JP5916241B2 - 妊娠成績についての高い能力を有するコンピテントな卵母細胞およびコンピテントな胚を選択するための方法 - Google Patents
妊娠成績についての高い能力を有するコンピテントな卵母細胞およびコンピテントな胚を選択するための方法 Download PDFInfo
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Description
本発明は、コンピテント(comptent)な卵母細胞またはコンピテントな胚を選択するための方法に関する。
補助生殖医療(ART)における体外受精(IVF)の試みの後の妊娠率および出生率は依然として低い。実際に、3回中2回のIVFサイクルが妊娠に失敗し(SART2004)、そして10中8を超える移植された胚が着床に失敗している(KovalevskyおよびPatrizio, 2005)。さらに、IVFにより生まれた赤ん坊の50%超が、多胎妊娠による(Reddy et al., 2007)。ARTによって引き起こされる多胎妊娠から生じる早産には、年間、約8億9000万米ドルの医療費がかかると推定されている(BromerおよびSeli, 2008)。
本発明は、卵母細胞を囲む卵丘細胞において10個の遺伝子の発現レベルを測定する工程を含む、受精すると妊娠に至る高い着床率を有する生存可能な胚を産生する卵母細胞を選択するための方法に関し、前記遺伝子は、ATF3、SIAT6、PRKACA、PLA2G5、GPC6、G0S2、RBMS1、NFIC、SLC40A1およびWNT6であり、そして前記卵母細胞は、前記卵丘細胞が前記の10個の遺伝子のいずれをも過剰発現しない場合に選択される。
本発明者らは、卵丘細胞において発現される10個の遺伝子のセットを、胚の能力および妊娠成績についてのバイオマーカーとして決定した。本発明者らは、卵母細胞を囲む卵丘細胞の遺伝子発現プロファイルが異なる妊娠成績に相関し、これが妊娠に向けて発達する胚の特異的な発現サインの同定を可能とすることを実証した。本発明者らの結果は、卵母細胞を囲む卵丘細胞の分析が、胚の選択のための非侵襲的なアプローチであることを示す。
本発明に関する全ての遺伝子はそれ自体公知であり、そして以下の表Aに列挙されている。表Aは、その合わせた発現プロファイルが、受精すると妊娠に至る高い着床率を有する生存可能な胚を産生する卵母細胞を選択するための、または妊娠に至る高い着床力を有するコンピテントな胚を選択するための情報となることが示された遺伝子セットを提示する。
i)前記の雌被験体から、少なくとも1つの卵母細胞をその卵丘細胞と共に提供する工程;
ii)本発明の方法によって、前記卵母細胞が、受精すると妊娠に至る高い着床率を有する生存可能な胚を産生する卵母細胞であるかどうかを決定する工程
を含む、雌の被験体における調節卵巣過剰刺激(COS)プロトコールの効力を評価するための方法にも関する。
i)前記女性から、天然サイクル、改変サイクルまたは刺激されたサイクル下において、少なくとも1つの卵母細胞をその卵丘細胞と共に単離する工程;
ii)本発明の方法によって、前記卵母細胞が、受精すると妊娠に至る高い着床率を有する生存可能な胚を産生する卵母細胞であるかどうかを決定する工程;
iii)そして、それが、受精すると妊娠に至る高い着床率を有する生存可能な胚を産生する卵母細胞を生じるかどうかに基づいて、COS処置の効力をモニタリングする工程
を含む、調節卵巣過剰刺激(COS)プロトコールの効力をモニタリングするための方法に関する。
i)卵母細胞をその卵丘細胞と共に準備する工程、
ii)前記卵母細胞を体外受精する工程、
iii)本発明の方法によって工程i)の前記卵母細胞が、受精すると妊娠に至る高い着床率を有する生存可能な胚を産生する卵母細胞であるかどうかを決定することによって、工程ii)から生じた胚がコンピテントであるかどうかを決定する工程
を含む、胚が、妊娠に至る高い着床率を有する胚であるかどうかを決定するための方法にも関する。
i)本発明の方法を実施することによって妊娠に至る高い着床率を有する胚を選択する工程、
ii)前記雌の子宮に工程i)において選択された胚を着床させる工程
を含む、雌の妊娠成績を増強するための方法にも関する。
表Aにおいて前記したような遺伝子の発現レベルの決定を、多種多様な技術によって実施することができる。一般的には、決定されたような発現レベルは、相対的な発現レベルである。
材料および方法:
患者およびIVF処置:この後向き研究において、30.9±2.5歳の年齢で、男性不妊要因のために本発明者らのICSI(卵細胞質内精子注入)センターに問い合わせられた通常の応答患者(n=30)を研究した。患者を、組換えFSH(GonalF, Puregon;それぞれMerck-SeronoおよびOrganon)またはhMG(Menopur, Ferring)とGnRHアゴニストまたはアンタゴニストの組合せを用いて刺激した。卵巣応答を、血清中エストラジオールレベルおよび超音波検査によって評価して、卵胞の発達をモニタリングした。卵母細胞の回収を、超音波の指針下で、hCG投与(5000IU)の投与から36時間後に実施した。
CC:卵丘細胞、P+:正の妊娠成績を有する胚からの卵丘細胞、p−:妊娠成績を有さない胚からの卵丘細胞、G1/2:グレード1〜2の胚からの卵丘細胞、G3/4:グレード3〜4の胚からの卵丘細胞、NT:移植せず
