First case of dichorionic diamniotic triplet pregnancy after single blastocyst transfer.

Multiple pregnancies are associated with significant maternal, fetal, and neonatal risks, including prematurity, low birth weight, pre-eclampsia, anemia, postpartum hemorrhage, intrauterine growth restriction, neonatal morbidity, and increased neonatal and infant mortality rates. Assisted reproductive technology (ART) treatments should prioritize efforts to reduce such events, resisting patient demand for the transfer of multiple embryos at each transfer to increase success rates. Extended culture, embryo selection, and single blastocyst transfer can mitigate the risk of high-order multiple pregnancies. Intriguingly, elective single-embryo transfer (eSET) greatly reduces, but does not completely eliminate, the likelihood of multiple gestations. The occurrence of monozygotic twinning (MZT) gives rise to identical twins. It is more prevalent in women undergoing in vitro fertilization (IVF) compared with natural conception. In fact, the reported risks of monozygotic twinning in IVF and natural conception are 1.7 and 0.4%, respectively. The factors suspected to increase the risk of MZT in IVF are multiple embryo transfer, micromanipulation, and extended in vitro culture. Determining chorionicity and amnionicity is crucial in the assessment of multiple pregnancies during the first-trimester ultrasound examination. Dichorionic twins result from embryo splitting within 3 days after fertilization, while monochorionic twins occur when the splitting takes place between 4 and 8 days after fertilization. These timings are suggested by observations carried out in natural pregnancies. In ART, there is evidence of dichorionic twins derived from single embryo transfer (SET). Here, we report a case of dichorionic diamniotic triplets after a single blastocyst transfer occurred in our center. To our knowledge, this is the first case documented so far.

Assisted Reproduction and Discussion of Rare Cases in Monozygotic Twinning.

Assisted reproductive technology is a crucial factor that increases the incidence of monozygotic twinning in humans. This article discusses the impact of various indicators in assisted reproductive technology studies on pregnancy outcomes, especially studies with a large number of clinical cases. Furthermore, three rare cases in multiples pregnancy are discussed: fetus papyraceous of a pair of male monozygotic twins in a set of triplets, two pairs of sesquizygotic twins with sex-discordance, and rare conjoined triplets.

Monozygotic twin rate among ART centers: a multicenter analysis of data from 18 Italian units.

PURPOSE: The risk of monozygotic twins (MZTs) is increased in couples undergoing assisted reproductive technology (ART) treatments. Several systematic reviews have investigated the possible determinants linked to ART, but results obtained have not been conclusive. The study aims to investigate whether the incidence of MZT differed among ART centers. METHODS: This is a multicenter retrospective cohort study using the Italian ART National Registry database and involving the centers reporting data from individual ART cycles from 2015 to 2019. To investigate the incidence of MZT, only single embryo transfer cycles were considered. Women who had sex-discordant deliveries were excluded. MZT rate was calculated as the number of multiple pregnancies (more than one gestational sac at first ultrasound) out of the total number of clinical pregnancies. A binomial distribution model was used to determine the 95% CI of the frequency of MZT. RESULTS: Eighteen centers were included, and they provided data on 10,433 pregnancies. The total number of MZT was 162, corresponding to an incidence of 1.5% (95% CI: 1.3-1.8%). The rate of MZT among centers varied between 0% (95% CI: 0.0-25.9%) and 3.2% (95% CI: 1.3-8.1%). All the 95% CIs included 1.5%, rejecting the hypothesis that the MZT rate may significantly differ among centers. CONCLUSIONS: The rate of MZT did not significantly vary among ART centers. Local factors are unlikely to explain the increased rate of MZT in ART pregnancies.

Identical twins carry a persistent epigenetic signature of early genome programming.

Monozygotic (MZ) twins and higher-order multiples arise when a zygote splits during pre-implantation stages of development. The mechanisms underpinning this event have remained a mystery. Because MZ twinning rarely runs in families, the leading hypothesis is that it occurs at random. Here, we show that MZ twinning is strongly associated with a stable DNA methylation signature in adult somatic tissues. This signature spans regions near telomeres and centromeres, Polycomb-repressed regions and heterochromatin, genes involved in cell-adhesion, WNT signaling, cell fate, and putative human metastable epialleles. Our study also demonstrates a never-anticipated corollary: because identical twins keep a lifelong molecular signature, we can retrospectively diagnose if a person was conceived as monozygotic twin.

Examining the Vanishing Twin Hypothesis of Neural Tube Defects: Application of an Epigenetic Predictor for Monozygotic Twinning.

