学会・論文発表

ヒト胚初期不規則分割と染色体解析(第22回生殖バイオロジー東京シンポジウム)

渡辺 真一

胚の初期不規則動態として、1細胞が短時間のうちに3細胞以上に分割するdirect cleavage(DC)が知られる。DCは染色体の異常分配を招き胚盤胞発生率を低下させるが、胚盤胞に到達すれば移植妊娠率は低下しないとの報告は多い。
当院で染色体解析を行った胚盤胞(研究使用の同意を得た廃棄胚)を初期分割様式によって分類したところ、DCが見られた胚盤胞の正倍数率は初期分割正常胚盤胞と同等であった。なぜそうなるのか? 理由を探るため、初期分割後の割球がそれぞれその後胚盤胞を形成しているか否かをタイムラプス動画で観察すると、第二分割まで正常な割球は9割以上が胚盤胞を形成していたのに対し、第二分割までのDCで形成された割球は約5割しか胚盤胞を形成しておらず、この「異常割球の除外」によって胚は正常性を保っていると考えられた(Watanabe et al. F&S Science 2023)。しかしDC胚はリペアのためのコストとして「除外される分の細胞量」を支払うことになり、発育速度と胚盤胞のグレードが低下する。DC胚盤胞の移植妊娠率は低いとする報告も散見されるが、凍結基準が緩い場合そうした結果となるかもしれない。
一旦分割した割球が融合するreverse cleavage(RC)にもしばしば遭遇するが、RCの胚発生能や妊孕能への影響についてコンセンサスは得られていない。我々の検討では、RCが見られた胚盤胞の正倍数率は初期正常分割胚盤胞よりも有意に高い、という結果が出て解釈に苦しんでいる。RCには、正常分割後の融合で4倍体を形成する場合と、DC後の融合があると考えられる。後者の場合、異常分割細胞が融合により「修復」されているかも知れないと考え、融合細胞の胚盤胞形成率を調べたが、そのような結果は得られなかった(2023受着)。現在のところRCは4倍体形成、もしくはDC後の無意味な挙動であると認識せざるを得ない。

Reverse cleavageは異常分割細胞の自己修復の意味を持つか?(第41回日本受精着床学会)

渡辺真一 鈴木篤智 冨田麻莉 松田有希野 吉貝香里 中野英子 澤田富夫

【目的】
胚の初期不規則動態として、一旦分割した割球が融合するReverse cleavage(RC)が知られているが、RCが胚発育や妊孕性にどのような影響を及ぼすかは意見の一致を見ていない。RCには正常分割後の融合とdirect cleavage(DC、1細胞が短時間で3細胞以上に分割する動態)後の融合があり、前者は4倍体形成を招くと考えられるが、後者は異常分割細胞が修復される可能性が考えられる。今回、DC由来割球と、DC後に融合した割球のその後の発育を比較し、RCの意義を検討した。

【対象と方法】
2020-2021年に採卵されEmbryoScope(Vitrolife)で5日間以上培養された835個の胚のうち、2細胞期となった後5時間以内に分割(以下早期分割)が起こり3細胞以上となった胚185個を対象とした。早期分割した割球について、その後融合しなかった場合をDC割球、融合した場合をRC割球として、それらのその後の発育を観察し、胚盤胞を形成したか否かを記録した(なお、有核の構造物を「割球」、完全に分割した割球が融合することを「RC」と定義した)。

【結果】
DC割球を持つ胚(DC胚)は174個、RC割球を持つ胚(RC胚)は11個であった。割球の胚盤胞形成率はDC割球21.6%、RC割球37.1%で有意差はなかったがRC割球が高い傾向が見られた。胚の良好胚盤胞(4BC以上)発生率はDC胚38.5%(67/174)、RC胚45.5%(5/11)で有意差はなかった。単一胚盤胞移植生産率はDC胚18.8%(3/16)、RC胚0%(0/1)であった。

【結論】
 早期分割した割球がその後融合することで割球の胚盤胞形成率が増加する傾向が見られたが有意差はなく、この融合が異常分割細胞の修復であることは示されなかったが、今後対象を増やすことで詳細を明らかにしたい。

The reason why direct cleavage and rapid cleavage should be differentiated(第39回ヨーロッパ生殖医学会(ESHRE))

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

Study question
Direct cleavage (DC) and Rapid cleavage (RaC) both appear to be 1 cell divided into 3 (or more) cells, should they be distinguished?

Summary answer
Since blastomeres by RaC had higher developmental potential than blastomeres by DC, and since many RaC embryos had “normal” blastomere, the two should be distinguished.

What is known already
DC, in which one cell divides into three (or more) cells without the two-cell stage, and RaC, in which one or two cells divide rapidly after the short two-cell stage (The former lead to segregation of chromosomes into three cells, while the latter leads to inadequate DNA replication), are both often recorded as “DC”, but when classified in detail, there are reports that the blastocyst development rate is higher in RaC embryos. However, the reason for this has not been clarified.

Study design, size, duration
This was a retrospective study of 643 embryos collected and cultured for at least 5 days in 2020 at our clinic. The embryos were time-lapse monitored by EmbryoScope (Vitrolife, Sweden) and classified into three groups according to the style of first division.

Participants/materials, setting, methods
The embryos were classified as follows: DC group: embryos that have divided into three (or more) cells without a two-cell stage; RaC group: embryos that rapidly (<5 hours) divided one (or both) cells after the 2-cell stage; Normal cleavage (NC) group: embryos that had a 2-cell stage for more than 5 hours.
The subsequent development of blastomeres of all embryos was observed and whether they participated in blastocysts or not was recorded.

Main results and the role of chance
Of the embryos, 63 were in the DC group and 199 were in the RaC group (173 of which had one rapidly dividing blastomere and 26 of which had two).
The blastocyst participation rate of blastomeres was 4.4% in the DC group, 19.5% of rapidly dividing blastomeres and 61.6% of normal blastomeres in the RaC group, with significant differences between all groups (p<0.01).
The good blastocyst (4BC or higher in Gardner grade) development rate was 4.8% (3/63) in the DC group, 40.5% (70/173) in the RaC group with one rapidly dividing blastomere (RaC1 group), 19.2% (5/26) in the RaC group with two such blastomeres (RaC2 group), showing a significant difference between the DC and RaC1 groups (P < 0.01).
The live birth rate for single blastocyst transfer was 50% (1/2) in the DC group, 27.8% (5/18) in the RaC1 group, and 100% (1/1) in the RaC2 group.
(Note that there were 381 in the NC group, and the above rates were 76.5%, 63.3% (241/381), and 29.3% (22/75), respectively)

Limitations, reasons for caution
This study examined only the style of first division and did not consider second and subsequent divisions.
In addition, as of 2020, the clinical use of PGT-A is not approved in principle in Japan, and the subject embryos have not undergone chromosome analysis.

Wider implications of the findings
The reason for the higher blastocyst development rate in RaC embryos was indicated by the difference in blastocyst participation rates of the respective blastomeres and the presence of normal blastomeres, but since live births were obtained in both embryos, so these irregular divisions would not completely compromise the blastocyst euploidy.

Early irregular division of human embryos changes in style between the first and second divisions(第39回ヨーロッパ生殖医学会(ESHRE))

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.

How does the short insemination method affect clinical outcomes and laboratory management?(第39回ヨーロッパ生殖医学会(ESHRE))

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.