胚の成績によるCCの遺伝子発現プロファイル:胚の成績に相関したCCの遺伝子発現プロファイルを同定するために、本発明者らは、各々の成績カテゴリーについての遺伝子発現サインを確立した:受精していない卵母細胞のCC、胚の発達をもたらすが広範なフラグメンテーションをもたらした(グレード3〜4)卵母細胞のCC、および全くフラグメンテーションがないかまたは限定されたフラグメンテーションを伴う胚の発達をもたらした(グレード1〜2)卵母細胞のCC。顆粒膜細胞試料を、リファレンス組織(対照)と捉えた。実際に、顆粒膜細胞は、他の成体組織とは異なり、CCに密接に関連した細胞である。このリファレンス組織の使用は、粗の系統差異に関連した異なって発現される遺伝子の数を低下させ、従って、CC/卵母細胞の相互作用における僅かな変化の同定を促進する。SAM分析は、受精されていない群において2605個の遺伝子が、グレード3/4群において2739個の遺伝子が、グレード1〜2群において2482個の遺伝子がFDR<5%でアップレギュレーションされていたことを示した。逆に、それぞれ4270個、4349個および4483個の遺伝子がダウンレギュレーションされた。その後、遺伝子のこれらのリストを交差させて、その重複を決定した。449個の上昇発現および890個の下降発現した遺伝子が、3つ全ての群において共通し、各カテゴリーは特異的な遺伝子発現プロファイルを示した。興味深いことに、860個のアップレギュレーションされた遺伝子(例えばガラニンおよびギャップジャンクションA5(GJA5)を含む)および1416個のダウンレギュレーションされた遺伝子(HLA−GおよびEGR1を含む)が、良好な形態学的な胚の品質を伴う卵丘細胞において特異的にモデュレーションされていた。グレード1〜2の群は、強力な遺伝子発現プロファイルを示したが、この群は妊娠成績に関して不均一であり、そして妊娠をもたらす胚に関連した18個のCC試料(4つの双胎妊娠を含む)を含んだが、妊娠をもたらすことに失敗した胚に関連した16個のCC試料も含んでいたことを注記しなければならない。
妊娠成績によるCCのSAM分析は、CC分析の遺伝子発現に基づいて、妊娠をもたらす胚を産生する卵母細胞と、妊娠をもたらさなかったものとの間で異なるであろう胚の能力についてのバイオマーカーである表Bの45個の遺伝子を同定した。QRT−PCRを使用してマイクロアレイデータを独立的に確認した。本発明者らは、妊娠を達成しなかったグレード1〜2の胚のCCと、妊娠を達成したグレード1〜2の胚のCCとの間の、36個のアップレギュレーションされた遺伝子および9個のダウンレギュレーションされた遺伝子の異なる発現を分析した。
45個の遺伝子のリストの信頼性を検査するために、男性不妊のために本発明者らのICSIセンターに問い合わせられた若い(36歳未満)通常の応答患者を含む、前向き研究を実施した。胚の選択は、CCの遺伝子発現プロファイルに従って(第1群)、または形態学的局面に従って(対照として使用した第2群)のいずれかで行なった。各群について2つの胚を戻した。最初の60人の患者について(1群につき30人の患者)、卵収集日に、第1群において、各CC試料を個々に収集し、そして遺伝子発現分析のために処理した。CC試料(n=267)を分析した。
ヒトを含む大半の哺乳動物種において、卵母細胞を囲む卵丘細胞は、依然として受精時に輸卵管に存在し、そして胚が着床するまで留まる。卵丘内および周辺における細胞外マトリックス再構築はおそらく、これらの両方の工程において重要な役割を果たしている。これに関して、本発明者らは、胚の能力および妊娠成績についてのバイオマーカーである卵丘細胞において発現されている10個の遺伝子を同定した。本発明者らの研究において、本発明者らは、卵母細胞を囲むCCの遺伝子発現プロファイルが異なる成績に関連し、これは、妊娠に向けて発達している胚の特異的な発現サインの同定を可能とすることを実証した。結論すると、本発明者らはICSIまたはIVFのために問い合わせられた患者の異なる妊娠成績をもたらす、卵母細胞に由来するヒト卵丘細胞間における異なる遺伝子発現を発見した。本発明者らの結果は、卵母細胞を囲む卵丘細胞の分析が、胚の選択のための非侵襲的なアプローチであることを示す。典型的には、CCを卵母細胞のピックアップ後直ちに収集することができ、CCをゲノム検査(G検査)を用いて分析して、胚の能力を評価することができ、そしてその後、G検査結果に基づいて新たに戻すための胚を選択することができる。
本出願全体を通して、種々の参考文献が、本発明が属する当技術分野の最新技術を記載する。これらの参考文献の開示は、本明細書において、本開示への参照によって組み入れられる。
Claims (2)
- 卵母細胞を囲む卵丘細胞における10個の遺伝子の発現レベルを測定する工程を含む、受精すると妊娠に至る高い着床率を有する生存可能な胚を産生する卵母細胞を選択するための方法であって、前記遺伝子は、ATF3、SIAT6、PRKACA、PLA2G5、GPC6、G0S2、RBMS1、NFIC、SLC40A1およびWNT6であり、そして前記卵母細胞は、前記卵丘細胞が前記の10個の遺伝子のいずれをも過剰発現しない場合に選択される、前記方法。
- 胚を囲む卵丘細胞における10個の遺伝子の発現レベルを測定する工程を含む、妊娠に至る高い着床率を有する胚を選択するための方法であって、前記遺伝子は、ATF3、SIAT6、PRKACA、PLA2G5、GPC6、G0S2、RBMS1、NFIC、SLC40A1およびWNT6であり、そして前記胚は、前記卵丘細胞が前記の10個の遺伝子のいずれをも過剰発現しない場合に選択される、前記方法。
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