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). METHODS: 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. RESULTS: 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. CONCLUSION: Advanced maternal age was associated with a lower rate of MZT. A threshold female age of 36 years existed for lower MZT.

Does assisted hatching affect live birth in fresh, first cycle in vitro fertilization in good and poor prognosis patients?

PURPOSE: To assess the effect of assisted hatching (AH) on live birth rate (LBR) in first cycle, fresh in vitro fertilization (IVF) in good and poor prognosis patients. METHODS: Retrospective cohort using cycles reported to the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System. Live birth rate was compared in women who underwent first cycle, autologous, fresh IVF cycles with (n = 48,858) and without (n = 103,413) AH from 2007 to 2015. RESULTS: The propensity-weighted LBR was 39.2% with AH versus 43.9% without AH in all patients. The rate difference (RD) with AH was - 4.7% ([CI - 0.053, - 0.040], P < 0.001) with the calculated number needed to harm being 22. AH affected live birth in both good prognosis and poor prognosis patients. The propensity-weighted monozygotic twinning (MZT) rate was 2.3% in patients treated with AH as compared to 1.2% patients that did not receive AH. The RD with AH on MZT in fresh, first IVF cycles was 1.1% ([0.008, 0.014], P < 0.001). CONCLUSION: AH may affect LBR across all patients and in poor prognosis patients in fresh IVF cycles. Caution should be exercised when applying this technology. More prospective research is needed.

Differences in blastomere totipotency in 2-cell mouse embryos are a maternal trait mediated by asymmetric mRNA distribution.

It is widely held that the first two blastomeres of mammalian embryos are equally totipotent and that this totipotency belongs to the group of regulative properties. However, this interpretation neglects an important aspect: evidence only came from successful monozygotic twins which can speak only for those pairs of half-embryos that are able to regulate in the first place. Are the frequently occurring incomplete pairs simply an artefact, or do they represent a real difference, be it in the imperfect blastomere's ability to regulate growth or in the distribution of any compound X that constrains regulation? Using the model system of mouse embryos bisected at the 2-cell stage after fertilization, we present evidence that the interblastomere differences evade regulation by external factors and are already latent in oocytes. Specifically, an interblastomere imbalance of epiblast production persists under the most diverse culture conditions and applies to the same extent in parthenogenetic counterparts. As a result, cases in which twin blastocysts continued to develop in only one member account for 65 and 57% of zygotic and parthenogenetic pairs, respectively. The interblastomere imbalance is related to the subcellular distribution of gene products, as documented for the epiblast-related gene Cops3, using mRNA FISH in super-resolution mode confocal microscopy. Blastomere patterns of Cops3 mRNA distribution are α-amanitin-resistant. Thus, the imbalance originates not from de novo transcription, but from influences which are effective before fertilisation. These data expose previously unrecognized limits of regulative capacities of 2-cell stage blastomeres and point to aspects of cytoplasmic organization of the mouse oocyte that segregate unequally to blastomeres during cleavage.

Prevalence and risk factors of zygotic splitting after 937 848 single embryo transfer cycles.

