Mari Tomida, Shinichi Watanabe, Shigenori Suzuki, Yukino Matsuda, Kaori Yoshikai,
Eiko Nakano, Tomio Sawada
Sawada Women’s Clinic, Nagoya, Japan

Study question
Is there a difference in irregular division kinetics between first and second division of human embryos?

Summary answer
The incidence of rapid cleavage was reduced in the second division, but the developmental potential of the blastomeres by this dynamics was unexpectedly low.

What is known already
The styles of irregular division of early human embryos are known: direct cleavage (DC), in which one cell becomes three (or more) cells without a two-cell phase, and rapid cleavage (RaC), in which one (or both) cells dividing rapidly after two-cell phase, both of which reduces the embryonic developmental potential. However, there are no reports that have examined in detail whether these division styles occur similarly in the first and second divisions, and how these dynamics affect the potential.

Study design, size, duration
This is a retrospective study of 635 embryos collected in 2020 at our clinic and cultured for at least 5 days. The embryos were time-lapse monitored by EmbryoScope (Vitrolife, Sweden) to observe the first and second divisions, and the blastomeres of each embryo were classified according to their style of division.

Participants/materials, setting, methods
It was observed whether there was a DC (division into 3 cells without a 2-cell phase) or RaC (One or two blastomeres divide within 5 hours after the 2-cell phase) at the first division. First dividing normal embryos were observed for the presence of a DC or RaC at the second division. Blastomeres by DC or RaC were observed for subsequent development and whether they participated in blastocysts or not was recorded.

Main results and the role of chance
Among the subject embryos, 63 embryos had DC and 199 embryos had RaC in the first division, and their blastomeres were classified into the DC1 and RaC1 groups, respectively. Of the 373 first division normal embryos, 39 had DC and 23 had RaC (there were 4 embryos with both DC and RaC) in the second division, and their blastomeres were classified into DC2 and RaC2 groups. The blastomeres blastocyst participation rate was 4.4% in the DC1 group and 19.5% in the RaC1 group, respectively, the former was significantly lower (P<0.01), however, in the DC2 and RaC2 groups, 23.3% and 4.2%, respectively, the latter was significantly lower (P<0.01). The incidence of DC was similar in the first (9.9%) and second (10.5%) divisions, but the incidence of RaC was 31.3% in the first division and 6.2% in the second division, the latter was significantly lower (P<0.01). The development rate of good blastocysts (≥4BC in Gardner grade) was 4.8% in embryos with DC1 and 19.5% in embryos with RaC1, with the former significantly lower (P<0.01), 37.0% in embryos with DC2, and 26.3% in embryos with RaC2, with no significant difference. Limitations, reasons for caution As of 2020, the clinical use of PGT-A is not approved in principle in Japan, and the subject embryos have not been chromosomally analyzed. Wider implications of the findings RaC would lead to inadequate DNA proliferation, but the incidence of this and the prognosis of the blastomeres were different in the first and second divisions. In human embryos, the mechanisms that control blastomeres from dividing may change as development progresses, but further studies are needed to elucidate this.

Yukino Matsuda, Shinichi Watanabe, Mari Tomida, Shigenori Suzuki, Kaori Yoshikai, Eiko Nakano, Tomio Sawada
Sawada Women’s Clinic, Nagoya, Japan

Study question
Does the short insemination method using a time-lapse monitoring system improve clinical outcomes and laboratory management?

Summary Answer
The method increased the normal fertilization rate of Conventional IVF and also increased the 3PN rate. The working environment of the laboratory staff was improved.

What is known already
Short insemination using a time-lapse monitoring system has been reported to reduce fertilization confirmation failures due to loss of pronuclei, but many reports indicate that the method was implemented to perform so-called ‘rescue ICSI’ for unfertilized oocytes on the day of egg retrieval and there are not many reports of clinical outcomes in conventional IVF without rescue ICSI. There are also few reports on the working environment and working hours in the laboratory.

