JP3693907B2 - Noninvasively quality evaluation method and apparatus for mammalian embryo - Google Patents

Noninvasively quality evaluation method and apparatus for mammalian embryo Download PDF

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JP3693907B2
JP3693907B2 JP2000312882A JP2000312882A JP3693907B2 JP 3693907 B2 JP3693907 B2 JP 3693907B2 JP 2000312882 A JP2000312882 A JP 2000312882A JP 2000312882 A JP2000312882 A JP 2000312882A JP 3693907 B2 JP3693907 B2 JP 3693907B2
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mammalian embryo
embryo
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single mammalian
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JP2002122568A (en
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博昭 大矢
宏良 星
智一 末永
仁 珠玖
卓夫 白石
宏之 阿部
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独立行政法人科学技術振興機構
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【0001】 [0001]
【発明の属する技術分野】 BACKGROUND OF THE INVENTION
本発明は、哺乳動物胚の無侵襲的品質評価方法及びその装置に関するものである。 The present invention relates to non-invasive quality evaluation method and apparatus of the mammalian embryo.
【0002】 [0002]
【従来の技術】 BACKGROUND OF THE INVENTION
近年、培養技術の目覚ましい進歩により、多くの哺乳動物胚(マウス、ラット、ハムスター、ウサギ、ヤギ、ヒツジ、ブタ、ウシ、ヒトなど)の体外培養が可能となってきた。 Recently, the remarkable advances in culture techniques have become many mammalian embryo (mouse, rat, hamster, rabbit, goat, sheep, swine, cattle, humans, etc.) and can extracorporeal culture. 体外培養された胚はレシピエント(雌仮腹)の子宮に移植され、産仔の誕生も可能となっている。 Vitro cultured embryos are implanted into the uterus of the recipient (female surrogate), it has become a possible birth of offspring.
【0003】 [0003]
しかしながら、作出した胚が生存し、しかも正常に発生が進行しているかどうかの判断基準は、一般的に形態観察のみに基づいている。 However, determination of whether the reference produced embryos survived, moreover normally generated is in progress, only the basis of general morphological observation. このような形態的評価では、低品質と判定した胚が移植後に受胎したり、高品質と判定した胚が受胎しない例が報告されている〔F. In such form assessment, embryos judged low quality or conception after transplantation, embryos determined that high quality have been reported an example in which no conception [F. Shea,Theriogenology,15,31−42(1981)〕。 Shea, Theriogenology, 15,31-42 (1981)]. また、形態的評価法と胚の生存性との間に相関関係が見られないことも報告されている〔D. It has also been reported that show no correlation between morphological evaluation method and embryo viability [D. S. S. Visser et al. Visser et al. ,Hum. , Hum. Reprod. Reprod. ,8,1719−1722(1993)〕。 , 8,1719-1722 (1993)]. これらのことは、形態的観察による胚評価法だけでは、必ずしも正確に胚の生存性や品質を判断できないことを示唆している。 These things alone embryo evaluation method according to morphological observation, suggesting that not always accurately determine the survival and quality of embryos.
【0004】 [0004]
胚の品質評価において、形態的評価法の他に、胚のグルコースやアミノ酸代謝活性を指標とした品質評価法の試みも行われてきた〔D. In the quality evaluation of the embryo, in addition to the morphological evaluation method, it has also been made attempts quality evaluation method as an index of glucose and amino acid metabolism activity of embryos [D. K. K. Gardner and H. Gardner and H. J. J. Leese,J. Leese, J. Exp. Exp. Zool. Zool. ,242,103−105(1987);M. , 242,103-105 (1987); M. Rondeau et al. Rondeau et al. ,Theriogenology,44,351−366(1995)〕。 , Theriogenology, 44,351-366 (1995)].
【0005】 [0005]
これらの方法では、計測にラジオアイソトープを用いなければならなかったり、複数個以上の胚が必要であるなどの欠点があった。 In these methods, or had to use radioisotopes in measurement, there is a drawback such as is required plurality or more embryos. 一方、細胞や胚などの代謝活性や特異的な生体反応を評価する手法として、少数の細胞数でも無侵襲に計測可能な電気化学的手法が注目されつつある。 On the other hand, as a method of evaluating the metabolic activity or specific biological reactions such as cells and embryos, while the electrochemical method capable of measuring the non-invasive in number few cells are noted there.
【0006】 [0006]
以下、その関連技術について説明する。 The following describes related art.
【0007】 [0007]
(1)クラーク型酸素電極をはじめとする、精密位置決め機能を有さない固定型/通常サイズの電極が細胞呼吸活性評価に広く適用されている。 (1) including Clark-type oxygen electrode, the electrode of the fixed / normal size are widely applied to cellular respiration activity evaluation without precise positioning function. 空間分解能を有さない従来型の酸素電極を工夫することにより、培養したままリアルタイムで細胞呼吸活性を計測可能な装置は既にある。 By devising a conventional oxygen electrode having no spatial resolution, measuring apparatus capable cellular respiration activity in real time while culturing is already. ウシ初期胚に関して、通常サイズのクラーク型酸素電極を用いて、単一の胚盤胞期胚の酸素消費量を定量した報告がある〔E. Respect bovine embryos, using a Clark-type oxygen electrode of normal size, reported there was quantified the oxygen consumption of a single blastocyst stage [E. W. W. Overstorm et al. Overstorm et al. ,Theriogenology,37,269(1992)〕。 , Theriogenology, 37,269 (1992)]. 胚盤胞期胚において、形態的に正常/異常に分類した2グループ間で、正常胚のグループが酸素消費量も多く、その後の胚発生能も高かったと報告している。 In blastocysts, have reported between morphologically normal / abnormal classified two groups, a group of normal embryo oxygen consumption much, even higher subsequent embryonic developmental competence. しかしこれは明らかに形態観察のみで判定可能な基準であり、酸素消費量計測の意義は極めて小さい。 But this is determinable reference only clearly morphological observation, the significance of oxygen consumption measurement is extremely small.
【0008】 [0008]
この手法は、桑実胚と胚盤胞期胚の酸素消費量の差を検出していない。 This approach does not detect the difference in oxygen consumption of morula and blastocyst stage embryos. しかも空間分解能を有さない計測法は、胚表面の濃度が場所により異なる場合に局所の濃度を正確に検出できない。 Moreover measurement method having no spatial resolution, can not be detected accurately the concentration of topically when the concentration of the embryo surface is different depending on the location. したがって、局所の濃度差情報を胚の品質判定に利用できないという欠点がある。 Therefore, there is a disadvantage that the concentration difference information of the local unavailable quality determination of the embryo.