Strong associations between neural tube defects (NTDs) and monozygotic (MZ) twinning have long been noted, and it has been suggested that NTD cases who do not present as MZ twins may be the survivors of MZ twinning events. We have recently shown that MZ twins carry a strong, distinctive DNA methylation signature and have developed an algorithm based on genomewide DNA methylation array data that distinguishes MZ twins from dizygotic twins and other relatives at well above chance level. We have applied this algorithm to published methylation data from five fetal tissues (placental chorionic villi, kidney, spinal cord, brain and muscle) collected from spina bifida cases (n = 22), anencephalic cases (n = 15) and controls (n = 19). We see no difference in signature between cases and controls, providing no support for a common etiological role of MZ twinning in NTDs. The strong associations therefore continue to await elucidation.

The mystery of monozygotic twinning II: What can monozygotic twinning tell us about Amyoplasia from a review of the various mechanisms and types of monozygotic twinning?

Monozygotic (MZ) twins ("identical twins") are essentially unique to human beings. Why and how they arise is not known. This article reviews the possible different types of MZ twinning recognized in the previous article on twins and arthrogryposis. There appear to be at least three subgroups of MZ twinning: spontaneous, familial, and those related to artificial reproductive technologies. Each is likely to have different etiologies and different secondary findings. Spontaneous MZ twinning may relate to "overripe ova." Amyoplasia, a specific nongenetic form of arthrogryposis, appears to occur in spontaneous MZ twinning and may be related to twin-twin transfusion.

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.

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.

Four-Generation Pedigree of Monozygotic Female Twins Reveals Genetic Factors in Twinning Process by Whole-Genome Sequencing.

Familial monozygotic (MZ) twinning reports are rare around the world, and we report a four-generation pedigree with seven recorded pairs of female MZ twins. Whole-genome sequencing of seven family members was performed to explore the featured genetic factors in MZ twins. For variations specific to MZ twins, five novel variants were observed in the X chromosome. These candidates were used to explain the seemingly X-linked dominant inheritance pattern, and only one variant was exonic, located at the 5'UTR region of ZCCHC12 (chrX: 117958597, G > A). Besides, consistent mitochondrial DNA composition in the maternal linage precluded roles of mitochondria for this trait. In this pedigree, autosomes also contain diverse variations specific to MZ twins. Pathway analysis revealed a significant enrichment of genes carrying novel SNVs in the epithelial adherens junction-signaling pathway (p = .011), contributed by FGFR1, TUBB6, and MYH7B. Meanwhile, TBC1D22A, TRIOBP, and TUBB6, also carrying similar SNVs, were involved in the GTPase family-mediated signal pathway. Furthermore, gene-set enrichment analysis for 533 genes covered by copy number variations specific to MZ twins illustrated that the tight junction-signaling pathway was significantly enriched (p < .001). Therefore, the novel changes in the X chromosome and the provided candidate variants across autosomes may be responsible for MZ twinning, giving clues to increase our understanding about the underlying mechanism.

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.

Monozygotic twinning after assisted reproductive technologies: a case report of asymmetric development and incidence during 19 years in an international group of in vitro fertilization clinics.

OBJECTIVE: To describe a case of monozygotic twinning with asymmetric development following a single fresh embryo transfer as part of an intracytoplasmic sperm injection (ICSI) treatment. Secondarily, to report the incidence of monozygotic twinning at the IVI (Instituto Valenciano de Infertilidad) clinics. DESIGN: Case report. SETTING: Private fertility centers. PATIENT(S): A 33-year-old woman with a 2-year history of primary infertility. INTERVENTION(S): Controlled ovarian hyperstimulation and ICSI treatment with single-embryo transfer. MAIN OUTCOME MEASURE(S): Incidence of monozygotic twinning at the IVI clinics. RESULT(S): We report a twin pregnancy after a single-embryo transfer. Twins were dichorionic and diamniotic. One fetus had a 6-day delay in its growth compared with the other when observed by ultrasound. Two female infants were delivered, and despite presenting congenital diseases, they were successfully treated and evolved correctly. A subsequent DNA analysis confirmed that the infants were monozygotic. Furthermore, we estimated a monozygotic twinning rate of 1.17% at the IVI clinics, taking into account those cases in which two or more embryos with heart beats were observed by ultrasound scanning after single-embryo transfers. CONCLUSION(S): Ultrasound scans performed during pregnancy suggested a possible dizygotic origin of the twins, but DNA analysis performed after birth established that they were monozygotic. Genetic analysis is the only valid tool to confirm if like-sex dichorionic twins are monozygotic or dizygotic.

High incidence of monozygotic twinning after assisted reproduction is related to genetic information, but not to assisted reproduction technology itself.