Design
This was a retrospective study of 3714 oocytes from 715 cycles in 595 patients who underwent Conventional IVF with egg retrieval in 2016-2022.
Long insemination (1577 oocytes) was performed in cycles up to May 2019 and short insemination (2137 oocytes) in later cycles.
In IVF, 40,000 motile sperm were placed in 1 ml of culture medium per oocyte.

Materials and Methods
Oocytes of the long insemination group (group L) were inseminated 5 hours after egg retrieval and denuded 19 hours later to observe their pronuclei.
Oocytes from the short insemination group (group S) were inseminated 2 hours after egg retrieval, denuded 4 hours later, and cultured in EmbryoScope (Vitrolife, Sweden) to observe the pronuclei.
Since the working hours set at our clinic are 8：00 A.m.-5:00 p.m., the time schedule for both groups followed this schedule.

Result
The 2PN rate was 60.9% in group L and 64.7% in group S. The 3PN rates were 7.5% and 10.2% respectively, both rates being significantly higher in group S (P<0.05). The non-fertilization rates were significantly higher in group L (25.3% and 21.7% respectively); the 1PN rate was not significantly different (3.6% vs. 3.4%). The rate of missed pronuclei was 2.8% in group L and 0% in group S. Early embryo transfer pregnancy rate (25.4% vs 27.8%), good blastocyst development rate (52.2% vs 63.6%) and blastocyst transfer pregnancy rate (41.1% vs 31.7%) in groups L and S respectively. The good blastocyst incidence was significantly higher in group S. In the case of the Conventional IVF cycle in which egg retrieval was performed at 8:00 a.m., the embryologists in group L were to observe the pronuclei at 8:00 a.m. the next day, but in reality the embryologists had to come to work earlier because of denudation, and even then pronuclear loss occurred. In contrast, the group S did not require an earlier attendance because denudation was not performed the next morning, and the time-lapse images did not fail to confirm the pronuclei at all. Limitations, reasons for caution Since the short insemination method was introduced at our clinic in June 2019, this study compared clinical outcomes before and after that date. Wider implications of the findings It has been reported in the past that early insemination time increases both 2PN and 3PN rates. The short insemination method in this study also used a faster insemination time, which may have led to similar insemination results.

冨田　麻莉　渡辺　真一　鈴木　篤智　松田　有希野　吉貝　香里　中野　英子
澤田　富夫

【目的】

2022年4月から生殖補助医療(ART)への保険適用が開始され臨床成績への影響が注目されている。今回、当院で行った保険適用開始以前の自費診療と保険診療で行われたARTについて臨床成績の比較検討を行った。

【対象と方法】

2021年6月-2022年3月に自費診療で行ったART(自費群、254周期)、2022年4-12月に保険診療で行ったART(保険群、225周期)を対象として、成熟卵子獲得数、受精・培養・単一胚移植成績を比較した。

【結果】

平均年齢は自費群38.1歳、保険群36.1歳、採卵1回あたりの平均成熟卵子獲得数はそれぞれ4.8個、6.0個でいずれも有意差が見られた(P＜0.01)。IVFとICSIの正常受精率は自費群50.2%、78.1%、保険群54.7%、80.4%でいずれも有意差はなかった。胚盤胞凍結率(ガードナー分類≧4BC)は、39歳以下で自費群45.6%、保険群37.2%で有意差が見られ(P<0.05)、40-42歳ではそれぞれ25.0%、18.9%で有意差はなかった。初期胚移植妊娠率、流産率は39歳以下で自費群34.1%、13.8%、保険群34.9%、11.1%、40-42歳は自費群13.3%、50.0%、保険群14.3%、0%、胚盤胞移植妊娠率、流産率は39歳以下で自費群31.6%、28.0%、保険群42.9%、12.8%、40-42歳は自費群25.0%、50.0%、保険群46.7%、14.3%となり、いずれも有意差はなかったが、保険群の妊娠率が高い傾向にあった。 【結論】 保険診療によって受精成績、移植成績が低下することはなかった。39歳以下の胚盤胞凍結率に有意差が認められたが、4CCで凍結されなかった胚が自費群11.8％と保険群19.2％で保険群の方が有意に高く(P<0.05)、保険診療の移植回数制限により凍結基準を厳しくしたためと考えられた。これに関して有意差はなかったが、保険診療で胚盤胞移植妊娠率が高い傾向にあったことに関連している可能性が考えられた。