【0009】 [0009]
(2)電気化学顕微鏡(Scanning electrochemical microscopy)は、精密位置決め機能を有し、マイクロ(微小)電極を探針とする計測装置であり、探針を試料表面に近接し走査することにより、試料の放出/吸収する分子種の検出や、分子種の分布の画像化を可能にする〔A. (2) Electrochemical Microscopy (Scanning Electrochemical microscopy) has a precision positioning function, a measuring device for the probe micro (micro) electrodes, by scanning proximate the probe to the sample surface, the sample detection and molecular species to release / absorption, to allow imaging of molecular species distribution [A. J. J. Bard et al. Bard et al. ,Anal. , Anal. Chem. Chem. ,61,132−138(1989);R. , 61,132-138 (1989); R. C. C. Engstrom et al. Engstrom et al. ,Anal. , Anal. Chem. Chem. ,58,844−848(1986)〕。 , 58,844-848 (1986)].
【0010】 [0010]
球状の単一細胞が特定の分子種を放出/吸収する際に、細胞近傍に形成される濃度分布を電気化学顕微鏡類似の装置で直接計測した研究が既にある。 When a single cell of the spherical releasing / absorbing a particular molecular species, research is already obtained by directly measuring the concentration distribution formed in the cell near the electrochemical microscope similar device. マイクロ電極を細胞表面に向かって近接/遠ざけることにより計測した電流プロファイルから、細胞表面濃度と細胞から十分離れた位置の濃度の差(ΔC)が求められている。 The microelectrode from current profile measured by the proximity / away towards the cell surface, the difference in concentration of a position sufficiently away from the cell surface concentration and the cells ([Delta] C) is required.
【0011】 [0011]
さらに、濃度の差(ΔC)より、試料1個あたりの特定分子種放出/吸収速度が定量されている〔T. Furthermore, from the difference in density ([Delta] C), a specific molecular species release / absorption rate per one sample is quantified [T. Yasukawa,et al. Yasukawa, et al. ,Biophys. , Biophys. J. J. ,76,1129−1135(1999)〕。 , 76,1129-1135 (1999)]. この方法で、酸素還元電流プロファイルを計測し、生体からの酸素放出/吸収量を定量することも可能となっている 〔T. In this way, the oxygen reduction current profiles measured, has become possible to quantify the oxygen release / absorption from biological [T. Yasukawa et al. Yasukawa et al. ,Denki Kagaku,66,660−661(1998)〕。 , Denki Kagaku, 66,660-661 (1998)]. しかし、この方法で体外培養胚の酸素消費量の定量化に成功した例は報告されていない。 However, examples of successful quantification of oxygen consumption vitro cultured embryos have been reported in this way.
【0012】 [0012]
(3)自己参照電極法(Self−referencing ion−selective microelectrode/Self−referencing polarographic oxygen−selective microelectrode)は、電気化学顕微鏡類似の計測装置である〔P. (3) self-reference electrode method (Self-referencing ion-selective microelectrode / Self-referencing polarographic oxygen-selective microelectrode) is an electrochemical microscope similar measuring device [P. J. J. S. S. Smith et al. Smith et al. ,Microsc. , Microsc. Res. Res. Tech. Tech. ,46,398−417(1999);S. , 46,398-417 (1999); S. C. C. Land et al. Land et al. ,J. , J. Exp. Exp. Biol. Biol. ,202,211−218(1999)〕。 , 202,211-218 (1999)]. 電極の位置を周期0.3Hz振幅10μm程度で振動させ、電流信号をロックイン検出することにより高感度化が図られている。 The position of the electrode is vibrated at about cycle 0.3Hz amplitude 10 [mu] m, high sensitivity is achieved by lock-in detection of the current signal.
【0013】 [0013]
この方法の欠点として、検出原理や出力信号の解釈が複雑であること、また、計測に余分な時間を要することが挙げられる。 The drawback of this method, it is interpreted the detection principle and the output signal is a complex, also include it takes extra time measurement.
【0014】 [0014]
この方法は、本来、イオン選択性電極を探針とする電位感応型の計測法であるが、近年では微小酸素電極を探針として電流検出も行っている。 This method is originally a potential-sensitive measuring method of the probe the ion-selective electrode, in recent years have done also current detect small oxygen electrode as the probe. 最近、自己参照電極法がマウス体外培養胚の酸素消費量の定量に適用された〔J. Recently, a self-reference electrode technique was applied for the determination of oxygen consumption in mice vitro cultured embryos [J. R. R. Trimarchi et al. Trimarchi et al. ,Biol. , Biol. Reprod. Reprod. ,62,1866−1874(2000)〕。 , 62,1866-1874 (2000)]. この報告では自己参照電極法が無侵襲であることに言及し、計測に供した胚がその後も正常に発生することも確認している。 In this report we mention that the self reference electrode method is noninvasive, embryos were subjected to the measurement is also confirmed that thereafter develop normally. しかし、酸素消費量と発生率の関係については相関が得られなかったと明言している。 However, it has declared that correlation was not obtained for the relationship between oxygen consumption and incidence. つまり、彼らの用いた計測法では、胚の酸素消費量からその後の発生過程を予測する基準を設定することができていない。 That is, in their use measurement methods have not been able to set a reference to predict the subsequent developmental processes from oxygen consumption of the embryo. さらに、彼らは、マウス初期胚の酸素消費量計測に際し胚細胞を包む透明帯の除去を施しており、このような損傷を与えた胚は継続して培養する目的に適さない。 Moreover, they are subjected to removal of the zona pellucida surrounding the embryo cells upon oxygen consumption measurement of early mouse embryos, embryos gave such damage is not suitable for the purpose of culturing continued.
【0015】 [0015]
(4)ヒトの体外培養胚の評価法として、個々の胚につき形態観察と酸素消費量の対応付けを試みた研究例がある〔C. (4) as an evaluation method of in vitro cultured human embryo, there is a study example in which an attempt to associate individual embryos per Morphology and oxygen consumption [C. Magnusson et al. Magnusson et al. ,Hum. , Hum. Reprod. Reprod. ,1,183−184(1986)〕。 , 1,183-184 (1986)]. 形態観察において品質の劣った胚と正常胚との間には、酸素消費量に有為な差が見られた。 Between the high and normal embryos inferior quality in morphological observation, significance difference was observed in oxygen consumption. しかしながら、形態観察で正常と判別された胚の中では、酸素消費量とその後の胚発生率の関係において相関性は見られなかった。 However, among the embryos is determined to be normal in morphological observation, the correlation in relation to oxygen consumption and subsequent embryo development rate was observed. また、酸素消費量計測の原理は、オキシヘモグロビンがヘモグロビンに変換する時の吸収スペクトル強度変化に因るものであり、空間分解能は期待できない。 Further, the principle of oxygen consumption measurement is due to the absorption spectrum intensity changes when oxyhemoglobin is converted to hemoglobin, the spatial resolution can not be expected. さらに、計測は、検出試薬の存在下でしかも、低酸素濃度で行う必要があり、胚を損傷する可能性も否定できない。 Furthermore, measurements, moreover in the presence of detection reagent should be conducted in a low oxygen concentration, undeniable possibility of damaging the embryo.
