Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Madigan Army Medical Center, Tacoma, WA 98431-1100, USA.
Department of Clinical Investigation, Madigan Army Medical Center, Tacoma, WA 98431-1100, USA.
Hum Reprod. 2021 Jan 25;36(2):367-380. doi: 10.1093/humrep/deaa333.
Are there phase-specific changes in the early secretory (ES) phase human tubal lavage proteome that can inform and potentially optimize IVF culture media?
The human tubal lavage proteome during the ES phase relative to the menstrual phase reveals substantial differential protein abundance in pathways such as glycolysis, redox homeostasis and activation of 14-3-3 zeta-mediated signaling.
The Fallopian tube is uniquely suited to the development of the preimplantation embryo as it transits the tube during the ES phase of the menstrual cycle. Euploid cleavage-stage embryo arrest may reflect incomplete recapitulation of in-vivo conditions by current media formulations.
STUDY DESIGN, SIZE, DURATION: Proteome-wide analysis of distal tubal lavage specimens collected from 26 healthy women undergoing open microtubal anastomosis surgery from January 2013 to January 2018 was performed. Specimens were grouped by menstrual cycle phase in order to analyze phase-specific differences in protein abundance. For the murine embryo assay, single-cell embryos (N = 482) were collected from superovulated wild type C57BL/6 female mice and cultured in microdrops over 5 days for the assessment of blastocyst development.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Human tubal lavage specimens were processed for label-free mass spectrometry. Reported menstrual cycle day was confirmed by measuring serum hormones. Key protein targets in the ES phase were validated via immunoblot. The ES phase-specific increase in 14-3-3 zeta protein was confirmed via ELISA of conditioned media obtained from primary human Fallopian tube epithelial cell culture. A murine embryo assay was performed to investigate the impact of graduated concentrations of 14-3-3 zeta on the blastocyst development rate.
Comparison of the ES and menstrual phase human tubal lavage proteomes revealed 74 differentially expressed proteins with enrichment of pathways and biological processes involved in the regulation of carbohydrate metabolism, oxidative stress and cell survival. The adapter-regulator protein 14-3-3 zeta was among the most significantly increased in the ES phase. Supplementation of embryo culture media with 14-3-3 zeta at concentrations tested did not significantly improve the murine blastocyst development.
LIMITATIONS, REASONS FOR CAUTION: Although select associations were recapitulated in the conditioned media from sex steroid exposed primary human tubal epithelial cells, cell culture represents an in-vitro approximation. Changes to embryo culture media, such as protein supplementation, must undergo rigorous preclinical safety testing prior to adoption for human use.
This study represents the first description of the human Fallopian tube lavage proteome across the menstrual cycle, revealing a unique proteomic signature during the ES phase. Although supplementation of culture media with 14-3-3 zeta at appropriate concentrations showed no significant impact on the murine blastocyst development rate, other biologically plausible candidate proteins for individual or high throughput testing strategies are identified.
STUDY FUNDING/COMPETING INTEREST(S): This work was funded in part by an Army Medical Department Advanced Medical Technology Initiative grant from the United States Army Medical Research and Materiel Command's Telemedicine and Advanced Technology Research Center. There are no competing interests.
N/A.
人输卵管灌洗液早期分泌（ES）期的蛋白质组是否存在特定阶段的变化，可以为 IVF 培养提供信息并可能优化培养？
与月经周期相比，ES 期人输卵管灌洗液蛋白质组在糖酵解、氧化还原稳态和激活 14-3-3 ζ 介导的信号通路等途径中显示出大量差异的蛋白丰度。
输卵管特别适合胚胎的早期发育，因为胚胎在月经周期的 ES 期通过输卵管。整倍体卵裂期胚胎阻滞可能反映了当前培养基配方不完全再现体内条件。
研究设计、大小和持续时间：对 2013 年 1 月至 2018 年 1 月期间接受开放微管吻合术的 26 名健康女性的远端输卵管灌洗标本进行了全蛋白质组分析。根据月经周期阶段对标本进行分组，以分析蛋白质丰度的特定阶段差异。对于小鼠胚胎试验，从超排卵的野生型 C57BL/6 雌性小鼠中收集单细胞胚胎（N=482），并在微滴中培养 5 天，以评估囊胚发育。
参与者/材料、设置、方法：对人输卵管灌洗液标本进行无标记质谱分析。通过测量血清激素来确认报告的月经周期日。通过免疫印迹验证 ES 期的关键蛋白靶标。通过 ELISA 验证了 14-3-3 ζ 蛋白在 ES 期的特异性增加，ELISA 是用人输卵管上皮细胞的条件培养基进行的。进行了小鼠胚胎试验，以研究不同浓度的 14-3-3 ζ 对囊胚发育率的影响。
比较 ES 期和月经周期的人输卵管灌洗液蛋白质组，发现 74 种差异表达的蛋白质，其丰度与碳水化合物代谢、氧化应激和细胞存活调节相关的途径和生物学过程有关。适配器调节剂蛋白 14-3-3 ζ 是 ES 期增加最多的蛋白之一。在测试浓度下，将 14-3-3 ζ 添加到胚胎培养基中并没有显著提高小鼠囊胚的发育率。
局限性、谨慎的原因：尽管在暴露于性激素的人输卵管上皮细胞的条件培养基中再现了一些选择关联，但细胞培养只是一种体外近似。胚胎培养基的改变，如蛋白质补充，在用于人体之前必须经过严格的临床前安全性测试。
这项研究首次描述了人输卵管灌洗液在整个月经周期的蛋白质组，揭示了 ES 期独特的蛋白质组特征。尽管在适当浓度下用 14-3-3 ζ 补充培养基对小鼠囊胚发育率没有显著影响，但鉴定出了其他具有生物学意义的候选蛋白，用于单独或高通量测试策略。
研究资金/利益冲突：这项工作部分得到了美国陆军军医部高级医疗技术倡议从美国陆军军医研究与物资司令部远程医疗和先进技术研究中心授予的赠款资助。没有竞争利益。
无。
