严重卵巢功能障碍患者的生育保护
Summary
我们为严重卵巢功能障碍患者提供卵巢卵泡无药物体外活化 （IVA） 的实验室程序详情。这种方法可以增加每次卵巢过度刺激的可检索卵母细胞的数量，并有利于这些患者的生育能力保护。
Abstract
卵巢功能在衰老期间和某些病理生理条件下逐渐下降，包括卡约型异常、自身免疫性疾病、化疗和放射疗法以及卵巢手术。在患有严重卵巢功能障碍的未婚妇女中，生育能力的保存对于未来的怀孕非常重要。虽然卵母细胞冷冻保存是保存生育力的既定方法，但这些患者即使在卵巢过度刺激后也只能保留数量有限的卵母细胞，导致反复刺激，以确保卵母细胞有足够的保证未来怀孕。为了解决这个问题，我们最近开发了一种无药物体外活化（IVA）程序，使我们能够刺激卵巢卵泡的早期发育到前额卵泡阶段。这些前额叶囊可以响应独特的性腺激素刺激协议，导致每次卵巢刺激的检索卵母细胞数量增加，用于冷冻保存。无药物IVA包括手术方法和卵巢刺激。在腹腔镜手术中，我们从一个或两个卵巢中取出了部分皮层。卵巢皮质组织被切成小块，以破坏河马信号通路，刺激早期卵泡的发展。这些立方体被移植到剩余的卵巢以及两个输卵管的血清下。我们已经发布了无药物IVA的外科手术和随后的卵巢刺激协议，但在这里我们介绍了无药物IVA所需的实验室方法的细节。
Introduction
卵巢功能在衰老期间逐渐下降，一些病理生理疾病，包括卡约型异常、自身免疫性疾病、化疗和放射治疗以及卵巢手术。生育力保护是患有严重卵巢功能障碍的未婚妇女保持未来怀孕潜力的最佳选择之一。在生育保护方面，目前有两种方法主要在生育领域。卵母细胞冷冻保存是一个成熟的生育保护程序，许多成功的病例已经报告1，2。另一方面，卵巢组织冷冻保存也建立了为癌症患者的生育保存，但它仍然是一个实验策略3，4。在这两种方法中，怀孕需要多个数量的成熟卵母细胞。一般来说，早产卵巢不足（POI）的患者在40岁之前成为闭经，卵巢储备低的中年妇女对卵巢刺激产生成熟的卵母细胞5、6、7的反应很差。此外，蚂蚁卵泡数量少的年轻患者也向卵巢刺激7显示POR。这些患者的可检索卵母细胞数量非常有限，即使经过适当的卵巢过度刺激，因此需要多个昂贵的程序，以确保足够的卵母细胞怀孕。
卵母细胞捐赠，其次是体外受精（IVF）与丈夫的精子和胚胎移植（ET）是这些POI和POOR患者谁很难获得自己的卵母细胞8，9，10的唯一选择。然而，卵母细胞捐赠是复杂的道德问题，以及自身免疫和怀孕并发症11，12，13，14。为了解决这些问题，需要建立使用患者自身卵母细胞的不孕症治疗。对于POI患者，我们开发了体外活化（IVA）方法，使卵泡生长成功，并产生成熟的卵母细胞，导致一些怀孕和分娩15。在IVA中，我们在腹腔镜手术切除卵巢后将卵巢皮层分割，并培养卵巢两天，用Akt刺激药物激活卵泡，然后进行异位移植回到第二次腹腔镜手术15下法洛皮管下的人工袋中。这个过程促进卵巢皮层分裂后原始、初级和次要卵泡的生长，促进河马信号中断16，随后两天培养与Akt信号刺激器17。
与严重的 POI 病例相比，卵巢储备减少的 POR 患者具有多个继发性毛囊。由于河马信号中断本身是有效的促进继发性毛囊生长16，我们最近证明成功的怀孕和分娩的POOR患者使用无药物IVA程序涉及皮质碎裂和正畸移植没有治疗Akt刺激药物。无药物IVA刺激卵巢卵泡的早期阶段，在仅进行一次手术后发育到前额卵泡阶段，并增加了为IVF-胚胎移植15，18检索卵母细胞的数量。无药物IVA方法与我们原来的IVA相比有几个优点，1） 避免培养过程中潜在的毛囊损失， 2） 尽量减少第二次手术的侵入性和成本， 3） 只涉及短期手术后卧床休息和 4） 由于正交移植而自发怀孕的可能性。我们最近发表了一篇视频文章，展示了无药物IVA19的外科手术和手术后卵巢刺激的详细协议20。在这里，我们介绍无药物IVA所需的实验室方法的详细信息。
Protocol
Representative Results
Discussion
在这份手稿中，我们展示了无药物IVA的详细实验室协议。无药物IVA是一种新的不孕症治疗方法，为POR患者与减少卵巢储备，以促进次要卵泡生长，导致产生更成熟的卵母细胞卵巢刺激后，并在成功怀孕20增加。在15名卵巢储备减少的POR患者中，这种方法实现了一次自发怀孕，体外受精-胚胎移植允许4例活产，以及1例持续妊娠19例。
为了准备?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢伊哈纳、桥本太郎、高桥正彦、吉泽裕久、麻生太郎、藤田健太郎、久藤美子、久本裕久和武松木美子支持无毒品IVA程序，以及Aaron J.W. Hsueh教授（斯坦福大学医学院，加州斯坦福）对手稿的批判性阅读和编辑。我们也感谢丽贝卡杜鲁门和格雷戈里杜鲁门插入英语旁白。这项研究得到了日本科学促进会（JSPS）、科学研究B（19H03801）和具有挑战性的探索研究（18K19624）的支持。
Materials
| 4.5 onz specimen container | FALCON | 354013 | Other products may also be suitable |
| 60mm dish | FALCON | 351007 | Other products may also be suitable |
| 50 x 50 cm sterile drape | HOGY Medical | SR-823 | Any type of sterile produsts may also be suitable |
| Disposable pippete | FALCON | 357575 | Other products may also be suitable |
| Fine scissors, Curved | WPI | #14224-G | Although other products may also be suitable, we strongly recommend use this products |
| Hot plate | TOKAI HIT | TPiE-SP | Use at operation room to maintain the temperature of dishes containing ovarian tissue before transplantation |
| Human Serum Albumin Solution | Irvine Scientific | 9988 | Medium for handling ovarian tissue |
| IVA cannule | KITAZATO | 446030 IVA-6030E | Specific cannula for tissue autografting |
| KAI medical Disposable scalpel | WPI | #5 10-A | Although other products may also be suitable, we strongly recommend use this products |
| Micro scissors, Curved | WPI | #503364 | Although other products may also be suitable, we strongly recommend use this products |
| Modified HTF Medium-HEPES | Irvine Scientific | 90126 | Medium for handling ovarian tissue |
| Sterile gauze | Osaki Medical | 15004 | Any type of sterile produsts may also be suitable |
| Swiss Tweezers, Curved Tips | KAI | #504505 | Although other products may also be suitable, we strongly recommend use this products |
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