In this study, the overall MZT rate was 2.46% of 9229 clinical pregnancies after single fresh and frozen-thawed blastocyst transfer in our center, which was generally in line with previous reports . The primary finding was the association between the risk of MZT and TE grade. Blastocysts with higher grade TE are more susceptible to resulting in MZT pregnancies. However, we failed to find the relation between MZT and embryo cryopreservation (fresh vs frozen-thawed ET), AH, and extended culture (day 5 vs day 6), compared to prior reports [15-17, 20]. Differences of these results might be caused by heterogeneity among these IVF centers, including the proportion of fresh and frozen-thawed cycles, blastocyst quality, and IVF-related laboratory manipulations, such as indications of AH and culture media.
Franasiak et al. found that the incidence of MZT did not increase after transfer at blastocyst stage compared with cleavage stage ET when controlling the quality of embryo cohort, suggesting embryo characteristics might contribute to MZT rates. Prior studies on blastocyst morphology were mainly conducted to evaluate the quality and their predictive value for clinical outcomes. Otsuki et al.  observed 71 transferred frozen-thawed blastocysts by time-lapse observations and originally noted the hypothesis that compared with a blastocyst with tightly packed ICM cells, loosely grouped one was more conductive to splitting. Since then, the inner link of inherent blastocyst features and embryo splitting began to be discussed.
In the study examining the effects of hyaluronan-enriched transfer medium on MZT pregnancies after single frozen embryo transfer , they reported no association between embryo expansion, TE, ICM grade and the risk of MZT, which contradicted ours. All MZT embryos underwent PGT and part of them were cultured in a hyaluronan-enriched transfer medium, whereas PGT cycles were excluded in our cohorts. Nevertheless, TE grade was indicated a significant independent factor affecting the occurrence of MZT (A vs C: aOR 5.46, 95%CI 1.48-20.16, P=0.011; B vs C: aOR 3.96, 95%CI 1.17-13.40, P=0.027), but not the arrangement of ICM cells in the study including 2863 pregnancies after frozen-thawed SBT . Combining the differences in serum hCG after ET between the MZT and singleton, they hypothesized that higher grade TE which was thought to be more well-developed might be associated with monozygotic splitting, potentially by increasing secretion of hCG. Our results agreed with their findings.
Meanwhile, in the study including 26254 clinical pregnancies after SBT from 4 centers conducted by Shi et al. , 402 pregnancies with sex concordance at birth were identified as MZT. Blastocysts with grade A ICM and grade B or C TE showed the lowest MZT rate, while counterparts with grade B or C ICM and grade A TE presented the highest. Blastocysts with lower grade ICM (B or C) and higher-grade TE (A) were with higher risk of MZT (aOR 2.62, 95%CI 1.60-4.43). They proposed a new hypothesis based on Otsuki's theory , which was that when loosely packed ICM cells might be more susceptible to splitting, higher grade TE with more number tightly arranged cells would provide further support in the development of splitting ICM resulting in MZT pregnancies.
Influenced by prior studies about the predictive power of blastocyst morphology for clinical outcomes [31-34], blastocysts with ICM score “C” are not routinely selected to transfer in our center for the higher efficiency of fertility treatment. Thus, our cohort included SBT cycles only with A and B grade ICM. Only TE grade was found to be associated with MZT, but not ICM grade. It could be related to the differences in the definition of MZT pregnancy, laboratory techniques and embryo culture system among different IVF centers. Our results were consistent with the conclusions about the role of TE grading in increasing MZT rate after IVF/ICSI of above reports considering blastocyst morphology. In addition, factors related to the susceptibility of ICM division were not found, and further scientific research is needed to explore the specific mechanism of ICM splitting.
The large sample size including 9229 clinical pregnancies after SBT from a single center is the main strength of this study. Although the assessment scores of blastocyst morphology based on Gardner scoring system were indeed influenced by subjectivity, our data was collected from a single center and evaluated by experienced embryologists followed by standard operating procedure, which has much reduced information bias. And there is a lack of more objective criteria for choice currently. Our study also has some limitations. First was the observational and retrospective nature. Second, data about ovarian stimulation procedures and fertilization methods of frozen-thawed cycles did not be recorded in our current CCRM/EMRCD. Therefore, the association between insemination methods and MZT occurrence was not analyzed in this study. Third, MZT pregnancy was mainly defined according to the presence of GSs and FHBs via ultrasound, but not confirmed with sex concordance at birth and the gold criteria of DNA profiling for zygosity, which might overlook the possibility of a concurrent natural conception, although this is a rare event.