Skory Robin M, Xu Yuanming, Shea Lonnie D, Woodruff Teresa K
Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA Center for Reproductive Science, Northwestern University, Evanston, IL 60208, USA.
Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, Evanston, IL 60201, USA.
Hum Reprod. 2015 Jun;30(6):1386-95. doi: 10.1093/humrep/dev052. Epub 2015 Mar 16.
Can cultured follicles model the ovarian cycle, including follicular- and luteal-phase hormone synthesis patterns and ovulation?
Under gonadotrophin stimulation, murine follicles grown in an encapsulated three-dimensional system ovulate in vitro and murine and human follicle hormone synthesis mimics follicular and luteal phases expected in vivo.
Studies of the human ovary and follicle function are limited by the availability of human tissue and lack of in vitro models. We developed an encapsulated in vitro follicle growth (eIVFG) culture system, which preserves 3D follicular structure. Thus far, the alginate system has supported the culture of follicles from mice, dog, rhesus macaque, baboon and human. These studies have shown that cultured follicles synthesize steroid hormones similar to those observed during the follicular phase in vivo.
STUDY DESIGN, SIZE, DURATION: Cultured murine follicles were treated with human chorionic gonadotrophin (hCG) and epidermal growth factor (EGF) and either assayed for luteinization or removed from alginate beads and assayed for ovulation. Human follicles were also cultured, treated with follicle-stimulating hormone (FSH), hCG and EGF to mimic gonadotrophin changes throughout the ovarian cycle, and culture medium was assayed for hormone production.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine and human follicles were cultured in alginate hydrogel and hormone production [17β-estradiol, progesterone, inhibin A, inhibin B, activin A and anti-Müllerian hormone (AMH)] was quantified in medium by enzyme-linked immuno assay (ELISA). Human ovarian tissue was acquired from females between 6 and 34 years of age with a cancer diagnosis. These participants were undergoing ovarian tissue cryopreservation at National Physicians Cooperative sites as part of the Oncofertility Consortium.
When grown in this system, 96% of mouse follicles ovulated in response to hCG and released meiotically competent eggs. Ovulated follicles recapitulated transcriptional, morphologic and hormone synthesis patterns post-luteinizing hormone (LH/hCG). In addition to rodent follicles, individual human follicles secreted steroid and peptide hormones that mimicked the patterns of serum hormones observed during the menstrual cycle.
LIMITATIONS, REASONS FOR CAUTION: This was a descriptive study of an in vitro model of ovulation and the ovarian hormone cycle. The ovulation studies were limited to murine tissue and further studies are needed to optimize conditions using other species.
The eIVFG system reliably phenocopies the in vivo ovarian cycle and provides a new tool to study human follicle biology and the influence of cycling female hormones on other tissue systems in vitro.
STUDY FUNDING/COMPETING INTERESTS: This work was supported by NIH U54 HD041857, NIH U54 HD076188, NIH UH2 E5022920, NIH UH3 TR001207 and F30 AG040916 (R.M.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
培养的卵泡能否模拟卵巢周期，包括卵泡期和黄体期的激素合成模式以及排卵过程？
在促性腺激素刺激下，在三维封装系统中培养的小鼠卵泡能够在体外排卵，且小鼠和人类卵泡的激素合成可模拟体内预期的卵泡期和黄体期。
对人类卵巢和卵泡功能的研究受到人体组织可用性和缺乏体外模型的限制。我们开发了一种体外卵泡生长（eIVFG）封装培养系统，该系统可保留卵泡的三维结构。到目前为止，藻酸盐系统已支持小鼠、犬、恒河猴、狒狒和人类卵泡的培养。这些研究表明，培养的卵泡合成的甾体激素与体内卵泡期观察到的类似。
研究设计、规模、持续时间：用人类绒毛膜促性腺激素（hCG）和表皮生长因子（EGF）处理培养的小鼠卵泡，然后检测其黄体化情况，或者从藻酸盐珠中取出检测排卵情况。人类卵泡也进行培养，用促卵泡激素（FSH）、hCG和EGF处理以模拟整个卵巢周期中促性腺激素的变化，并检测培养基中的激素产生情况。
参与者/材料、环境、方法：将小鼠和人类卵泡培养在藻酸盐水凝胶中，通过酶联免疫吸附测定（ELISA）对培养基中的激素产生情况[17β-雌二醇、孕酮、抑制素A、抑制素B、激活素A和抗苗勒管激素（AMH）]进行定量分析。人类卵巢组织取自6至34岁被诊断患有癌症的女性。这些参与者作为生育力保护联盟的一部分，正在国家医师合作中心进行卵巢组织冷冻保存。
在该系统中培养时，96%的小鼠卵泡对hCG有反应并排卵，释放出具有减数分裂能力的卵子。排卵后的卵泡重现了促黄体生成素（LH/hCG）作用后的转录、形态和激素合成模式。除了啮齿动物卵泡外，单个人类卵泡分泌的甾体和肽类激素模拟了月经周期中观察到的血清激素模式。
局限性、谨慎原因：这是一项关于排卵和卵巢激素周期体外模型的描述性研究。排卵研究仅限于小鼠组织，需要进一步研究以优化其他物种的实验条件。
eIVFG系统可靠地模拟了体内卵巢周期，并为研究人类卵泡生物学以及体外循环女性激素对其他组织系统的影响提供了新工具。
研究资金/利益冲突：这项工作得到了美国国立卫生研究院（NIH）U54 HD041857、NIH U54 HD076188、NIH UH2 E5022920、NIH UH3 TR001207和F30 AG040916（R.M.S.）的支持。内容仅由作者负责，不一定代表NIH的官方观点。资助者在研究设计、数据收集和分析、决定发表或撰写稿件方面没有参与。