【0016】 [0016]
(5)計測技術が空間分解能を有する利点は、上記(1)で述べた「局所の濃度差情報を胚の品質判定に利用できる」他に、「試料近傍に形成される酸素濃度勾配を直接計測できる」、「複数の試料でも画像化等により1本のプローブで定量的計測が可能である」、「任意に電極の位置を胚から十分離れた位置に移動し、ベースラインの濃度を参照できる」こと等が挙げられる。 (5) The advantage of measuring technique has a spatial resolution, the mentioned (1) "the local concentration difference information of the available quality judgment embryo" to another, an oxygen concentration gradient formed in the "near sample directly measurement can be ", by" imaging like in a plurality of samples are possible quantitative measurements in one probe "," the position of the arbitrarily electrode moves to a position sufficiently away from the embryos, see the concentration of baseline It can "be, and the like.
【0017】 [0017]
【発明が解決しようとする課題】 [Problems that the Invention is to Solve
(1)現行では形態観察に頼るところが大きい胚の品質評価に際し、1個1個の胚毎に代謝活性を数値化し情報を加えることにより、品質評価の精度向上を実現する。 (1) upon the quality evaluation of the embryo it is largely relying on morphological observation in the current, by the addition of one single quantify metabolic activity per embryo information, to improve the accuracy of the quality evaluation.
【0018】 [0018]
(2)代謝活性を評価する指標として、単一胚毎の酸素消費量を計測する。 (2) as an index for evaluating the metabolic activity, to measure the oxygen consumption of each single embryo. ただし、胚の代謝活性を正確に把握するために、計測は培養条件あるいはそれに限り無く近い条件で行われる必要がある。 However, in order to accurately grasp the metabolic activity of the embryo, the measurement has to be carried out under conditions as close as possible to it or culture conditions. さらに、測定後の胚を利用するために、計測は短時間にかつ無侵襲で行われる必要がある。 Furthermore, in order to utilize the embryo after the measurement, the measurement has to be performed in a short time and non-invasive. また、局所の濃度差情報を品質判定に利用するために、空間分解能を有する計測装置の適用が望ましい。 In order to use the concentration difference information of the local quality determination, application of the measuring device with a spatial resolution is desired. そのような条件を満たす計測手段として、例えば電気化学顕微鏡は適用可能性が高い。 Such conditions are satisfied measuring means, for example, an electrochemical microscope has a high applicability.
【0019】 [0019]
(3)しかしながら、個々の動物種により胚の発生過程、代謝活性、培養条件は大きく異なるため、実用可能性を示すためには、正常な胚がたどる発生過程をあらかじめ把握しておくことが不可欠である。 (3) However, the process embryonic development by the individual animal species, metabolic activity, differ culture conditions large, in order to show the feasibility is essential to advance understand developmental processes normal embryo followed it is.
【0020】 [0020]
(4)電極位置決め装置付きの微小電極や電気化学顕微鏡類似の計測装置を生存した細胞に対して適用した例は既にある〔T. (4) Example of application of the electrode positioner with a microelectrode and an electrochemical microscope similar surviving cells measuring apparatus is already [T. Yasukawa et al. Yasukawa et al. ,Denki Kagaku,66,660−661(1998);P. , Denki Kagaku, 66,660-661 (1998); P. J. J. S. S. Smith et al. Smith et al. ,Microsc. , Microsc. Res. Res. Tech. Tech. ,46,398−417(1999);S. , 46,398-417 (1999); S. C. C. Land et al. Land et al. ,J. , J. Exp. Exp. Biol. Biol. ,202,211−218(1999)〕。 , 202,211-218 (1999)].
【0021】 [0021]
さらに、体外培養胚の代謝活性を無侵襲に計測することを目的とした研究も近年報告されつつある〔D. Furthermore, research is being reported recently for the purpose of measuring the metabolic activity of in vitro cultured embryos noninvasively [D. K. K. Gardner and H. Gardner and H. J. J. Leese,Hum. Leese, Hum. Reprod. Reprod. ,1,25−27(1986);D. , 1,25-27 (1986); D. K. K. Gardner and H. Gardner and H. J. J. Leese,J. Leese, J. Exp. Exp. Zool. Zool. ,242,103−105(1987);J. , 242,103-105 (1987); J. R. R. Trimarchi et al. Trimarchi et al. ,Biol. , Biol. Reprod. Reprod. ,62,1866−1874(2000)〕。 , 62,1866-1874 (2000)]. これらの報告の中には、計測の後も胚が損傷を受けないで正常に発生することを確認したという報告もある。 Some of these reports, there are also reports that the embryo even after the measurement, it was confirmed that occur normally not damaged. しかし、胚のある時期において計測された個々の検体の代謝活性から、被計測検体のその時期以降の発生過程を予測し、判定基準を明確に示した例はない。 However, the metabolic activity of individual samples was measured at some point in the embryo, to predict the course of the generation after that time of the measurement specimen, examples showed clearly criteria not. これまでの報告例では、単一胚毎の形態観察と酸素消費量の対応が明確化されておらず、形態観察で判断可能な異常と酸素消費量に基づく異常の区別が極めて曖昧である。 In the previous reported cases, the corresponding morphological observation and oxygen consumption per single embryo has not been clarified, the distinction between abnormal based on the abnormality and oxygen consumption can be determined by morphological observation is extremely ambiguous.
【0022】 [0022]
本発明は、上記状況に鑑みて、哺乳動物胚の形態観察と代謝活性の指標である酸素消費量の計測を行い、個々の哺乳動物胚の生存性や正常性を、早期に正確に把握できる哺乳動物胚の無侵襲的品質評価方法及びその装置を提供することを目的とする。 The present invention is, in view of the above situation, performs measurement of indicators in which the oxygen consumption of morphological observation and metabolic activity of the mammalian embryo, the viability and health of the individual mammalian embryo, can be accurately grasped early and to provide a non-invasive quality evaluation method and apparatus of the mammalian embryo.
