Gameto Inc., New York, NY, USA.
Ruber Juan Bravo University Hospital, Eugin Group, Madrid, Spain.
Hum Reprod. 2023 Dec 4;38(12):2456-2469. doi: 10.1093/humrep/dead205.
Can in vitro maturation (IVM) and developmental competence of human oocytes be improved by co-culture with ovarian support cells (OSCs) derived from human-induced pluripotent stem cells (hiPSCs)?
OSC-IVM significantly improves the rates of metaphase II (MII) formation and euploid Day 5 or 6 blastocyst formation, when compared to a commercially available IVM system.
IVM has historically shown highly variable performance in maturing oocytes and generating oocytes with strong developmental capacity, while limited studies have shown a positive benefit of primary granulosa cell co-culture for IVM. We recently reported the development of OSCs generated from hiPSCs that recapitulate dynamic ovarian function in vitro.
STUDY DESIGN, SIZE, DURATION: The study was designed as a basic science study, using randomized sibling oocyte specimen allocation. Using pilot study data, a prospective sample size of 20 donors or at least 65 oocytes per condition were used for subsequent experiments. A total of 67 oocyte donors were recruited to undergo abbreviated gonadotropin stimulation with or without hCG triggers and retrieved cumulus-oocyte complexes (COCs) were allocated between the OSC-IVM or control conditions (fetal-like OSC (FOSC)-IVM or media-only IVM) in three independent experimental design formats. The total study duration was 1 April 2022 to 1 July 2023.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte donors between the ages of 19 and 37 years were recruited for retrieval after informed consent, with assessment of anti-Mullerian hormone, antral follicle count, age, BMI and ovarian pathology used for inclusion and exclusion criteria. In experiment 1, 27 oocyte donors were recruited, in experiment 2, 23 oocyte donors were recruited, and in experiment 3, 17 oocyte donors and 3 sperm donors were recruited. The OSC-IVM culture condition was composed of 100 000 OSCs in suspension culture with hCG, recombinant FSH, androstenedione, and doxycycline supplementation. IVM controls lacked OSCs and contained either the same supplementation, FSH and hCG only (a commercial IVM control), or FOSCs with the same supplementation (Media control). Experiment 1 compared OSC-IVM, FOSC-IVM, and a Media control, while experiments 2 and 3 compared OSC-IVM and a commercial IVM control. Primary endpoints in the first two experiments were the MII formation (i.e. maturation) rate and morphological quality assessment. In the third experiment, the fertilization and embryo formation rates were assessed with genetic testing for aneuploidy and epigenetic quality in blastocysts.
We observed a statistically significant improvement (∼1.5×) in maturation outcomes for oocytes that underwent IVM with OSCs compared to control Media-IVM and FOSC-IVM in experiment 1. More specifically, the OSC-IVM group yielded a MII formation rate of 68% ± 6.83% SEM versus 46% ± 8.51% SEM in the Media control (P = 0.02592, unpaired t-test). FOSC-IVM yielded a 51% ± 9.23% SEM MII formation rate which did not significantly differ from the media control (P = 0.77 unpaired t-test). Additionally, OSC-IVM yielded a statistically significant ∼1.6× higher average MII formation rate at 68% ± 6.74% when compared to 43% ± 7.90% in the commercially available IVM control condition (P = 0.0349, paired t-test) in experiment 2. Oocyte morphological quality between OSC-IVM and the controls did not significantly differ. In experiment 3, OSC-IVM oocytes demonstrated a statistically significant improvement in Day 5 or 6 euploid blastocyst formation per COC compared to the commercial IVM control (25% ± 7.47% vs 11% ± 3.82%, P = 0.0349 logistic regression). Also in experiment 3, the OSC-treated oocytes generated blastocysts with similar global and germline differentially methylated region epigenetic profiles compared commercial IVM controls or blastocysts after either conventional ovarian stimulation.
N/A.
LIMITATIONS, REASONS FOR CAUTION: While the findings of this study are compelling, the cohort size remains limited and was powered on preliminary pilot studies, and the basic research nature of the study limits generalizability compared to randomized control trials. Additionally, use of hCG-triggered cycles results in a heterogenous oocyte cohort, and potential differences in the underlying maturation state of oocytes pre-IVM may limit or bias findings. Further research is needed to clarify and characterize the precise mechanism of action of the OSC-IVM system. Further research is also needed to establish whether these embryos are capable of implantation and further development, a key indication of their clinical utility.