Shinichi Watanabe, Kaori Yoshikai, Yukino Matsuda, Shunsuke Miyai,

Yuki Sawada, Hiroki Kurahashi, Tomio Sawada

https://www.fertstertscience.org/article/S2666-335X(22)00075-1/fulltext

Abstract

Objective: To investigate whether blastocysts that divide irregularly reduce subsequent blastocyst euploidy.

Design: Retrospective study.

Setting: Private clinic.

Patient(s): 122 blastocysts for which consent for disposal and research use were obtained.

Intervention(s): None.

Main Outcome Measure(s): Results of NGS analysis of the blastocysts and whether blastomeres by normal or irregular divisions subsequently participated in blastocyst formation or not.

Results: The embryos were classified according to their dynamics until the second cleavage. The blastocyst euploidy was 33.3% (19/57) in the normal cleavage (NC) group, 38.3% (18/47) in the direct cleavage (embryos with one cell dividing into three cells) (DC) group, and 72.2% (13/18) in the reverse cleavage (embryos with fused cells once divided) (RC) group. The rate of the RC group was significantly higher than that of the NC (P<0.05).

The blastocyst participation rate of the blastomeres were 95.6% in the NC group and 56.5% in that derived from DC of the first cleavage, and 91.7% in that of blastomeres derived from normal division of the second cleavage and 53.6% in that derived from DC of the second cleavage, both of which were significantly lower in the latter (P<0.01), and in the RC group, the rates of fused and non-fused blastomeres were 62.1% and 87.5%, respectively, with no significant difference.

Conclusion: The blastomeres generated by DC were often excluded from blastocyst formation, and we speculate that this is one reason why their division does not reduce blastocyst euploidy. The association between RC and euploidy of blastocysts merits further study.

渡辺真一　松田有希野　冨田麻莉　鈴木篤智　吉貝香里　中野英子　澤田富夫

【目的】

Direct cleavage（1細胞が3細胞以上に分割する現象、以下DC）を呈した胚は染色体分配異常により発生能が低下するが、そのような胚でも胚盤胞に発育し、健児が得られる可能性があることも知られている。その理由の一つとして、DCには2細胞期を短時間経た後の3分割（以上）と、2細胞期を経ない直接的な3分割（以上）があり、前者では妊孕性が保たれている可能性が報告されている。今回、第一分割DCを詳細に分類し、分割様式の違いによる胚発生能の差異を検討した。

【対象と方法】

2020年1-4月にEmbryoScope（Vitrolife）を用いて胚盤胞培養され、第一分割で3細胞以上に分割した胚のうち、2細胞となってから早期（5時間以内）に片方の割球が2個以上に分割した胚（A群：44個）と、2細胞期を経ず3細胞以上に分割した胚（B群：23個）について、それぞれの割球がその後胚盤胞を形成したか否かを記録した。

【結果】

割球の胚盤胞形成率は、A群の早期分割割球21.4%、非早期分割割球59.3%で後者が有意に高かった（P<0.01）。B群は3.6％で、A群の2種の割球のいずれと比較しても有意に低かった（P<0.01）。

なお、A、B群の良好胚盤胞（4BC以上）発生率は43.2%（19/44）、4.3%（1/23）でA群が有意に高く（P<0.01）、単一胚移植での生産率は33.3%（1/3）、0%（0/1）であった。

【結論】

2細胞期を短時間経た後に3分割以上となった胚では、早期分割しなかった割球が胚盤胞発生能を保持しており、染色体正常である可能性が示唆された。同様の胚で早期分割した割球、および2細胞期を経ず直接3分割以上となった割球は多くが胚盤胞を形成せず、染色体分配異常が示唆された。これらの分割は全てDCと評価されることが多いが、分類すべきと考えられた。