【0023】 [0023]
【課題を解決するための手段】 In order to solve the problems]
本発明は、上記目的を達成するために、 The present invention, in order to achieve the above object,
〔1〕哺乳動物胚の無侵襲的品質評価方法において、(a)顕微鏡により単一哺乳動物胚の大きさの計測と、形態観察を行う工程と、(b)前記顕微鏡による単一哺乳動物胚の大きさの計測と形態観察に基づいて、前記単一哺乳動物胚に関して、すみやかに酸素消費量を計測する工程と、(c)上記工程(a)および工程(b)により、あらかじめ単一哺乳動物胚の大きさ・形態と、 単一哺乳動物胚の溶液中と単一哺乳動物胚表面の酸素濃度差に基づく酸素消費量の関係、さらに単一哺乳動物胚のその後の胚半径、胞胚腔形成率/脱出胚盤胞到達率発生過程を記録するとともに、判定基準を設定する工程と、(d)上記工程(c)を参照し、単一哺乳動物胚の品質を判定する工程とを施すことを特徴とする。 [1] In the non-invasive quality evaluation method of a mammalian embryo, (a) and performing the magnitude of the measurement of a single mammalian embryo, the morphological observation by a microscope, (b) a single by the microscope mammalian embryo based on the magnitude of the measurement and morphological observation of, with respect to the single mammalian embryo, a step of measuring promptly oxygen consumption, by (c) the steps (a) and (b), advance a single mammalian and size-form of the animal embryo, solutions and single mammalian embryo surface oxygen concentration difference oxygen consumption relationship based on the single mammalian embryo, further subsequent embryo radius of a single mammalian embryo, blastocoelic records the developmental process of the formation rate / exit scutellum胞到our rate, and setting criteria, see (d) is the step (c), and determining the quality of a single mammalian embryo and characterized by applying.
【0024】 [0024]
〔2〕哺乳動物胚の無侵襲的品質評価装置において、単一哺乳動物胚の大きさの計測と形態観察を行うための顕微鏡と、前記顕微鏡による単一哺乳動物胚の大きさの計測と形態観察に基づいて、単一哺乳動物胚の溶液中と単一哺乳動物胚表面の酸素濃度差に基づく酸素消費量の計測を行う電気化学顕微鏡計測装置と、培養液に準ずる計測溶液と、(d)上記(a),(b),(c)を用いて単一哺乳動物胚近傍の酸素濃度変化を高空間分解能で定量する手段と、(e)上記(d)によりあらかじめ作出した単一哺乳動物胚の大きさ・形態と酸素消費量の関係および単一哺乳動物胚のその後の胚半径、胞胚腔形成率/脱出胚盤胞到達率発生結果を集計する手段と、(f)上記(e)の集計データとの比較により、単一哺乳動物胚の正常性を判 [2] The non-invasive quality evaluation unit of the mammalian embryo, and the microscope for performing size measurement and morphological observation of a single mammalian embryo, the size of the measurement and the form of a single mammalian embryo by the microscope based on the observation, an electrochemical microscope measuring device to measure the solution and the oxygen consumption amount based on the oxygen concentration difference of a single mammalian embryo surface of a single mammalian embryo, and measuring the solution equivalent to the culture, (d ) above (a), (b), means for quantifying the oxygen concentration changes in single mammalian embryo vicinity at a high spatial resolution using (c), a single mammal was previously produced by (e) (d) above subsequent embryo radius relationship and single mammalian embryo size and morphology and oxygen consumption of animal embryos, and means for aggregating the occurrence results of blastocoel formation rate / exit scutellum胞到our rate, (f) above ( by comparison with aggregated data e), determine the health of a single mammalian embryo する手段とを具備することを特徴とする。 Characterized by comprising a means for.
【0025】 [0025]
〔3〕上記〔2〕記載の哺乳動物胚の無侵襲的品質評価装置において、前記電気化学顕微鏡計測装置は、顕微鏡ステージ上に設置した微小電極、電極位置決め装置、微小電流計測装置、温度制御装置、培養気相条件(酸素、二酸化炭素)制御装置を含むことを特徴とする。 [3] In the above-mentioned [2], noninvasive quality evaluation unit of a mammalian embryo, wherein said electrochemical microscope measurement device microelectrodes placed on the microscope stage, the electrode positioner, small current measuring device, the temperature control device culture gas phase conditions (oxygen, carbon dioxide), characterized in that it comprises a control device.
【0026】 [0026]
【発明の実施の形態】 DETAILED DESCRIPTION OF THE INVENTION
以下、本発明の実施の形態を図を参照しながら説明する。 It will be described with the embodiment of the present invention with reference to FIG.
【0027】 [0027]
体外培養胚の移植は、通常胚盤胞期胚を使用する。 Transplantation of in vitro culture embryos, using a normal blastocyst stage embryos. 受胎率を上げるためには、この時期に品質の優れた正常胚を1個づつ正確に判別することが必要である。 To increase the conception rate, it is necessary to determine the superior normal embryo quality at this time one by one accurately.
【0028】 [0028]
本発明では、まず、形態観察で品質評価した胚の酸素消費量の定量化を行い、胚の品質に対する形態と酸素消費量の関係について検討した。 In the present invention, firstly, we perform the quantification of the oxygen consumption of embryos quality evaluation in the form observed and investigated the relationship between morphology and oxygen consumption relative to the quality of the embryo. 形態観察のみでは胚の品質評価が難しいので、酸素消費量を計測し、その後、胚を培養し再び形態観察と酸素消費量を計測した。 Since only the morphological observation is difficult quality evaluation of the embryo, the oxygen consumption is measured, then cultured embryos were measured morphological observation and oxygen consumption again.
【0029】 [0029]
以下、具体的な実施例について述べる。 Hereinafter will be described specific examples.
【0030】 [0030]
体外培養のウシ卵子体外成熟/体外受精は、定法により行った〔S. Bovine oocytes in vitro maturation / IVF vitro culture was carried out by a conventional method [S. Yamashita et al. Yamashita et al. ,Cytotechnology,31,121−129(1999)〕。 , Cytotechnology, 31,121-129 (1999)]. 体外受精後の胚の発生培養には、無血清培養液(IVD101,機能性ペプチド研究所製)を用い、低酸素条件(5%酸素、5%二酸化炭素、90%窒素、湿度100%、38.5℃)で培養した。 The occurrence culturing embryos after IVF, serum-free medium (IVD101, functional peptides Laboratory Ltd.) using a hypoxic conditions (5% oxygen, 5% carbon dioxide, 90% nitrogen, 100% humidity, 38 They were cultured in .5 ℃).
【0031】 [0031]
定法により単一胚毎に形態観察に基づく判定を行った。 It was determined based on morphological observation every single embryo by a conventional method. 胚の形状、大きさ(透明帯を含む半径r s )を記録した。 The shape of the embryo was recorded size (radius r s which includes a transparent band). 具体的には胚の光学顕微鏡写真を撮影した。 Specifically it was taken optical micrograph of the embryo. 単一胚の酸素消費量の計測にあたっては、電気化学顕微鏡を応用した。 Of when the measurement of oxygen consumption of a single embryo, by applying the electrochemical microscope.