Together, these findings demonstrate a novel approach to IVM with broad applicability to modern ART practice. The controls used in this study are in line with and have produced similar to findings to those in the literature, and the outcome of this study supports findings from previous co-culture studies that found benefits of primary granulosa cells on IVM outcomes. The OSC-IVM system shows promise as a highly flexible IVM approach that can complement a broad range of stimulation styles and patient populations. Particularly for patients who cannot or prefer not to undergo conventional gonadotropin stimulation, OSC-IVM may present a viable path for obtaining developmentally competent, mature oocytes.
STUDY FUNDING/COMPETING INTEREST(S): A.D.N., A.B.F., A.G., B.P., C.A., C.C.K., F.B., G.R., K.S.P., K.W., M.M., P.C., S.P., and M.-J.F.-G. are shareholders in the for-profit biotechnology company Gameto Inc. P.R.J.F. declares paid consultancy for Gameto Inc. P.C. also declares paid consultancy for the Scientific Advisory Board for Gameto Inc. D.H.M. has received consulting services from Granata Bio, Sanford Fertility and Reproductive Medicine, Gameto, and Buffalo IVF, and travel support from the Upper Egypt Assisted Reproduction Society. C.C.K., S.P., M.M., A.G., B.P., K.S.P., G.R., and A.D.N. are listed on a patent covering the use of OSCs for IVM: U.S. Provisional Patent Application No. 63/492,210. Additionally, C.C.K. and K.W. are listed on three patents covering the use of OSCs for IVM: U.S. Patent Application No. 17/846,725, U.S Patent Application No. 17/846,845, and International Patent Application No.: PCT/US2023/026012. C.C.K., M.P.S., and P.C. additionally are listed on three patents for the transcription factor-directed production of granulosa-like cells from stem cells: International Patent Application No.: PCT/US2023/065140, U.S. Provisional Application No. 63/326,640, and U.S. Provisional Application No. 63/444,108. The remaining authors have no conflicts of interest to declare.
人类诱导多能干细胞（hiPSC）来源的卵巢支持细胞（OSC）共培养能否提高人类卵母细胞的体外成熟（IVM）和发育能力？
与商业可用的 IVM 系统相比，OSC-IVM 可显著提高中期 II（MII）形成和第 5 或 6 天的整倍体囊胚形成率。
IVM 在卵母细胞成熟和产生具有强大发育能力的卵母细胞方面表现出高度可变的性能，而有限的研究表明初级颗粒细胞共培养对 IVM 有积极的益处。我们最近报道了从 hiPSC 中开发的 OSC，这些 OSC 在体外再现了动态卵巢功能。