STUDY QUESTION: What is the prevalence of multiple pregnancy with zygotic splitting after single embryo transfer (SET)? SUMMARY ANSWER: The prevalence of multiple pregnancy with zygotic splitting after SET was 1.36%. WHAT IS KNOWN ALREADY: In 2008, the Japan Society of Obstetrics and Gynaecology (JSOG) recommended the adoption of SET to reduce multiple births. Since then, to improve the clinical pregnancy rate, elective SET using blastocyst transfer and frozen-warmed ET has increased. Blastocyst culture and zona pellucida manipulation, including ICSI and AH, have been widely reported as risk factors for monozygotic twinning. However, all these studies may have included cases with dizygotic pregnancies produced by a transferred embryo and a spontaneous conception. STUDY DESIGN, SIZE, DURATION: A retrospective observational study was performed, based on 937 848 SET cycles in registered ART data from the JSOG between 2007 and 2014. The study was approved by the Registration and Research Subcommittee of the JSOG and Juntendo University Ethics Committee. PARTICIPANTS/MATERIALS, SETTING, METHODS: To identify possible factors affecting the prevalence of zygotic splitting, we identified pregnancies, in which the number of foetuses exceeded the number of gestational sacs (GSs), to restrict our analysis to 'true' zygotic splitting. Multiple logistic regression analysis was performed using singleton pregnancy after SET, as control. P < 0.05 was considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: Fresh and frozen-warmed SET produced 276 934 clinical pregnancies (29.5%/SET), including 4310 twins (1.56% of pregnancies) and 109 triplets (0.04% of pregnancies). Based on sex analysis of dichorionic twins after SET, the prevalence of multiple pregnancy with zygotic splitting was 1.36%. Statistical analysis revealed that compared to singleton pregnancies zygotic splitting pregnancies were associated with frozen-warmed ET cycles (odds ratio [OR] = 1.34; 95% CI: 1.16-1.55), blastocyst culture (OR = 1.79; 95% CI: 1.54-2.09) or AH (OR = 1.21; 95% CI: 1.08-1.35). In fresh ET cycles, the prevalence rate of zygotic splitting pregnancy after single blastocyst transfer was significantly higher than that after SET cycles with cleavage embryos (OR = 2.20; 95% CI: 1.83-2.66). However, no significant difference in ovarian stimulation and fertilization methods was recognized. LIMITATIONS, REASONS FOR CAUTION: In the current Japanese ART registry system, data regarding frozen-warmed ET do not include information about ovarian stimulation and fertilization methods. Registration for AH only began in 2010. There is no way of validating if data submitted by clinics is correct. WIDER IMPLICATIONS OF THE FINDINGS: Clinicians should consider whether to counsel couples about the small increase in the risk of zygotic splitting associated with some embryo manipulations. STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for the study. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: None.

Evaluation of placental vascularization indices in monochorionic diamniotic and dichorionic diamniotic twin pregnancies.

OBJECTIVES: We aimed to investigate and compare placental vascularization indices between monochorionic-diamniotic, dichorionic-diamniotic normal twin pregnancies, and normal singular pregnancies. We hypothesized that there is correlation between placental three-dimensional power Doppler vascularization indices and birth weight in case of twin pregnancies, and that normal singular pregnancies have higher placental vascularization indices than normal twin pregnancies. STUDY DESIGN: Placental three-dimensional power Doppler vascularization indices, such as vascularization index, flow index, and vascularization-flow index were measured in monochorionic-diamniotic (N = 15) and dichorionic-diamniotic (N = 36) normal twin pregnancies, and in normal singular (N = 109) pregnancies. Correlations were analyzed between vascularization indices, and birth weight, APGAR score, umbilical pH, umbilical venous bicarbonate, lactate, and base excess. RESULTS: Vascularization indices and birth weight were significantly (p < 0.01) higher in normal singular gestations (vascularization index = 10.36, flow index = 46.08, vascularization-flow index = 4.08, average birth weight = 3377 g at 38.2 weeks average gestational age) compared to monochorionic-diamniotic and dichorionic-diamniotic normal twin pregnancies. No significant differences were found in vascularization indices between monochorionic-diamniotic and dichorionic-diamniotic normal twins. There were no significant differences in APGAR score, umbilical pH, umbilical venous bicarbonate, lactate, and base excess between groups examined (p < 0.01). We found strong linear correlations between placental vascularization indices and birth weight in both twin groups. CONCLUSION: Placental three-dimensional power Doppler vascularization indices seem appropriate for predicting birth weight in monochorionic-diamniotic and dichorionic-diamniotic normal twin pregnancies. Our pilot study revealed reference values for vascularization indices in case of twin pregnancies examined.

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

BACKGROUND: 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. OBJECTIVE AND RATIONALE: 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. SEARCH METHODS: 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. OUTCOMES: 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. WIDER IMPLICATIONS: 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.

High incidence of monozygotic twinning in infertility treatment.

BACKGROUND: Monozygotic twinning is associated with increased perinatal morbidity and mortality. There is evidence that the number of monozygotic twins increases after assisted reproductive techniques. METHODS: We searched PUBMED, MEDLINE, and Scopus from 1987 to 2015 for studies analyzing the incidence and possible etiology of monozygotic twinning in infertility patients and critically reviewed the current state of knowledge. RESULTS AND CONCLUSIONS: Monozygotic twinning is a rare in natural conception but occurs around twice the normal rate after assisted reproduction. Factors associated with this phenomenon remain speculative, though there is some evidence that micromanipulation techniques, prolonged culture, and genetics are involved. In view of the possible complications, adequate pre-conception counselling is advocated.

Monochorionic quadramniotic and triamniotic pregnancies following single embryo transfers: two case reports and a review of the literature.