【0032】 [0032]
電気化学顕微鏡計測においては、探針である微小(マイクロ)電極を胚近傍に近接させながら酸素還元電流を観測し、溶液中の酸素濃度と胚表面酸素濃度の差(ΔC)を記録した。 In electrochemical microscope measurement, the micro (micro) electrodes is a tip to observe the oxygen reduction current while close to the embryo near, the difference between the oxygen concentration and the embryo surface oxygen concentration in solution ([Delta] C) was recorded.
【0033】 [0033]
図1は本発明の実施例を示す哺乳動物胚の無侵襲的品質評価装置の構成図であり、図1(A)はその無侵襲的品質評価装置の全体構成図、図1(B)はその無侵襲的品質評価装置の部分拡大図である。 Figure 1 is a block diagram of a non-invasive quality evaluation unit of the mammalian embryo showing an embodiment of the present invention, FIG. 1 (A) is an overall configuration diagram of the non-invasive quality evaluation device, FIG. 1 (B) its is a partially enlarged view of a non-invasive quality evaluation unit.
【0034】 [0034]
これらの図において、1は微小(マイクロ)電極、2は参照極/対極、3は培地/計測溶液、4はディッシュ、5は胚試料、6は電極位置決め装置(精密位置決め装置)、7はコンピュータ、8は微小電流計測装置(ポテンショスタット)、9は試料保持装置、10は温度制御装置、11はガスボンベ、12はバブラー、13はグローブボックス、14は倒立顕微鏡、15は顕微鏡ステージ、16は電極シール部、17は電極ディスク面である。 In these drawings, 1 is very small (micro) electrodes, the reference electrode / counter electrode 2, the medium / measuring solution 3, 4 dishes, 5 embryo sample, 6 electrode positioning apparatus (fine positioning device), 7 computer , the minute current measuring device (potentiostat), 9 sample holding apparatus, 10 is a temperature controller, 11 gas cylinder 8, 12 a bubbler, 13 a glove box, the inverted microscope 14, 15 microscope stage, 16 electrodes seal portion, 17 is an electrode disk surface.
【0035】 [0035]
電気化学計測系は、マイクロ電極1、参照極/対極電極2、微小電流計測装置(ポテンショスタット)8で構成される。 Electrochemical measuring system is composed of microelectrodes 1, a reference electrode / counter electrode 2, a minute electric current measurement apparatus (potentiostat) 8. 電極の駆動は、電極位置決め装置6により行った。 Drive electrodes was conducted by electrode positioning device 6. 電気化学計測系および電極駆動系はコンピュータ7により制御した。 Electrochemical measuring system and the electrode driving system is controlled by the computer 7. 35mmディッシュ(Nunc社製)中で、胚試料5が顕微鏡視野の中心に位置するよう、試料保持装置(マニピュレータおよびマイクロインジェクタ、ナリシゲ社製)9で操作した。 Among 35mm dish (Nunc Co.), so that the embryo sample 5 is located at the center of the microscope field of view, the sample holding device (manipulator and microinjector, Narishige Co., Ltd.) was operated at 9.
【0036】 [0036]
培地/計測溶液3は胚発生培養液に準ずる無血清培養液(IVMD101,機能性ペプチド研究所製)を用いた。 Medium / Measurement solution 3 was used serum-free medium pursuant to embryogenic culture medium (IVMD101, functional peptides Laboratory Ltd.). 温度制御装置10には恒温循環槽を用いた。 With constant temperature circulation bath to a temperature controller 10. 培養気相条件は(5%二酸化炭素、95%空気、湿度100%)ガスを流し制御した。 Culture gas phase conditions were controlled flow (5% carbon dioxide, 95% air, 100% humidity) gas. 諸装置を倒立顕微鏡ステージ15上に設置した。 Was installed various devices on an inverted microscope stage 15. マイクロ電極1は、電極半径が0.92μm、シール部を含めた全体半径が4.0μmのものを用いた。 Microelectrode 1, electrode radius 0.92 .mu.m, whole including the seal portion radius used was the 4.0 .mu.m. 酸素還元電流の記録にあたり、電極電位を銀/塩化銀参照電極に対し、−0.6Vに印加した。 Recording Upon the oxygen reduction current, the electrode potential against a silver / silver chloride reference electrode was applied to -0.6 V.
【0037】 [0037]
マイクロ電極1を胚試料5の表面に最近接させた地点では、マイクロ電極1の先端が胚試料5の中心と同じ高さで表面に接するように配置した。 The microelectrode 1 in point was closest to the surface of the embryo sample 5, the tip of the microelectrode 1 is placed in contact with the surface at the same height as the center of the embryo sample 5. この際、マイクロ電極1の先端の電極ディスク面17と胚試料5の表面はほぼ直交する配置になるため、マイクロ電極1−胚試料5の表面間の最近接距離は、電極シール半径(4.0μm)と同値である。 At this time, since the arrangement surface of the tip of the electrode disk surface 17 and the embryos sample 5 microelectrode 1 is substantially perpendicular, the closest distance between the surface of the microelectrode 1 embryos sample 5, the electrode sealing radius (4. 0μm) to be equivalent. 電極走査速度は14.7μm/sとした。 Electrode scanning rate was 14.7μm / s. 電極走査範囲としてマイクロ電極1−胚試料5の最近接地点とその点より500μm離れた地点を往復した。 And reciprocally recently a point distant 500μm from the ground point and the point of the microelectrode 1 embryo sample 5 as an electrode scanning range.
【0038】 [0038]
図2は本発明の実施例を示すマイクロ電極で記録された酸素還元電流より求められた特性図であり、図2(A)はその酸素濃度プロファイルと胚の表面から電極までの距離(r)の関係を示す図、図2(B)は酸素濃度プロファイルと、胚の中心から電極までの距離と胚半径の比〔(r s /(r+r s )〕の関係を示す図である。図中の線aは、受精後6日目のウシ単一桑実胚に対するプロファイルを示す。計測後48時間継続培養し胚盤胞期に到達した同一胚に対して、電極を栄養芽細胞(TRP,trophoblast)側〔線b〕および内部細胞塊(ICM,inner cell mass)側に向かって近接した場合〔線c〕のプロファイルも図中に示した。 Figure 2 is a characteristic diagram obtained from recorded oxygen reduction current at microelectrodes showing an embodiment of the present invention, the distance from FIG. 2 (A) the surface of the oxygen concentration profile and the embryo to the electrode (r) shows the relationship, FIG. 2 (B) is a diagram showing the oxygen concentration profile, the relationship between the ratio of the distance and embryos radius from the center of the embryo to the electrode [(r s / (r + r s) ]. figure the lines a, for the same embryos reached shows the profile for bovine single morula 6 days after fertilization. 48 hours after the measurement continued culturing the blastocyst stage, the electrode trophoblasts (TRP, trophoblasts) side [line b] and inner cell mass (ICM, even profile when adjacent towards the inner, cell mass) side [line c] shown in FIG.
