in vitro fertilization of egg cell man_at_mouse/iStock/Thinkstock

N&H TOPLINE: Live-cell imaging improves selection of viable bovine IVF embryos

Live-cell imaging technique could be useful for analyzing bovine IVF embryos' chromosomal dynamics and development to help increase pregnancy success rate.

Scientists in Japan have demonstrated a new technique for improving the selection of embryos for transfer. Embryo transfer via in vitro fertilization (IVF) has become an important innovation in sectors of agriculture like cattle production. Approximately half of all bovine embryos produced worldwide were derived from IVF. However, the pregnancy success rate of IVF embryos transplanted into recipients remains low.

To increase the chances for pregnancy success, key technological issues affecting the in vitro production of embryos and the assessment of viable embryos must be addressed, according to a news release from Tokyo University of Agriculture & Technology in Japan. This is also true for human artificial reproductive technology (ART).

Generally, the assessment of bovine embryo quality is performed by morphological grading on days 7-8 post-insemination, as recommended by the International Embryo Technology Society (IETS), but the pregnancy success rate of embryos judged as transferable is only 30-50%. Hence, there is a need for novel technology for non-invasively and reliably selecting viable IVF embryos.

Research groups in Japan led by Dr. Satoshi Sugimura at Tokyo University of Agriculture & Technology, Dr. Kazuo Yamagata at Kindai University, Tatsuma Yao with FUSO Pharmaceutical Industries Ltd. and Dr. Satoko Matoba with NARO have reported successful non-visible selection of bovine IVF embryos without chromosomal abnormalities by long-term "live-cell imaging" with fluorescence confocal microscopy. Their results were published in the journal Scientific Reports.

The chromosomal abnormality was detected by injection of messenger RNA encoding histone H2B-mCherry and EGFP-alpha-tubulin. The live-cell imaging revealed that about half of embryos judged as morphologically transferable by IETS criteria had nuclear/chromosomal abnormalities, such as an abnormal number of pronuclei (PN) and abnormal chromosome segregation, which may lead to abortion. All but two recipients that received embryos without any nuclear/chromosomal abnormalities got pregnant. The method is expected to improve the pregnancy success rate by selecting bovine IVF embryos with the live-cell imaging.

Research, experiment details

In this study, the researchers succeeded in performing non-invasive, long-term, live-cell imaging of bovine IVF embryos with fluorescence confocal laser microscopy. This technique allowed for visualization of the nuclear/chromosomal dynamics of bovine embryos for eight days and determination of various biological factors involved in the relationship between nuclear/chromosomal abnormalities and subsequent in vitro embryonic development and morphological embryo quality.

In human ART, the number of pronuclei (PN) is the most important prerequisite for predicting the developmental competence of embryos. However, the lipid-rich dark cytoplasm of bovine embryos has inhibited observation of PN. In the present study, the researchers found that embryos with an abnormal number of PN had impaired embryo development compared with embryos with two PN.

Abnormal chromosome segregation (ACS) during in vitro embryonic development was demonstrated as a promising indicator of embryo viability in mouse ART. When okadaic acid induced severe ACS in bovine IVF embryos, subsequent development decreased. ACS was also observed during the first cleavage in embryos under normal bovine IVF conditions, which exhibited lower blastocyst competence as well.

It has been well documented that the delayed timing of first cleavage is involved in blastocyst formation and pregnancy success. The Japanese researchers also observed a relationship between delayed timing of first cleavage and ACS.

A previous study reported that embryos that cleaved directly into three to four cells (multi-division) at first cleavage have a high incidence of chromosomal abnormalities and a low viability after transfer. The present study confirmed that a multi-PN number was involved in multi-division.

The lag-phase, which occurs in the fourth or fifth cell cycle in which the longer Gap 2 phase is inserted, corresponds to embryonic genome activation in cattle. Previously, the researchers showed that a small number of blastomeres at the lag-phase was related to a higher incidence of apoptosis in blastocysts. Live-cell imaging revealed a relationship between ACS and a low blastomere number at the lag-phase. It has been reported that DNA damage, such as double-strand breaks, may cause ACS and induce apoptosis in embryos, which could be a reason for the high incidence of apoptosis observed.

Scientific Reports

Schematic representation of experimental design from Tokyo University of Agriculture Technology, Tokyo, Japan, of embryos collected from bovine ovaries and evaluated by live-cell imaging. In vitro fertilization was conducted for six hours; mRNA was injected into oocytes, and live-cell imaging was performed for eight days. The number of pronuclei, timing, chromosome segregation, cytokinesis at first cleavage, number of blastomeres at the end of first cleavage and number of blastomeres at the onset of lag-phase were retrospectively observed.

In this study, nuclear/chromosomal abnormalities, such as ACS and an abnormal number of PN, were observed in embryos that were graded as morphologically transferable, regardless of IETS criteria and morphokinetic indicators (MKIs). Thus, it may be difficult to judge nuclear/chromosomal abnormalities based on morphological evaluation. Indeed, karyotyping of blastocysts revealed that 14.3% of in vivo embryos and 12.5% of embryos selected by live-cell imaging contained mixoploids, whereas embryos selected by IETS and MKIs contained mixoploids at 42.9% and 27.6%, respectively. Interestingly, since multi-PN embryos were not included in the MKI-selected embryos, monitoring the embryo development with time-lapse cinematography instead of the morphological “snapshot” evaluation used in accordance with IETS criteria may reduce the risk of selecting embryos with multi-PN, which are clinically discarded from transferrable embryos in human ART.

In conclusion, this live-cell imaging technique could be useful for analyzing the association between nuclear/chromosomal dynamics and embryo development in bovine embryos whose nuclei/PN are not observable by visible light microscopy, the news release said.

The research was funded by the JSPS KAKENHI and Itokinen Research Foundation.

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