PURPOSE: The purpose of this study is to report two cases of monozygotic quadruplet and triplet pregnancies following single embryo transfer (ET). METHODS: A 29-year-old woman and a 34-year-old woman underwent ART treatment in two affiliated University based ART units. The first woman underwent ICSI with day 3 embryo biopsy for pre-implantation genetic diagnosis (PGD) followed by day 4 transfer, which resulted in a monochorionic quadramniotic (MCQA) quadruplet pregnancy. The second woman underwent conventional IVF with transfer of a single blastocyst, which resulted in a monochorionic triamniotic (MCTA) triplet pregnancy. RESULTS: The first patient underwent successful selective foetal reduction at 16 + 3 and 17 + 4 weeks of gestation. Two healthy twin girls were delivered by elective caesarean section at 35 + 6 weeks of gestation. The second patient underwent successful selective foetal reduction at 14 + 1 weeks of gestation. The remaining monochorionic diamniotic (MCDA) twins are well at the time of writing this article. CONCLUSIONS: To our knowledge, these cases represent the first case of viable MCQA pregnancy following single ET in the world and the third case of a viable MCTA pregnancy following conventional IVF with single ET. Several factors including blastocyst stage transfer and zona pellucida manipulation have been thought to contribute to monozygotic twinning in the context of ART. These two cases add to the growing literature of monozygotic multiple pregnancies following ART.

Gestación gemelar monocigótica asociada a técnicas de reproducción asistida / Monozigotic twinning associated with assisted reproductive technologies

La frecuencia de gestaciones gemelares monocigóticas se estima entre 0,4-0,45% de las gestaciones espontáneas. Sin embargo, tras la utilización de técnicas de reproducción asistida (TRA) numerosos estudios han demostrado un aumento del riesgo de gestaciones monocigóticas. A propósito de 2 casos presentados recientemente en nuestro servicio, planteamos una revisión de los posibles factores que se pueden asociar entre las TRA y la gestación múltiple monocigótica (AU)

Monozygotic twin pregnancies account for about 0.4%-0.45% of nonstimulated in vivo conceptions. However, numerous studies have demonstrated that the risk of these pregnancies is increased after the use of assisted reproductive technologies. We report two cases of monozygotic twin pregnancies that occurred recently in our hospital and review the possible factors that may be involved in the association between assisted reproductive technologies and monozygotic twin pregnancies (AU)

The timing of monozygotic twinning: a criticism of the common model.

In the dominant model, monozygotic (MZ) twinning is universally accepted as a post-fertilization event resulting from splitting of the embryo along its first 2 weeks of development. The stage at which splitting occurs determines chorionicity and amnionicity. A short history on how the model was built is presented, stressing the role played by some embryologists, in particular George Corner, in its completion and final success. Strikingly, for more than 60 years no deep criticisms have been raised against the model, which, in virtue of its rational and plausible character, enjoys the status of undisputed truth. At close examination, the embryological support of the model shows some important weak points, particularly when dealing with late splitting. In the author's view, the model not only has contributed to 'suspend' our knowledge on the timing of MZ twinning, but seems indefensible and claims to be substituted. That factor could imply relevant consequences for embryology and bioethics. As an alternative to the model, a new theory to explain the timing of MZ twinning is proposed. It is based on two premises. First, MZ twinning would be a fertilization event. In that case, due to an alteration of the zygote-blastomere transition, the first zygotic division, instead of producing two blastomeres, generates twin zygotes. Second, monochorionicity and monoamnionicity would not depend on embryo splitting, but on fusion of membranes. Some support for this theory can be found in recent embryological advances and also in some explanations of old.

The teeth and faces of twins: providing insights into dentofacial development and oral health for practising oral health professionals.