【0039】 [0039]
図2より、本発明の計測が無侵襲的に行われ、計測後の発生過程に影響を与えないこと、胚表面の局所濃度変化を検出可能であることが示された。 From FIG. 2, the measurement of the present invention is carried out noninvasively, it does not affect the generation process after measurement, it has been shown that can detect local changes in the concentration of the embryo surface. すなわち、図2(B)においてプロットが良好な直線性を示すことは、胚試料近傍の濃度勾配が球面拡散の理論式によく従うことを意味する。 That is, it shows a plot good linearity in FIG. 2 (B), means that the concentration gradient in the vicinity of the embryo samples follow well the theoretical equation of the spherical diffusion.
【0040】 [0040]
この図2(B)より、r s /(r+r s )=0および1における酸素濃度は、各々胚から十分離れた地点および胚試料表面における酸素濃度に相当する。 From this FIG. 2 (B), the oxygen concentration at r s / (r + r s ) = 0 and 1 corresponds to the oxygen concentration in sufficiently far point and embryos sample surface from each embryo. 従って、両地点の酸素濃度の差がΔCに相当する〔T. Thus, the difference in oxygen concentration between both points corresponds to ΔC [T. Yasukawa,et al. Yasukawa, et al. ,Biophys. , Biophys. J. J. ,76,1129−1135(1999)〕。 , 76,1129-1135 (1999)]. 上記ΔCの決定には、酸素還元電流のプロファイルが往路(電極を胚に近接)と復路(電極を胚から遠ざける)で対称性が良いことを確認し、往路/復路各々の電流プロファイルを解析した。 The determination of the ΔC, the profile of the oxygen reduction current (the electrode away from the embryo) backward and forward (near the electrode embryos) Check that good symmetry was analyzed forward / backward each current profile . 1個の胚に対して3往復以上電流プロファイルを記録し、ΔCの平均およびその標準偏差を得た。 3 records the round trip over current profile for one embryo, to obtain a mean and standard deviation of [Delta] C. ΔCとr sから、次式を用いて、単一胚あたりの酸素消費量(F)を算出することもできる。 From ΔC and r s, using the following equation, oxygen consumption per single embryos (F) it can also be calculated.
【0041】 [0041]
F=4πDr s ΔC F = 4πDr s ΔC
ここで、Dは酸素の拡散係数、r sは透明帯を含む胚の半径である。 Here, D is the diffusion coefficient of oxygen, r s is the radius of the embryo containing the zona pellucida. この式から明らかなように、ΔCは酸素消費量の定量化や、個々の胚における酸素消費量を比較する際に指標となる数値である。 As it is apparent from this equation, [Delta] C is a numerical value serving as an index when comparing and quantifying the oxygen consumption, oxygen consumption in individual embryo. 特に桑実胚においては胚半径(r s )がほぼ同一であるため、ΔCにより酸素消費量を比較可能である。 Particularly in the morula for embryo radius (r s) is substantially the same, it is possible to compare the oxygen consumption by the [Delta] C.
【0042】 [0042]
上記した図2の線a、線b、線cに対して求められたΔCは、各々6.90±1.35、9.14±1.67、16.40±1.83μMであった。 Line a in FIG. 2 described above, [Delta] C was determined for the line b, a line c were respectively 6.90 ± 1.35,9.14 ± 1.67,16.40 ± 1.83μM.
【0043】 [0043]
桑実胚(r s =74μm)に関しては、胚表面の酸素濃度は均一であるので直ちにF=1.40±0.27×10 -14 mols -1が求まる。 For the morula (r s = 74μm), the oxygen concentration of the embryo surface immediately F = 1.40 ± 0.27 × 10 -14 mols -1 determined because it is uniform. 胚盤胞期胚(r s =100μm)に関しては、TRP側とICM側で濃度差があるため、Fを求めることは難しい。 For the blastocyst stage (r s = 100μm), because of the density difference TRP side and ICM side, it is difficult to determine the F. しかし、TRP側とICM側に近接させた際のΔCを用いて、近似的に、2.50±0.46×10 -14 mols -1 <F<4.49±0.50×10 -14 mols -1と表すことは可能である。 However, using the ΔC when brought close to the TRP side and ICM side, approximately, 2.50 ± 0.46 × 10 -14 mols -1 <F <4.49 ± 0.50 × 10 -14 it is possible to represent the mols -1.
【0044】 [0044]
上記と同一条件で、受精後6日の桑実胚29個に対し、形態観察と酸素消費量計測を実施し、その後の発生過程を記録した。 In the same conditions, with respect to 29 pieces morula 6 days after fertilization, performed morphological observation and oxygen consumption measurements were recorded subsequent developmental processes. 発生過程の指標としては、表中の計測後48時間継続培養した胚の半径(r s )、胞胚腔形成率、脱出胚盤胞到達率を調査し、結果を表1にまとめた。 As an indicator of the development process, of embryos for 48 hours after the measurement continued culture in the table radius (r s), blastocoel formation rate, to investigate the escape blastocyst胞到our rate, and the results are summarized in Table 1.
【0045】 [0045]
【表1】 [Table 1]
【0046】 [0046]
表1は受精後6日のウシ桑実胚(n=29)の、溶液中の酸素濃度と胚表面酸素濃度の差(ΔC)と、受精後8日目の胚半径(r s )、胞胚腔形成率、脱出胚盤胞到達率の関係を示している。 Table 1 of bovine morula 6 days after fertilization (n = 29), the difference between the oxygen concentration and the embryo surface oxygen concentration in the solution and ([Delta] C), the embryo radius (r s) of 8 days after fertilization, the blastocyst lumen formation rate, which shows the relationship between the escape blastocyst 胞到 our rate.
【0047】 [0047]
受精後6日の桑実胚のうち、溶液中の酸素濃度と胚表面酸素濃度の差(ΔC)が5.0μM未満の胚(ΔC<5.0μM)は、受精後8日目の胚半径80μm未満(r s <80μm)が78%、胞胚腔形成率33%、脱出胚盤胞到達率0%であったのに対し、ΔCが5.0μM以上の胚(ΔC≧5.0μM)は、受精後8日目の胚半径80μm以上(r s ≧80μm)が80%、胞胚腔形成率95%、脱出胚盤胞到達率68%であった。 Of morula 6 days after fertilization, the oxygen concentration and the difference between the embryo surface oxygen concentration ([Delta] C) is the embryo of less than 5.0μM (ΔC <5.0μM) in solution, embryos radius of 8 days after fertilization less than 80μm (r s <80μm) 78%, blastocoel formed 33%, whereas the 0% escape scutellum胞到our rate, [Delta] C is 5.0 [mu] M or more embryos (ΔC ≧ 5.0μM) is , 8 day embryos radius 80 [mu] m or more after fertilization (r s ≧ 80μm) 80%, blastocoel formed of 95% and a 68% escape scutellum胞到our rate. 即ち、受精後6日の桑実胚において、判定基準として「ΔCが5.0μM以上」を設定した場合、基準を満たす胚と満たさない胚では、その後の胚発生過程に著しい違いがあることが分かった。 That is, in morula 6 days after fertilization, when set to "ΔC more than 5.0μM" as a criterion, the embryos that do not meet the embryos meet the criteria, that there are significant differences in the subsequent embryonic developmental processes I understood.
