Purpose To examine how differences in trophectoderm biopsy techniques affect the frequency of mosaic embryos and sequencing results. Methods We examined differences in next‐generation sequencing (NGS) analysis results among operators or according to biopsy technique. Additionally, we determined the cut‐off for the number of collected cells to predict the occurrence of mosaicism. We collected cells according to the cut‐off value and examined whether there was a difference in the NGS analysis results between the pulling and flicking methods. Results There was no difference in the NGS analysis results among the operators. Regarding re‐biopsy, changes in the mosaic were observed in all specimens. The cut‐off value for the number of collected cells was five, and when more than five cells were collected, there was no difference in the NGS analysis results between the two methods. Conclusions We demonstrated that if trophectoderm biopsy techniques and NGS are stable, the cell collection location has a greater effect on NGS results than the biopsy technique.

Purpose To compare the clinical outcomes of embryo transfers that were cryopreserved and thawed two or three times with those cryopreserved and thawed once by CryoTip. Methods Data for 388 single cryopreserved‐thawed blastocyst transfer cycles, performed from April 2012 to March 2014, were assessed. The blastocysts were classified into three groups: blastocysts (A) cryopreserved once, (B) cryopreserved twice, and (C) cryopreserved three times. Results The pregnancy rate was 43.8% (134/306) in group A and 46.3% (38/82) in group B, while the miscarriage rate was 29.1% (39/134) in group A and 23.7% (9/38) in group B. The rate of improvement/maintenance of blastocyst grade was 84.0% (257/306) in group A and 80.5% (66/82) in group B. The pregnancy and miscarriage rates of the blastocysts that showed improvement/maintenance in the grade were 45.9% (118/257) and 29.7% (35/118) in group A and 48.5% (32/66) and 21.9% (7/32) in group B, respectively. The pregnancy rate was 33.3% (2/6), while the miscarriage rate was 0.0% (0/2) in group C. Conclusions Pregnancy rates achieved with re‐cryopreserved and rethawed blastocyst transfer were comparable to those achieved with single cryopreserved‐thawed blastocyst transfer.

Purpose This study was conducted to assess the effectiveness of a new trophectoderm (TE) biopsy method that does not require prior opening of the zona pellucida at the blastocyst stage. Methods TE biopsy was conducted using a modified extrusion method for embryos during the cleavage stage. In this method, culture medium was injected into the perivitelline space to help extrude TE cells from the zona pellucida before TE biopsy. Results Our extrusion method preserves the embryo culture environment until immediately before biopsy because it does not require opening of the zona pellucida prior to TE biopsy. Furthermore, this method does not require a waiting time for blastocyst hatching after laser irradiation, thereby minimizing damage to the embryos and maintaining the time schedule of culture operations. Conclusions TE biopsy using this novel extrusion method may be useful in various applications, including the collection of TE cells for next‐generation sequencing analysis.

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In preimplantation genetic testing for aneuploidy, various opinions exist regarding the handling of mosaic embryos. This study aimed to investigate the impact of freeze-thawing, number of cells obtained, and number of laser irradiation cycles on the degree of embryonic mosaicism. There were 3 parts to the study. First, we classified specimens into thaw-biopsy (TB) (17 patients, 49 blastocysts) and nonthawed (B) (119 patients, 304 blastocysts) groups. In Study I, groups TB and B were classified into four categories (euploidy, low mosaic, high mosaic, and aneuploidy) according to next-generation sequencing (NGS) results, and the number of cells collected and laser irradiation cycles were compared for each category. In Study II, the effects of differences in the number of cells collected and laser irradiation cycles on NGS results were investigated in groups TB and B. Lastly, in Study III, the data on cell collection and laser irradiation cycles and NGS analysis results were compared between groups. In Studies I and II, no intergroup differences were observed. In Study III, group TB had a significantly higher incidence of chromosomal mosaicism than group B. Interestingly, group TB showed a significantly higher rate of low degree mosaicism than group B. Neither the number of cells collected norlaser irradiation cycles affected the percentage of chromosomal mosaicism. However, the freeze-thaw process increased the occurrence of mosaicism.

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