The continuing studies of the teeth and faces of Australian twins and their families in the Craniofacial Biology Research Group in the School of Dentistry at the University of Adelaide began 30 years ago. Three main cohorts of twins have been recruited, enabling various objectives and specific hypotheses to be addressed about the roles of genetic, epigenetic and environmental influences on human dentofacial growth and development, as well as oral health. This paper highlights some key findings arising from these studies, emphasizing those of direct relevance to practising oral health professionals. We also draw on published literature to review the significant developments in relation to the use of precision 2D and 3D imaging equipment, the application of modern molecular techniques, and the development of sophisticated computer software for analysing genetic relationships and comparing complex shapes. Such developments are valuable for current and future work. Apart from the classical or traditional twin model, there are several other twin models that can be used in research to clarify the relative contributions of genetic, epigenetic and environmental contributions to phenotypic variation. The monozygotic (MZ) co-twin model is one particularly valuable method, given that examination of only one pair of MZ twins can provide considerable insights into underlying causes of observed variation. This model can be used in a dental practice environment, with oral health professionals having the opportunity to explore differences in orofacial structures between MZ co-twins who are attending as patients. As researchers have become more aware of the complexities of the interactions between the genome, the epigenome and the environment during development, there is the need to collect more phenotypic data and define new phenotypes that will better characterize variations in growth processes and health status. When coupled with powerful new genetic approaches, including genome-wide association studies and linkage analyses, exciting opportunities are opening up to unravel the causes of problems in craniofacial growth and common oral diseases in human populations.

Twin-to-twin transfusion syndrome: a case study.

Twin-to-twin transfusion syndrome (TTTS) is a disease that affects roughly 15% of monochorionic twins. Although TTTS is not extremely prevalent, the rate of mortality and morbidly approaches 100% without early detection and treatment. The following case study is a triplet pregnancy that included a set of monochorionic twins affected by TTTS. Typically, it is assumed that monochorionicity can identify the zygosity of twins, which was disproven in this case. Research indicates that there has been an increased rate of monochorionic twins with in vitro fertilization. There is great debate about the most appropriate treatment modality for TTTS. Perinatal treatment followed by neonatal management is the standard of care for TTTS. Implications for the neonatal nurse practitioner and care for the patients are discussed.

Blastocyst culture is associated with an elevated incidence of monozygotic twinning after single embryo transfer.

In a 7-year (2002-2008) retrospective study of a large IVF program based on minimal ovarian stimulation and single ET (47,841 single ETs), monozygotic twinning occurred in 1.01% of 14,956 clinical pregnancies. Blastocyst culture was associated with a significantly increased monozygotic twinning risk (adjusted odds ratio, 2.04; 95% confidence interval, 1.29-4.48), whereas embryo freezing, type of stimulation protocol used, intracytoplasmic sperm injection fertilization, or zona removal did not influence its incidence.

Monozygotic pregnancies conceived by in vitro fertilization: understanding their prognosis.

OBJECTIVE: To analyze the outcomes and particular characteristics of monozygotic (MZ) pregnancies conceived by in vitro fertilization (IVF). DESIGN: Retrospective data analysis. SETTING: Large private-academic fertility center. PATIENT(S): IVF-conceived MZ pregnancies. INTERVENTION(S): Statistical analysis of MZ pregnancy outcomes depending on fetal order and pregnancy reductions status. MAIN OUTCOME MEASURE(S): Spontaneous pregnancy reduction, pregnancy loss, take-home baby rate, perinatal mortality, gestational age at delivery, and birth weight. RESULT(S): A total of 72 of 3,426 pregnancies (2.1%) were MZ, and 70 were included in the study. Of these, 34 cases (48.5%) were high-order multiple pregnancies (HOMP), and 36 (51.5%) were non-HOMP. In the HOMP group, only 2.9% (1 of 34) had a complete pregnancy loss while 38.8% (14 of 36) of the non-HOMP were lost by 20 weeks' gestation. Of the HOMP patients, 73.1% therapeutically reduced the MZ component, and a statistically significant difference in gestational age of delivery (37.8 ± 3.2 vs. 28.1 ± 7.7) and birth weight (2796 ± 865.8 vs. 1110.0 ± 731.6) was seen when compared with nonreduced HOMP. CONCLUSION(S): Twinning with MZ is encountered in a small but important number of pregnancies derived from assisted reproduction. The prognosis for these patients is unfavorable, particularly for single-implantation MZ pregnancies and for nonreduced HOMP. Patients who do not spontaneously reduce a MZ-HOMP by 12 weeks may benefit from therapeutically reducing the MZ component of the pregnancy.

Transcript profiling of individual twin blastomeres derived by splitting two-cell stage murine embryos.