【0048】 [0048]
ここで重要なことは、このような代謝活性の数値化に基づく判定基準は、形態観察のみから設定することが不可能な点にある。 It is important that the decision criteria based on digitizing such metabolic activity is that is not possible to set only from morphological observation.
【0049】 [0049]
図3は基準値近傍の桑実胚のうち典型的な試料の光学顕微鏡写真を示す図である。 Figure 3 is a diagram showing an optical micrograph of a typical sample of the morula reference value neighborhood.
【0050】 [0050]
図中A、BはΔCが5.0μM未満、CからFはΔCが5.0μM以上であった。 Figure A, B has ΔC less than 5.0 [mu] M, F is ΔC is equal to or more than 5.0 [mu] M from C. 図3より、本発明により設定された基準を、形態観察のみに基づき判別することは極めて困難であることが分かる。 Than 3, the set reference according to the invention, be determined based solely on morphological observation it can be seen is extremely difficult.
【0051】 [0051]
例えば、ウシを含む哺乳動物胚が正常胚か異常胚であるかは、胚の凍結保存時の耐凍能や受胎率に大きな影響を与える。 For example, mammalian embryo containing bovine is normal embryo or abnormal embryos, greatly affects the freezing ability and conception rates during cryopreservation of embryos. 従って、胚の品質を生存したまま(無侵襲的に)しかも発生の早い時期に評価できる技術が求められている。 Thus, while the survival of the quality of the embryos (non-invasively) And technology that can be evaluated early in the occurrence has been demanded. 通常形態的観察による評価法が一般的に用いられているが、この方法では正確な正常胚の判別が難しいという欠点があった。 Although evaluation method by conventional morphological observation is generally used, a determination is accurate normal embryo has a disadvantage that it is difficult in this method. 本発明では、上記したように、電気化学顕微鏡を用いて、単一胚の酸素消費量を定量的に計測可能であることを示し、さらに、形態的には見分けの困難な胚の品質評価をより正確に行えることを特徴としている。 In the present invention, as described above, using an electrochemical microscope, shown to be quantitatively measurable oxygen consumption of a single embryo, further quality evaluation difficult embryos distinguished morphologically It is characterized in that performed more accurately. この結果、移植や凍結保存に適した胚の選択により信頼性の高い品質評価法としての意義は大きい。 As a result, the significance of a reliable quality assessment by the selection of embryos suitable for implantation or cryopreservation is large.
【0052】 [0052]
以下、本発明の効果について説明する。 Hereinafter, effects of the present invention will be described.
【0053】 [0053]
平成9年度の国内でのウシ受精卵移植の受胎率は、体内受精卵新鮮胚移植で51%、体内受精卵凍結胚移植で45%、体外受精卵移植では34%となっている〔農林水産省畜産局統計資料〕。 Conception rate of cattle embryo transfer in Japan for the fiscal year 1997 is 51% in the body fertilized egg fresh embryo transfer, 45% in the body fertilized eggs frozen embryo transfer, in vitro embryo transfer and has a 34% [of Agriculture, Forestry and Fisheries saving livestock Bureau statistics]. 本発明によれば、それぞれの胚の品質をより正確に判定できることにより、受胎率を10%向上させるだけでも畜産業に与える経済的利益は多大なものとなる。 According to the present invention, by being able to determine the quality of each embryo more precisely, economic benefits that provide a conception rate in livestock just increase 10% becomes enormous. さらに、家畜胚のみならずヒト胚の品質評価も可能と考えられ、ヒト体外受精における不妊治療に対しても大きな期待が寄せられる。 Furthermore, the quality evaluation of becoming not human embryos only livestock embryos also believed possible, high expectations Asked against infertility in humans IVF. 具体的には、ヒト体外受精で得られた胚の妊娠率は約20%と低く、治療には複数個の胚(3個程度まで)が移植される。 Specifically, pregnancy rates of embryos obtained in human IVF is as low as about 20%, (up to 3 or so) a plurality of embryos in the treatment is transplanted. その結果、双子以上多胎妊娠の例も多く報告され、妊娠や出産後の子育ての大きな問題となっている。 As a result, the example of twins or higher multiple pregnancies many been reported, it has become a major problem of child care after pregnancy and childbirth. 移植前に正確な胚の品質評価ができれば、移植に用いる胚を1個に減らすことが可能となり、多胎妊娠を防止することで母体への負担等が著しく軽減する。 If the quality evaluation of the correct embryo prior to implantation, it becomes possible to reduce the embryo to be used for transplantation into one, the burden like maternal is reduced significantly by preventing multiple pregnancies.
【0054】 [0054]
また、妊娠率の向上は、患者の経済的負担を減らすことが期待される。 In addition, improvement of the pregnancy rate is expected to reduce the economic burden of patients.
【0055】 [0055]
単一胚の酸素消費量を高感度に計測可能な本発明は、胚の酸素消費量の計測のみならず、細菌などの前核性生物、植物細胞、動物細胞など多種多様な細胞の酸素消費量をごく少量の細胞数で計測できることが考えられる。 The oxygen consumption of a single embryo measurable present invention with high sensitivity, not only the measurement of oxygen consumption of embryos, pronuclear organisms such as bacteria, oxygen consumption of a wide variety of cells such as plant cells, animal cells It is considered to be able to measure the amounts small amount of cell number. さらに、酸素消費量だけでなく、プローブを代えることにより、他の分子種(Ca,Kなど)の細胞内変化を鋭敏に定量化することも可能である。 Furthermore, not only the oxygen consumption, by changing the probe, it is possible to sensitively quantify intracellular changes in other species (Ca, K, etc.).
【0056】 [0056]
なお、本発明は上記実施例に限定されるものではなく、本発明の趣旨に基づいて種々の変形が可能であり、それらを本発明の範囲から排除するものではない。 The present invention is not limited to the above embodiments, but various modifications are possible based on the spirit of the present invention are not excluded from the scope of the present invention.
【0057】 [0057]
【発明の効果】 【Effect of the invention】
以上、詳細に説明したように、本発明によれば、以下のような効果を奏することができる。 As described above in detail, according to the present invention can exhibit the following effects.