OBJECTIVE: To study the incidence of monozygotic twinning (MZT) in patients using in vitro fertilization, relative to their age, genetic background, ovarian function, and assisted reproductive techniques used. DESIGN: Analysis of a collected database. SETTING: Infertility treatment center. PATIENT(S): A total of 1,876 patients receiving infertility treatment between 2000 and 2012. Pregnancies with monozygotic twins (A: 23) were compared with deliveries of dizygotic twins (B: 423), singleton pregnancies (C: 880), and aborted pregnancies (D: 389). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): A genetic survey on multiple pregnancies in the extended family. Measures were micromanipulation technique, the length of embryo cultivation, type of cultivation media, basal follicle-stimulating hormone level, estradiol level on the day of human chorionic gonadotropin administration, number of oocytes, total consumption of gonadotropins, and consumption of gonadotropins needed for recovery of 1 oocyte. RESULT(S): No differences were found between the incidence of MZT in cycles that did vs. did not use micromanipulation techniques. In addition, the length of embryo cultivation or type of cultivation media used did not affect the results. Estradiol levels and implantation rates were significantly higher in group A. The incidence of MZT in families in group A was significantly higher than that in groups B and C. CONCLUSION(S): We propose that the high incidence of MZT in infertility-clinic patients is conditioned by hereditary factors, and good ovarian function only facilitates the expression. The resulting recommendation is that young women with a positive family history and good ovarian function undergo elective single-embryo transfer, and proper counseling is advisable.

Discordant sex in monozygotic XXY/XX twins: a case report.

We report a case of discordant phenotypic sex in monozygotic twins mosaic 47,XXY/46,XX: monozygotic heterokaryotypic twins. The twins presented with cognitive and comprehension delay, behavioural and language disorders, all symptoms frequently reported in Klinefelter syndrome. Molecular zygosity analysis with several markers confirmed that the twins are in effect monozygotic (MZ). Array comparative genomic hybridization found no evidence for the implication of copy number variation in the phenotypes. Ultrasound scans of the reproductive organs revealed no abnormalities. Endocrine tests showed a low testosterone level in Twin 1 (male phenotype) and a low gonadotrophin level in Twin 2 (female phenotype) which, combined with the results from ultrasound examination, provided useful information for potentially predicting the future fertility potential of the twins. Blood karyotypes revealed the presence of a normal 46,XX cell line and an aneuploïd 47,XXY cell line in both patients. Examination of the chromosome constitutions of various tissues such as blood, buccal smear and urinary sediment not surprisingly showed different proportions for the 46,XX and 47,XXY cell lines, which most likely explains the discordant phenotypic sex and mild Klinefelter features. The most plausible underlying biological mechanism is a post-zygotic loss of the Y chromosome in an initially 47,XXY zygote. This would result in an embryo with both 46,XX and 47,XXY cells lines which could subsequently divide into two monozygotic embryos through a twinning process. The two cell lines would then be distributed differently between tissues which could result in phenotypic discordances in the twins. These observations emphasize the importance of regular paediatric evaluations to determine the optimal timing for fertility preservation measures and to detect new Klinefelter features which could appear throughout childhood in the two subjects.

Sex ratios provide evidence for monozygotic twinning in the ring-tailed lemur, Lemur catta.

Monozygotic (MZ) twinning is generally considered to be rare in species other than human. We inspected sex ratios in European zoo-bred ring-tailed lemurs (Lemur catta), revealing a significant excess of same-sex twins. Of 94 pairs, 60 (64%) were either both males or both females (p = .004). Application of the Weinberg differential rule argues that 27% of all twins in this species are MZ pairs. In this protected species, where twinning is commonplace (~50% of newborns are twins), the probable existence of frequent MZ twinning has ramifications for breeding programs aimed to maximize genetic diversity, and suggests that twin studies in a species other than human could have potential as a medical research tool.

Why are monozygotic twins different?

Although popularly designated as "identical", monozygotic (MZ) twins are rarely identical. Much has been speculated on the origin of MZ twins and several theories have been proposed. Post-fertilization events, such as chromosomal mosaicism, skewed X-inactivation and imprinting mechanisms, as well as other epigenetic mechanisms are responsible for the differences between MZ twins. Numerous discordant MZ twins have been reported including discordance for lateral asymmetry, major malformation, growth and intrauterine death of the co-twin. This discrepancy may have long-term implications on complex diseases and their predisposition, organ transplantation and interpretation of twin-based studies. We reviewed the genotypic and phenotypic differences between MZ twins and discuss their main causes.

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.

Monozygotic triplet pregnancies after single blastocyst transfer: two cases and literature review.

Following IVF, single blastocyst transfer has been thought to reduce the risks of high-order multiple pregnancies. This is a report of two cases of monozygotic triplet pregnancies after single blastocyst transfer and a review of the current concepts of the pathogenesis of multiple monozygotic pregnancies as well as the options for managing these high-risk pregnancies. Both cases were reduced to a twin pregnancy by selective cord coagulation at 15-16 weeks. Whereas one patient had uneventful pregnancy until labour was induced for growth arrest and cord Doppler abnormalities in one twin, the other developed a severe twin-to-twin transfusion syndrome which required fetoscopic laser surgery at 21 weeks. In both cases, healthy twins were delivered by Caesarean section at 34.5 and 34 weeks, respectively. As the predictors of their occurrence are not fully understood, patients should be informed of the risks of monozygotic pregnancies after single blastocyst transfer.