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Medicine

通过卵母细胞维他化保存生育能力：临床和实验室视角

Published: September 16, 2021
doi:
10.3791/61963
, , , , , , ,
1Clinica Valle Giulia,GeneraLife IVF

Summary

卵母细胞冷冻保存被一些国际科学协会确认为后普生妇女生育保护的黄金标准。适当的临床和实验室策略可确保生育保护治疗的最大疗效、效率和安全性。

Abstract

在照顾患者未来生活质量的多功能医疗保健系统中，保持女性生育能力至关重要。卵母细胞冷冻保存被一些国际科学协会确认为后普生妇女生育保护的黄金标准，用于医疗和非医学适应症。主要医学适应症是结节性疾病、严重子宫内膜异位症等妇科疾病、影响卵巢储备的全身性疾病以及涉及早年更年期的遗传疾病。本文通过概述客观和循证咨询的建议，描述了生育保护治疗的整个临床和实验室工作。此外，它侧重于程序的有效性，并描述了充分利用卵巢储备和在尽可能短的时间内检索卵母细胞数量的最适当策略。卵巢储备的评估、理想刺激协议的定义以及卵母细胞检索、鉴定和病毒化程序以及最大限度地提高卵巢的疗效、效率和安全性的方法都得到了详细阐述。

Introduction

开发和实施有效的人类卵母细胞冷冻保存方案是生殖医学的重大突破。根据最近的证据，病毒化是冷冻保存元相II（MII）卵母细胞的最有效策略，因为它导致统计学上更高的存活率相比，缓慢冻结，独立于患者群体（不孕不育患者或卵母细胞捐赠计划）1，2，3。卵母细胞化的显著成就导致美国生殖医学学会（ASRM）和辅助生殖技术协会（SART）的实践委员会宣布这项技术对于选择性生育保护在后普生妇女中最为有效，无论是医疗还是非医学适应症4，5，6。维持生育力的医学适应症包括（一） 癌症和自身免疫性疾病，需要治疗7，如放疗、细胞毒性化疗和内分泌治疗（其对卵巢储备的有害影响与产妇年龄以及治疗的类型和剂量有关）：（二） 需要重复或彻底手术的卵巢疾病（如子宫内膜异位症）8：和 （iii） 遗传条件（如 X 脆弱）或卵巢过早衰竭。此外，对于所有因非医疗原因（也称为社会冻结）而未能完成父母目标的妇女来说，生育保护已成为一个有价值的选择。

无论生育保护的迹象如何，根据关于生育保护的主要国际准则，所有愿意使卵母细胞变质的患者都应接受适当的咨询，了解其成功的实际机会、费用、风险和程序9、10、11、12、13的局限性。最重要的是，应该清楚的是，对一组MII卵母细胞进行视网化并不能确保怀孕，但如果有必要的话，它为未来的体外受精（IVF）治疗提供了更高的成功机会。在这方面，女性在卵母细胞化时的年龄无疑是最重要的限制因素15作为晚期产妇年龄（AMA：>35岁）是女性不育的主要原因16。除了卵巢储备的逐步减少外，AMA还与卵母细胞能力受损有关，原因是有缺陷的生理途径，如新陈代谢、表观遗传调节、细胞周期检查点和美血隔离17。因此，卵子的合理数量主要取决于产妇的年龄。在36岁以下的女性中，至少需要8-10个MII卵母细胞18个，以最大限度地提高成功的机会。一般来说，卵母细胞的数量越多，成功的可能性就越大。因此，根据卵巢储备标记（如抗梅勒激素 （AMH） 水平或蚂蚁卵泡计数 （AFC） 定制卵巢刺激对于在尽可能短的时间内充分利用卵巢储备至关重要。

在整个程序的安全性是另一个关键问题，当注册患者为生育保护。临床医生应采用最佳策略，通过使用安全的方法，如腺激素释放激素 （GnRH） 拮抗协议，然后使用 GnRH 激动剂触发19和 （ii） 远程， 来最大限度地降低风险并预防 （i） 卵巢过度刺激综合征 （OHSS）。 然而，在卵母细胞检索过程中，腹膜出血、骨盆结构（尿管、肠道、附录、神经）受伤或骨盆感染的风险是可能的。最后，（三） 刺激的传统方案与超生理血清雌二醇有关，因此，在雌激素敏感疾病（如乳腺癌）中不建议使用。涉及芳香酶抑制剂（如莱罗佐尔或塔莫西芬）的协议更适合在这些情况下20，21。在实验室环境中，最广泛的卵母细胞化方案仍然是Kuwayama和同事2，23首先描述的，其中包括一个循序渐进的程序，涉及逐步添加低温保护剂（CMA）。在第一阶段（均衡/脱水），卵母细胞暴露在包含 7.5% v/v 乙二醇和 7.5% v/v 二甲基硫化物 （DMSO） 的 CPA 溶液中，而在第二阶段， 卵母细胞被转移到一个葡萄化解决方案与15%v/v乙二醇和15%v/v DMSO，加上0.5摩尔/L蔗糖。在病毒化介质中短暂孵化后，卵母细胞可以放置在专门设计的开放式低温中，最后在 -196 °C 下沉入液氮中储存，直至使用。

在这里，（一） 概述了客观和循证咨询的建议，（二） 侧重于手术的成本效益，（三） 描述了充分利用卵巢储备和在最短时间内检索卵母细胞数量的最适当策略。卵巢储备的评估、理想刺激协议的定义以及卵母细胞检索、鉴定和病毒化程序将与最大限度地提高卵巢疗效、效率和安全性的方法一起进行详细评估。由于文献中存在本协议的其他协议或改编，因此本手稿的代表性结果和讨论部分仅适用于此程序。

Protocol

1. 工作和临床咨询 注：如因不合逻辑原因需要保育的病人，确保没有预约会诊的等候名单，并尽快提供预约。 检查病史和以前的文件，并评估患者的一般健康状况。 在关系数据库中记录所有信息（包括肿瘤学家批准在癌症患者的情况下接受卵巢刺激）。 为患者提供有关手术可行性的具体咨询。解释手术的步骤（卵巢刺激、卵母细胞检索、卵母细胞病?…

Representative Results

中心生育保护计划概述 在12年期间（2008-2020年），285名妇女至少接受了一次卵母细胞检索，使收集的成熟卵子全部被检出。这些妇女中的大多数（n=250）接受了一次检索，35名妇女接受了多次检索。接受卵母细胞检索的原因归纳为4类：医疗（癌症除外）、癌症、非医学等。在250名接受卵母细胞检索的250名妇女中，8%有癌症以外的医学原因（10例子宫内膜异位症、3例…

Discussion

临床考虑

虽然已经探索了卵巢组织冷冻保存和体外成熟等新兴策略，但COS后卵母细胞体质化是保存生育力的黄金标准技术。在这种情况下，应在最短的时间内最大限度地增加检索和冷冻保存的卵母细胞数量，因为大多数癌症患者在开始癌症治疗之前可能仅从一个卵巢周期中受益。因此，适当的卵巢刺激方案对于充分利用卵巢储备和增加未来活产28</s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

没有

Materials

Collection
Equipment
Hot plate IVF TECH
Lab Markers Sigma Aldrich
Laminar Flow Hood IVF TECH Grade A air flow
Stereomicroscope Leica Leica M80
Thermometer
Test tube Warmer
Tri-gas incubator Panasonic MCO-5M-PE 02/CO2
Vacuum Pump Cook K-MAR-5200
Consumables
CSCM (Continuos single culture complete) medium Fujifilm Irvine Scientific 90165 IVF culture medium supplemented with HSA
Mineral Oil for embryo culture Fujifilm Irvine Scientific 9305
Ovum Aspiration Needle (Single lumen) Cook K-OSN-1730-B-60
Primaria Dish Corning 353803 Corning Primaria Dish 100×20 mm style standard cell culture dish
Round- bottom tubes Falcon 352001 Falcon 14ml Round Bottom Polystyrene Test tube with snap cap
Round- bottom tubes Falcon 352003 Oocyte collection tubes/ Falcon 5ml 12×75 Round Bottom Polipropilene Test tube with snap cap
Rubber Bulb Sigma Aldrich Z111589-12EA
Sterile glass Pasteur pipettes Hunter Scientific PPB150-100PL Pipette Pasteur Cotonate, 150mm, MEA e CE
Denudation
Equipment
CO2 incubator Eppendorf Galaxy 14S
Flexipet adjustable handle set Cook G18674 Stripper  holder
Gilson Pipetman Gilson 66003 p20
k-System Incubator Coopersurgical G210Invicell
Lab Markers Sigma Aldrich
Laminar Flow Hood IVF TECH Grade A air flow
Stereomicroscope Leica Leica M80
Consumables
Biopur epTIPS Rack Eppendorf 30075331 Micropipettes epTIPS Biopur 2-200 µl
Human Serum Albumin thermoFisher Scietific 9988
Hyaluronidase Fujifilm Irvine Scientific 90101 80 IU/mL of hyaluronidase enzyme in HEPES-buffered HTF
IVF culture dish (60 x 15mm) Corning 353802 Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish
IVF dish 4-well plate with sliding lid ThermoFisher Scietific 176740 Multidishes 4 wells (Nunc)
IVF One well dish Falcon 353653 Falcon 60 x 15 mm TC treated center-well IVF
Mineral Oil for embryo culture Fujifilm Irvine Scientific 9305
Modified HTF Medium Fujifilm Irvine Scientific 90126 HEPES-Buffered medium
Rubber Bulb Sigma Aldrich Z111589-12EA 1 mL for pasteur pipettes
Sterile glass Pasteur pipettes Hunter Scientific PPB150-100PL Pipette Pasteur Cotonate, 150 mm, MEA e CE
stripping pipette  tips (140 µm) Cook K-FPIP-1140-10BS-6 PIPETTE FLEXIPETS PER DENUDING
stripping pipette tips (130 µm ) Cook K-FPIP-1130-10BS-7 PIPETTE FLEXIPETS PER DENUDING
stripping pipette tips (170 µm) Cook K-FPIP-1170-10BS-5 PIPETTE FLEXIPETS PER DENUDING
Vitrification
Equipment
Electronic Timer
Flexipet adjustable handle set Cook G18674 Stripper  holder
Gilson Pipetman Gilson F123601 p200
Lab Markers Sigma Aldrich
Laminar Flow Hood IVF TECH Grade A air flow
Stainless Container for Cooling Rack Kitazato Liquid nitrogen container for vitrification
Stereomicroscope Leica Leica M80
Consumables
Biopur epTIPS Rack Eppendorf 30075331 Micropipettes epTIPS Biopur 2-200 µL
Human Serum Albumin Fujifilm Irvine Scientific 9988
IVF culture dish (60 x 15 mm) Corning 353802 Corning Primaria Falcon Dish 60 x 15 mm TC Primaria standard cell culture dish
IVF dish 6-well Oosafe OOPW-SW02 OOSAFE 6 WELL DISH WITH STRAW HOLDER
Modified HTF Medium Fujifilm Irvine Scientific 90126 HEPES-Buffered medium
stripping pipette tips (170 µm) Cook K-FPIP-1170-10BS-5 PIPETTE FLEXIPETS PER DENUDING
Vitrification Freeze kit Fujifilm Irvine Scientific 90133-so 2 Vials of ES (Equilibration Solution, 2 x 1 mL) and 2 Vials of VS (Vitrification Solution, 2 x 1 mL)
Vitrifit Coopersurgical Origio 42782001A VitriFit  Box
Warming
Equipment
Electronic Timer
Flexipet adjustable handle set Cook G18674 Stripper  holder
Gilson Pipetman Gilson F123601 p200
k-System Incubator Coopersurgical G210Invicell
Lab Markers Sigma Aldrich
Laminar Flow Hood IVF TECH Grade A air flow
Stainless Container for Cooling Rack Kitazato Liquid nitrogen container for vitrification
Stereomicroscope Leica Leica M80
Consumables
Biopur epTIPS Rack Eppendorf 30075331 Micropipettes epTIPS Biopur 2-200 µL
CSCM (Continuos single culture complete) medium Fujifilm Irvine Scientific 90165 IVF culture medium supplemented with HSA
IVF culture dish (60 x 15 mm) Corning 353802 Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish
IVF dish 4-well plate with sliding lid ThermoFisher Scietific 176740 Multidishes 4 wells (Nunc)
IVF dish 6-well Oosafe OOPW-SW02 OOSAFE® 6 WELL DISH WITH STRAW HOLDER
Mineral Oil for embryo culture Fujifilm Irvine Scientific 9305
SAtripping pipette tips (300µm) Cook K-FPIP-1300-10BS-5 PIPETTE FLEXIPETS PER DENUDING
Vitrification Thaw kit Fujifilm Irvine Scientific 90137-so 4 Vials of TS (Thawing Solution, 4 x 2 mL) + 1 Vial of DS (Dilution Solution, 1 x 2 mL) +1 Vial of WS (Washing Solution, 1 x 2 mL)
DOWNLOAD MATERIALS LIST

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Oocyte VitrificationFertility PreservationCryopreservationOocyte WarmingIVFCryoprotectantsEquilibration SolutionVitrification SolutionCryo DeviceHEPES Buffer MediumHuman Serum AlbuminStripper PipetteCentral Well IVF Dish

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Maggiulli, R., Vaiarelli, A., Cimadomo, D., Giancani, A., Tacconi, L., Fabozzi, G., Ubaldi, F. M., Rienzi, L. Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives. J. Vis. Exp. (175), e61963, doi:10.3791/61963 (2021).
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