【0058】 [0058]
電気化学顕微鏡を用いて、単一胚の酸素消費量を定量的に計測可能であり、さらに、形態的には見分けの困難な胚の品質評価をより正確に行うことができる。 Using an electrochemical microscope, a quantitatively measurable oxygen consumption of a single embryo, further, the morphological can perform quality evaluation difficult embryos distinguish more precisely. その結果、移植や凍結保存に適した胚の選択により信頼性の高い品質評価を行うことができる。 As a result, it is possible to perform reliable quality evaluation by the selection of embryos suitable for implantation or cryopreservation.
【図面の簡単な説明】 BRIEF DESCRIPTION OF THE DRAWINGS
【図1】 本発明の実施例を示す哺乳動物胚の無侵襲的品質評価装置の構成図である。 1 is a configuration diagram of a non-invasive quality evaluation unit of the mammalian embryo showing an embodiment of the present invention.
【図2】 本発明の実施例を示すマイクロ電極で記録された酸素還元電流より求められた特性図である。 2 is a characteristic diagram obtained from recorded oxygen reduction current at microelectrodes showing an embodiment of the present invention.
【図3】 基準値近傍の桑実胚のうち典型的な試料の光学顕微鏡写真を示す図である。 3 is a diagram showing an optical micrograph of a typical sample of the morula reference value neighborhood.
【符号の説明】 DESCRIPTION OF SYMBOLS
1 マイクロ電極 2 参照極/対極 3 培地/計測溶液 4 ディッシュ 5 胚試料 6 電極位置決め装置(精密位置決め装置) 1 microelectrode 2 reference electrode / counter electrode 3 medium / measuring solution 4 dish 5 embryos sample 6 electrode positioner (fine positioning device)
7 コンピュータ 8 微小電流計測装置(ポテンショスタット) 7 computer 8 minute current measuring device (potentiostat)
9 試料保持装置 10 温度制御装置 11 ガスボンベ 12 バブラー 13 グローブボックス 14 倒立顕微鏡 15 顕微鏡ステージ 16 電極シール部 17 電極ディスク面 9 sample holding device 10 temperature controller 11 gas cylinder 12 bubbler 13 glove box 14 inverted microscope 15 microscope stage 16 electrode sealing portion 17 the electrode disk surface

Claims (3)

  1. 哺乳動物胚の無侵襲的品質評価方法において、 In the non-invasive quality evaluation method of the mammalian embryo,
    (a)顕微鏡により単一哺乳動物胚の大きさの計測と、形態観察を行う工程と、 And performing the measurement of the size of a single mammalian embryo, the morphological observation by (a) a microscope,
    (b)前記顕微鏡による単一哺乳動物胚の大きさの計測と形態観察に基づいて、前記単一哺乳動物胚に関して、すみやかに酸素消費量を計測する工程と、 (B) on the basis of the measurement and morphological observation of the size of a single mammalian embryo by microscopy, with respect to said single mammalian embryo, a step of measuring promptly oxygen consumption,
    (c)上記工程(a)および工程(b)により、あらかじめ単一哺乳動物胚の大きさ・形態と、 単一哺乳動物胚の溶液中と単一哺乳動物胚表面の酸素濃度差に基づく酸素消費量の関係、さらに単一哺乳動物胚のその後の胚半径、胞胚腔形成率/脱出胚盤胞到達率発生過程を記録するとともに、判定基準を設定する工程と、 The (c) the steps (a) and (b), oxygen and size-form in advance a single mammalian embryo, based on the oxygen concentration difference between the solution and a single mammalian embryo surface of a single mammalian embryo consumption related, yet subsequent embryo radius of a single mammalian embryo, records the developmental process of the blastocoel formation rate / exit scutellum胞到our rate, and setting criteria,
    (d)上記工程(c)を参照し、単一哺乳動物胚の品質を判定する工程とを施すことを特徴とする哺乳動物胚の無侵襲的品質評価方法。 (D) the reference to step (c), no invasive quality evaluation method of a mammalian embryo, wherein the applying and determining the quality of a single mammalian embryo.
  2. 哺乳動物胚の無侵襲的品質評価装置において、 In non-invasive quality evaluation unit of the mammalian embryo,
    (a)単一哺乳動物胚の大きさの計測と形態観察を行うための顕微鏡と、 (A) a microscope for performing the magnitude of the measurement and morphological observation of a single mammalian embryo,
    (b)前記顕微鏡による単一哺乳動物胚の大きさの計測と形態観察に基づいて、単一哺乳動物胚の溶液中と単一哺乳動物胚表面の酸素濃度差に基づく酸素消費量の計測を行う電気化学顕微鏡計測装置と、 (B) on the basis of the measurement and morphological observation of the size of a single mammalian embryo using a microscope, the measurement of the solution and the oxygen consumption amount based on the oxygen concentration difference of a single mammalian embryo surface of a single mammalian embryo electrochemical microscope measuring apparatus which performs,
    (c)培養液に準ずる計測溶液と、 (C) a measuring solution equivalent to the culture,
    (d)上記(a),(b),(c)を用いて単一哺乳動物胚近傍の酸素濃度変化を高空間分解能で定量する手段と、 Means for quantifying the oxygen concentration change in the vicinity of a single mammalian embryo at a high spatial resolution using (d) is above (a), (b), (c),
    (e)上記(d)によりあらかじめ作出した単一哺乳動物胚の大きさ・形態と酸素消費量の関係および単一哺乳動物胚のその後の胚半径、胞胚腔形成率/脱出胚盤胞到達率発生結果を集計する手段と、 (E) (d) above by preliminarily produced the subsequent embryo radius blastocoel formation rate / exit scutellum胞到our rate of a single mammalian embryo relationship size and morphology and oxygen consumption and a single mammalian embryo and means for aggregating the generation results,
    (f)上記(e)の集計データとの比較により、単一哺乳動物胚の正常性を判定する手段とを具備することを特徴とする哺乳動物胚の無侵襲的品質評価装置。 (F) above by comparison with aggregated data (e), noninvasive quality evaluation unit of the mammalian embryo, characterized by comprising means for determining the normality of a single mammalian embryo.
  3. 請求項2記載の哺乳動物胚の無侵襲的品質評価装置において、前記電気化学顕微鏡計測装置は、顕微鏡ステージ上に設置した微小電極、電極位置決め装置、微小電流計測装置、温度制御装置、培養気相条件(酸素、二酸化炭素)制御装置を含むことを特徴とする哺乳動物胚の無侵襲的品質評価装置。 In non-invasive quality evaluation unit of the mammalian embryo of claim 2, wherein said electrochemical microscope measurement device microelectrodes placed on the microscope stage, the electrode positioner, small current measuring device, a temperature controller, a culture gas phase conditions (oxygen, carbon dioxide) non-invasive quality evaluation unit of the mammalian embryo, characterized in that it comprises a control device.
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