Cellular mechanisms of monozygotic twinning: clues from assisted reproduction

Abstract

Background: Monozygotic (MZ) twins are believed to arise from the fission of a single fertilized embryo at different stages. Monochorionic MZ twins, who share one chorion, originate from the splitting of the inner cell mass (ICM) within a single blastocyst. In the classic model for dichorionic MZ twins, the embryo splits before compaction, developing into two blastocysts. However, there are a growing number of ART cases where a single blastocyst transfer results in dichorionic MZ twins, indicating that embryo splitting may occur even after blastocyst formation.

Objective and rationale: For monochorionic MZ twins, we conducted a comprehensive analysis of the cellular mechanisms involved in ICM splitting, drawing from both ART cases and animal experiments. In addition, we critically re-examine the classic early splitting model for dichorionic MZ twins. We explore cellular mechanisms leading to two separated blastocysts in ART, potentially causing dichorionic MZ twins.

Search methods: Relevant studies including research articles, reviews, and conference papers were searched in the PubMed database. Cases of MZ twins from IVF clinics were found by using combinations of terms including 'monozygotic twins' with 'IVF case report', 'ART', 'single embryo transfer', or 'dichorionic'. The papers retrieved were categorized based on the implicated mechanisms or as those with unexplained mechanisms. Animal experiments relating to MZ twins were found using 'mouse embryo monozygotic twins', 'mouse 8-shaped hatching', 'zebrafish janus mutant', and 'nine-banded armadillo embryo', along with literature collected through day-to-day reading. The search was limited to articles in English, with no restrictions on publication date or species.

Outcomes: For monochorionic MZ twins, ART cases and mouse experiments demonstrate evidence that a looser ICM in blastocysts has an increased chance of ICM separation. Physical forces facilitated by blastocoel formation or 8-shaped hatching are exerted on the ICM, resulting in monochorionic MZ twins. For dichorionic MZ twins, the classic model resembles artificial cloning of mouse embryos in vitro, requiring strictly controlled splitting forces, re-joining prevention, and proper aggregation, which allows the formation of two separate human blastocysts under physiological circumstances. In contrast, ART procedures involving the transfer of a single blastocysts after atypical hatching or vitrified-warmed cycles might lead to blastocyst separation. Differences in morphology, molecular mechanisms, and timing across various animal model systems for MZ twinning can impede this research field. As discussed in future directions, recent developments of innovative in vitro models of human embryos may offer promising avenues for providing fundamental novel insights into the cellular mechanisms of MZ twinning during human embryogenesis.

Wider implications: Twin pregnancies pose high risks to both the fetuses and the mother. While single embryo transfer is commonly employed to prevent dizygotic twin pregnancies in ART, it cannot prevent the occurrence of MZ twins. Drawing from our understanding of the cellular mechanisms underlying monochorionic and dichorionic MZ twinning, along with insights into the genetic mechanisms, could enable improved prediction, prevention, and even intervention strategies during ART procedures.

Registraiton number: N/A.

Keywords: assisted hatching; assisted reproduction; blastocyst cavitation; chorion; embryo development; inner cell mass; monozygotic twins; zona pellucida.

Splitting of the inner cell mass within a blastocyst leads to monochorionic monozygotic twins; if one embryo splits into two blastocysts, dichorionic monozygotic twins develop.

Figure 1.

Classic model for different types of monozygotic (MZ) twinning (left) and cellular model for monochorionic MZ twin formation (right). (A) During human embryo development, the two cell lineages at the blastocyst stage, the inner cell mass (ICM, yellow) and the trophectoderm (TE, gray), primarily develop into the fetus and the chorion, respectively. (B and C) Monochorionic MZ twins share one chorion, and in most cases, they have their own amnion (third row) but in rare case, they can also share one amnion (second row). Classically, monochorionic MZ twins are formed when the ICM undergoes splitting before or after the blastocyst hatching. (D) Dichorionic MZ twins result from early embryo splitting before the morula stage. (E) Looseness of the ICM can occur during (F) the multi-point initiation of cavitation and there is subsequent accumulation into a single-dominant blastocoel (inset). (G) The 8-shaped hatching blastocysts are likely to undergo ICM separation when the ICM is positioned near the hatching point.

Figure 2.

Formation of dichorionic monozygotic (MZ) twins. (A) The classic model of dichorionic MZ twin formation proposed that it occurs when blastomeres split before the morula stage. (B) The divided blastomeres of zona pellucida (ZP)-free mouse embryos at the two-cell stage can be separately cultured in U-shaped or V-shaped bottom wells, eventually developing into small blastocysts. (C) In an ART case, one of the blastomere emerged from the ZP through a breach at the two-cell stage, with each blastomere forming an individual blastocyst. (D) Dichorionic MZ twins can result from the separation of the ICM and trophectoderm during atypical 8-shaped hatching, forming two individual small blastocysts. (E) In certain species such as sheep, cattle, goat, and pig, embryos can be replicated by splitting a blastocyst into two halves using a sharp needle, each containing a similar number of ICM and TE cells. (F) Blastocyst separation was observed in an ART case in a vitrified-warmed cycle, leading to a dichorionic MZ twin pregnancy.

Figure 3.

Animals that can produce monozygotic (MZ) twins or quadruplets. (A) The zebrafish janus mutant exhibits the separation of two groups of blastomeres prior to the eight-cell stage, resulting in the development of two distinct spheres (yellow) that ultimately lead to the formation of conjoined fish. If there is limited space between the two groups of cells, the blastomeres and marginal zones (dark blue) will merge during development. (B) To generate MZ quadruplets, zygotic splitting of nine-banded armadillo embryos happens after implantation. The substantial enlargement of the exocelom cavity (blue) functions as a physical force, causing the division of the embryonic shields that develop from the epiblastic plate (yellow), and effectively separating them into distinct spaces that remain unconnected.