In invertebrates and amphibians, informational macromolecules in egg cytoplasm are organized to provide direction to the formation of embryonic lineages, but it is unclear whether vestiges of such prepatterning exist in mammals. Here we examined whether twin blastomeres from 2-cell stage mouse embryos differ in mRNA content. mRNA from 26 blastomeres derived from 13 embryos approximately mid-way through their second cell cycle was subjected to amplification. Twenty amplified samples were hybridized to arrays. Of those samples that hybridized successfully, 12 samples in six pairs were used in the final analysis. Probes displaying normalized values >0.25 (n = 4573) were examined for consistent bias in expression within blastomere pairs. Although transcript content varied between both individual embryos and twin blastomeres, no consistent asymmetries were observed for the majority of genes, with only 178 genes displaying a >1.4-fold difference in expression across all six pairs. Although class discovery clustering showed that blastomere pairs separated into two distinct groups in terms of their differentially expressed genes, when the data were tested for significance of asymmetrical expression, only 39 genes with >1.4-fold change ratios in six of six blastomere pairs passed the two-sample t-test (P < 0.05). Transcripts encoding proteins implicated in RNA processing and cytoskeletal organization were among the most abundant, differentially distributed mRNA, suggesting that a stochastically based lack of synchrony in cell cycle progression between the two cells might explain at least some and possibly all of the asymmetries in transcript composition.

Impact of selective reduction of the monochorionic pair in in vitro fertilization triplet pregnancies on gestational length.

In this retrospective cohort of IVF-conceived triplets complicated by a monochorionic pair (n = 21), selective reduction of the pair to a singleton pregnancy was associated with a significantly greater likelihood of delivery at ≥34 weeks. On average, reduction of the pair was associated with 52 days longer gestation.

Twinning in the offspring of parents with chronic radiation exposure from nuclear testing in Kazakhstan.

The population of the Semipalatinsk region of Kazakhstan was chronically exposed to radioactive fallout from above-ground nuclear tests conducted during 1949-1956 by the Soviet Union. We investigated the effect of radiation exposure and other factors on risks of twinning overall and of same- and different-sex twinning and hence estimated dizygotic and monozygotic twinning rates in 11,605 deliveries around Semipalatinsk, 141 of which were twin, to 3992 mothers exposed to fallout during 1949-1956. Overall, the same-sex twinning rate was 7.85 [95% confidence interval (CI): 6.24, 9.47] per 1000 and the opposite-sex twinning rate was 4.45 (95% CI: 3.23, 5.67). Twinning rates did not differ significantly between radiation exposure categories, parental age at main radiation exposure, or year of birth. Different-sex, but not same-sex, twinning increased with maternal age (P(trend) = 0.04) but not with other demographic factors and was increased soon after radiation exposure [OR = 4.08 (95% CI: 1.11, 15.07)] for births occurring within 5 years compared with more than 20 years after exposure; this effect was similar in villages with low and high radiation exposure, however, so interpretation is uncertain.

Monozygotic twinning is not increased after single blastocyst transfer compared with single cleavage-stage embryo transfer.

OBJECTIVE: To compare the incidence of monozygotic twinning between cleavage-stage and blastocyst-stage embryo transfer in a large cohort of patients undergoing single embryo transfer. DESIGN: Retrospective study. SETTING: Dutch-speaking Free University of Brussels. PATIENT(S): This study covered the period between July 2003 and December 2005. 1,951 fresh IVF/ICSI cycles in which single embryo transfer was performed were retrospectively reviewed. Only the first cycle of each patient was included. INTERVENTION(S): Five hundred eighty seven (n = 587) cycles that resulted in clinical pregnancies were identified; 308 after single day-3 embryo transfer and 271 after single blastocyst transfer. MAIN OUTCOME MEASURE(S): The incidence of monozygotic twinning. RESULT(S): Overall, 13 cases (2.2%) of monozygotic twinning were observed, 2.6% in the cleavage-stage group (n = 8/308) and 1.8% in the blastocyst group (n = 5/271). No statistically significant differences were observed in the probability of monozygotic twinning between the Cleavage-stage and the Blastocyst group (difference: +0.8%; 95% CI, -1.97 to +3.41). All of these pregnancies resulted in the delivery of 24 healthy babies. The crude odds ratio for the incidence of monozygotic twinning after day-5 embryo transfer was calculated to be 0.71 (95% CI, 0.23-2.18). CONCLUSION(S): To investigate the potential association between the day of embryo transfer (day 3 or 5) and the incidence of monozygotic twinning, the clinical pregnancies analyzed should have been established after single embryo transfer. The current study represents the first methodologically appropriate study attempting to investigate the above research question. Our findings support that opting for blastocyst transfer does not increase the probability for monozygotic twins.