Abstract
Increased risk of monozygotic twinning (MZT) has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM) splitting in human ‘8’-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i) in vivo developed blastocysts and (ii–iii) in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that ‘8’-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P < 0.01). Moreover, Oct4 immunofluorescence staining was performed to identify the ICM in the hatching and hatched blastocysts. Scattered and split distribution of ICM cells was observed around the small zona opening of ‘8’-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in ‘8’-shaped hatching and hatched blastocysts than in ‘U’-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P < 0.01). Therefore, our results demonstrate that extended in vitro culture can cause high frequencies of ‘8’-shaped hatching, and ‘8’-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.
Highlights
Monozygotic twinning (MZT) is an uncommon phenomenon that occurs in about 0.4–0.45% of natural in vivo conceptions [1]
These results provide new insights into the origin of inner cell mass (ICM) splitting during blastocyst culture and may provide a potential explanation for the increased occurrence of human MZT when transferring in vitro-produced blastocysts
We found that the blastocysts that underwent ‘8’-shaped hatching struggled to achieve complete hatching due to the small zona opening
Summary
Monozygotic twinning (MZT) is an uncommon phenomenon that occurs in about 0.4–0.45% of natural in vivo conceptions [1]. The etiology of MZT is not clear. The use of assisted reproductive technology (ART) has been reported to increase the incidence of MZT by 2–12-fold compared with that of natural conception [2,3,4]. The factors that contribute to the increased frequency of MZT following ART are not entirely clear. When embryos are cultured to the blastocyst stage before embryo transfer, the incidence of MZT pregnancy increases dramatically [13, 14]. Extended embryo culture is one of the primary factors of ART most frequently associated with MZT
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