with partially or fully hatched blastocysts, and attempted to verify whether this phenomenon increases the incidence of MZT pregnancy or negatively impact newborns.MethodsThis retrospective study included 2059 patients undergoing PGD/PGS between March 1, 2013, and December 31, 2015. Clinical and neonatal outcomes were only collected from patients who received a single blastocyst transfer after PGD/PGS (n = 992). A 25- to 30-μm hole was made in the zona of day 3 embryos by laser. The blastocysts were biopsied and vitrified on day 6. The biopsied trophectoderm (TE) cells were analyzed using different genetic methods. One tested blastocyst was thawed and transferred to each patient in the subsequent frozen embryo transfer cycle. All the biopsied blastocysts were divided into three types: 8-shaped with ICM incarceration (type I), partially hatched without ICM incarceration (type II), and fully hatched (type III). ICM/TE grading, clinical and neonatal outcomes were compared between the groups.ResultsThe percentage of grade A ICMs in type I blastocysts (22.2%) was comparable to that in type III blastocysts (20.1%) but higher than that in type II blastocysts (4.5%). The percentage of grade A TEs in type I blastocysts (4.2%) was comparable to that in type II (3.6%) but lower than that in type III (13.5%). There were no significant differences in clinical pregnancy, MZT pregnancy, miscarriage, live birth, MZT births, and neonatal outcomes between the groups.ConclusionsCompared to partially and fully hatched blastocysts, 8-shaped blastocysts with ICM incarceration showed relatively higher ICM and lower TE grades. ICM incarceration in 8-shaped blastocysts does not increase the incidence of MZT and has no negative effects on newborns in PGD/PGS patients.

Do ARTs affect the incidence of monozygotic twinning?

Does the manipulation of gametes or embryos duringARTs increase the risk for monozygotic twinning (MZT)? Frozen embryo transfer (ET) is associated with a lower MZT rate, while blastocyst culture is associated with an increased risk of monozygotic pregnancy. Monozygotic twins have a higher risk for perinatal complications. Although an increased incidence of monozygotic pregnancies after ART has been previously reported, data regarding the possible impact of different laboratory procedures are conflicting. All clinical pregnancies after single ETcarried out in our centre between 2004 and 2013 (n=6096) were retrospectively analysed for the incidence of MZT. The effect of different laboratory procedures on the incidence of MZT was evaluated. The following ART risk factors were assessed: maternal age, type of ET (fresh versus frozen), zona pellucida (ZP) manipulation (specifically, ICSI, embryo biopsy and assisted hatching), use of donor oocytes, embryo stage at time of ET (cleavage, compaction, early or advanced blastocyst) and culture media. The overall MZT rate was 2.2% (136/6096). Frozen ET was associated with a significant reduction in MZT incidence (adjusted odds ratio (aOR) 0.48, 95% CI 0.29-0.80), while blastocyst transfer (early or advanced blastocyst) was associated with a significant increase in MZT risk (aOR 2.70, 95% CI 1.36-5.34; aOR 2.05, 95% CI 1.29-3.26, respectively). No significant differences were found between the MZT and singleton (non-MZT) groups regarding maternal age, the use of different ZP manipulation techniques, not type of culture media used. This study is limited by its retrospective nature and the fact that monozygosity was not confirmed by genetic testing. Furthermore, since monozygotic pregnancy is a rare event, other ART parameters that may influence its incidence could not be assessed during our analysis. Our findings warrant future studies designed to investigate the association between specific ART procedures and MZT, namely the potential risk of blastocyst transfer to increase MZT. No external funding was used for this study. There are no conflicts of interest.

Monozygotic Pregnancies Following Assisted Reproductive Technology: A Review

Introduction: Assisted reproductive technology (ART) is associated with an increased risk of monozygotic twinning. This narrative review attempts to summarise the known literature regarding the aetiology, incidence, risk factors, diagnosis, and prognosis of monozygotic twinning following ART. Aetiology: Monozygotic twinning is caused by the splitting of the early embryo during the peri-implantation phase. According to the classical hypothesis, the timing of the split determines the chorionicity and amnionicity, however this has been questioned in recent literature. Incidence and risk factors: The incidence of monozygotic twinning in natural conception appears to be independent of extrinsic factors such as ethnicity and age. The incidence of monozygotic twinning is increased from 0.4% of natural conceptions to around 0.9–2.24% of pregnancies following ART. The available literature supports a role of ovarian stimulation and extended culture to the blastocyst stage in increasing the risk of monozygotic twinning. The impact of maternal age and micromanipulation techniques such as assisted hatching and intra-cytoplasmic sperm injection appear to depend on the stage of the embryo being transferred leading to significant heterogeneity between studies. Diagnosis: The gold standard for diagnosing monozygotic twinning is genetic testing but its cost precludes it from routine widespread use. Most epidemiological studies utilise statistical estimates such as Weinberg’s differential rule and tailored questionnaires. Most studies from ART units have utilised transvaginal sonography for counting the number of gestational sacs or assessing the chorionicity. Prognosis: The prognosis of twins appears to be dependent on the chorionicity and amnionicity and is largely independent of the zygosity.

Clustering of monozygotic twinning in IVF.

The aim of this analysis was to study whether monozygotic twinning (MZT) events occur in clusters after IVF and, if so, to explore possible explanations for this clustering. This is a retrospective cohort study carried out in a single, large university-affiliated reproductive medicine practice. Medical records of all patients who had undergone fresh IVF cycles, resulting in a viable clinical pregnancy, from Jan 2002 to Dec 2013 were reviewed. The incidence of MZT in 6-month intervals and the association with independent risk factors such as maternal age, extended embryo culture, PGD/intracytoplasmic sperm injection (ICSI)/assisted hatching performed were analyzed. Over the 12-year study period, 25,502 fresh IVF cycles were performed, resulting in 8598 clinical pregnancies. Ninety-five cycles (1.1 %) resulted in MZ twins. The percentage of MZT was >2 standard deviations (SD) higher than the overall percentage of MZT in 4 of the 24 6-month intervals. PGD, extended embryo culture (≥4 days), and more recent cycles (2005 or later) were independent risk factors for MZT. The use of multivariable logistic regression modeling to control for risk factors for MZT did not correct for this clustering effect, with both high-risk interval (clustering) and extended embryo culture remaining significant. This study supports our hypothesis that MZT occurs in clusters and that this clustering effect could not be explained by demographics and cycle characteristics alone. Although we are unable to explain the clustering phenomenon, this study is important as it highlights high-risk intervals for MZT and opens the door to performing a more detailed investigation, to identify the mechanisms responsible for the spikes of MZT incidence.