title: Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture.
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.
authors.affiliation: Department of Assisted Reproduction, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011, PR China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Yan
authors.affiliation: Guangxi High Education key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530004, PR China.
authors.forename: Hongxing
authors.initials: H
authors.lastname: Liang
authors.affiliation: National Laboratory of Medical Genetics, Xiangya Hospital, Central South University, Changsha, 410078, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Deng
authors.affiliation: Department of Assisted Reproduction, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Long
authors.affiliation: Department of Assisted Reproduction, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011, PR China.
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Assisted Reproduction, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011, PR China.
authors.forename: Weiran
authors.initials: W
authors.lastname: Chai
authors.affiliation: Department of Assisted Reproduction, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011, PR China.
authors.forename: Lun
authors.initials: L
authors.lastname: Suo
authors.affiliation: Shanghai Key Laboratory of Reproductive Medicine, Shanghai 200025, PR China.
authors.forename: Chen
authors.initials: C
authors.lastname: Xu
authors.affiliation: Department of Assisted Reproduction, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011, PR China.|Shanghai Key Laboratory of Reproductive Medicine, Shanghai 200025, PR China.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Kuang
authors.affiliation: National Laboratory of Medical Genetics, Xiangya Hospital, Central South University, Changsha, 410078, PR China.
authors.forename: Lingqian
authors.initials: L
authors.lastname: Wu
authors.affiliation: Guangxi High Education key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530004, PR China.
authors.forename: Shengsheng
authors.initials: S
authors.lastname: Lu
authors.affiliation: Department of Assisted Reproduction, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011, PR China.
authors.forename: Qifeng
authors.initials: Q
authors.lastname: Lyu
mesh_terms: D000818:Animals; D053624:Blastocyst Inner Cell Mass / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D002455:Cell Division; D002478:Cells, Cultured; D046149:Embryo Culture Techniques / Q000379:methods*; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000378:metabolism
chemical_list: D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse