Isolation of pre-antral follicles from human ovarian medulla tissue
Isolation of pre-antral follicles from human ovarian medulla tissue
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DOI：
10.1093/humrep/deq318
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发表时间：
2011-01-01
影响因子：
6.1
通讯作者：
Andersen, Claus Yding
中科院分区：
文献类型：
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作者：
Kristensen, Stine Gry;Rasmussen, Annette;Andersen, Claus Yding
背景技术背景：冷冻保存卵巢组织以保存生育能力是基于卵巢皮质包含绝大多数卵泡储备，而剩余的组织髓质被丢弃。本研究描述了一种温和的方法，分离前腔卵泡从人卵巢髓质和评估其卵泡content.METHODS：髓质收集40名女孩/妇女年龄3-35岁进行冷冻保存的卵巢皮质的发展。对所有患者进行卵泡密度评估，并从22例患者中分离出腔前卵泡。中性红（NR）染色的基础上，用胶原酶IV和Liberase嗜热菌蛋白酶培养基的混合物酶消化，分离存活的腔前卵泡。髓质的卵泡密度从0到9824个卵泡/克髓质不等，3-9岁年龄组的卵泡密度显著高于年龄较大的年龄组（10-35岁）（P < 0.001）。采用酶消化结合NR鉴定卵泡的方法，从髓质中获得了大量的分离的、有活力的腔前卵泡，共收集到3607个卵泡并进行了分类。原始卵泡和生长卵泡的比例随年龄的增长而降低和升高（P < 0.0001和P < 0.0001），
BACKGROUND: Cryopreservation of ovarian tissue for fertility preservation is based on the ovarian cortex that contains the vast majority of the follicular reserve, while the remaining tissue, the medulla is discarded. The present study describes the development of a gentle method for isolating pre-antral follicles from human ovarian medulla and evaluating its follicular content.METHODS: Medulla was collected from 40 girls/women aged 3-35 years undergoing cryopreservation of the ovarian cortex. Follicle density was assessed for all patients and pre-antral follicles were isolated from 22 patients. On the basis of the neutral red (NR) staining of follicles and enzymatic digestion with a mixture of Collagenase IV and Liberase Thermolysin Medium, viable pre-antral follicles were isolated.RESULTS: NR accumulated in follicles resulting in a distinct red staining within the medulla. Follicle density of the medulla varied from 0 to 9824 follicles/gram of medulla and was significantly higher (P < 0.001) in the 3-9-year age group when compared with older groups (10-35 years). Enzymatic digestion combined with follicle identification by NR yielded a high output of isolated and viable pre-antral follicles from medulla, of which, 3607 follicles were collected and classified. The percentage of primordial and growing follicles decreased and increased, respectively, with age (P < 0.0001 and