研究设计、大小、持续时间：该研究设计为基础科学研究，采用随机同卵卵母细胞标本分配。使用初步研究数据，后续实验使用至少 65 个卵母细胞或每个条件下至少 20 个供体的前瞻性样本量。共招募了 67 名卵母细胞供体接受促性腺激素刺激，并在三个独立的实验设计格式中分配卵丘-卵母细胞复合物（COC）进行 OSC-IVM 或对照条件（胎儿样 OSC（FOSC）-IVM 或仅培养基 IVM）。总研究持续时间为 2022 年 4 月 1 日至 2023 年 7 月 1 日。
参与者/材料、设置、方法：在知情同意后，招募年龄在 19 至 37 岁之间的卵母细胞供体进行采集，使用抗苗勒管激素、窦卵泡计数、年龄、BMI 和卵巢病理学来评估纳入和排除标准。在实验 1 中，招募了 27 名卵母细胞供体，在实验 2 中，招募了 23 名卵母细胞供体，在实验 3 中，招募了 17 名卵母细胞供体和 3 名精子供体。OSC-IVM 培养条件由悬浮培养中的 100,000 个 OSC 组成，添加 hCG、重组 FSH、雄烯二酮和多西环素。IVM 对照物缺乏 OSC，并含有相同的补充剂、仅 FSH 和 hCG（商业 IVM 对照物）或具有相同补充剂的 FOSC（培养基对照物）。实验 1 比较了 OSC-IVM、FOSC-IVM 和培养基对照物，而实验 2 和实验 3 比较了 OSC-IVM 和商业 IVM 对照物。前两个实验的主要终点是中期 II（即成熟）形成率和形态质量评估。在第三个实验中，通过遗传测试评估胚胎的受精和形成率以及囊胚的非整倍体和表观遗传质量。
我们观察到与对照组培养基-IVM 和 FOSC-IVM 相比，OSC-IVM 显著提高了卵母细胞成熟（即成熟）结果，在实验 1 中约为 1.5 倍。更具体地说，OSC-IVM 组的中期 II 形成率为 68%±6.83%SEM，而培养基对照组为 46%±8.51%SEM（P=0.02592，未配对 t 检验）。FOSC-IVM 产生的中期 II 形成率约为 51%±9.23%SEM，与培养基对照组无显著差异（P=0.77 未配对 t 检验）。此外，与商业上可用的 IVM 对照条件（P=0.0349，配对 t 检验）相比，OSC-IVM 组还产生了统计学上显著约 1.6 倍的更高平均中期 II 形成率，为 68%±6.74%，在实验 2 中。OSC-IVM 和对照组之间的卵母细胞形态质量没有显著差异。在实验 3 中，与商业 IVM 对照物相比，OSC-IVM 组第 5 或第 6 天整倍体囊胚形成率显著提高（25%±7.47%比 11%±3.82%，P=0.0349 逻辑回归）。同样在实验 3 中，与商业 IVM 对照物或常规卵巢刺激后获得的囊胚相比，经 OSC 处理的卵母细胞产生的囊胚具有相似的全局和生殖系差异甲基化区域表观遗传特征。
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局限性、谨慎的原因：尽管这项研究的结果令人信服，但队列规模仍然有限，并且是基于初步的试点研究进行的，与随机对照试验相比，基础研究性质限制了其普遍性。此外，使用 hCG 触发的周期会导致卵母细胞队列异质，IVM 前卵母细胞的潜在成熟状态差异可能限制或偏向发现。进一步的研究需要阐明和描述 OSC-IVM 系统的确切作用机制。进一步的研究还需要确定这些胚胎是否能够着床和进一步发育，这是其临床应用的关键指标。
总的来说，这些发现展示了一种新的 IVM 方法，具有广泛的现代 ART 实践应用。本研究中使用的对照物与文献中的发现一致，并且该研究的结果支持以前的共培养研究的发现，这些研究发现初级颗粒细胞对 IVM 结果有好处。OSC-IVM 系统显示出作为一种高度灵活的 IVM 方法的前景，可以补充广泛的刺激方式和患者群体。特别是对于不能或不愿接受常规促性腺激素刺激的患者，OSC-IVM 可能为获得具有发育能力的成熟卵母细胞提供了一种可行的途径。
研究资金/利益冲突：A.D.N.、A.B.F.、A.G.、B.P.、C.A.、C.C.K.、F.B.、G.R.、K.S.P.、K.W.、M.M.、P.C.、S.P.和 M.-J.F.-G.是营利性生物技术公司 Gameto Inc. 的股东。P.R.J.F. 为 Gameto Inc. 担任顾问。P.C. 还担任 Gameto Inc. 科学顾问委员会成员。D.H.M. 曾为 Granata Bio、Sanford Fertility and Reproductive Medicine、Gameto 和 Buffalo IVF 提供咨询服务，并为埃及上埃及辅助生殖学会提供旅行支持。C.C.K.、S.P.、M.M.、A.G.、B.P.、K.S.P.、G.R.和 A.D.N. 在一项涵盖 OSC 用于 IVM 的专利中列出：美国临时专利申请号 63/492,210。此外，C.C.K. 和 K.W. 还在三篇涵盖 OSC 用于 IVM 的专利中列出：美国专利申请号 17/846,725、美国专利申请号 17/846,845 和国际专利申请号：PCT/US2023/026012。C.C.K.、M.P.S. 和 P.C. 还在三篇涵盖 OSC 用于 IVM 的专利中列出：国际专利申请号 PCT/US2023/065140、美国临时专利申请号 63/326,640 和美国临时专利申请号 63/444,108。C.C.K.、M.P.S. 和 P.C. 还在三篇涵盖 OSC 用于 IVM 的专利中列出：国际专利申请号 PCT/US2023/065140、美国临时专利申请号 63/326,640 和美国临时专利申请号 63/444,108。其余作者没有利益冲突需要声明。
