Binder N K, Evans J, Gardner D K, Salamonsen L A, Hannan N J
Department of Zoology, University of Melbourne, Parkville 3010, Australia Translational Obstetrics Group, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Heidelberg, VIC 3084, Australia.
MIMR-PHI Institute for Medical Research, 27-31 Wright St, Clayton 3168, Australia Department of Physiology, Monash University, Clayton, Australia.
Hum Reprod. 2014 Oct 10;29(10):2278-86. doi: 10.1093/humrep/deu211. Epub 2014 Aug 14.
Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation?
VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development.
Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth.
STUDY DESIGN, SIZE, DURATION: Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment).
PARTICIPANTS/MATERIALS, SETTING, METHODS: Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and fetal development.
Western blot analysis revealed the presence of VEGF121 and 165 isoforms in human uterine fluid. Time-lapse microscopy analysis revealed that VEGF (n = 22) and VEGF121 (n = 23) treatment significantly reduced the preimplantation mouse embryo time to cavitation (P < 0.05). VEGF and VEGF165 increased both blastocyst cell number (VEGF n = 27; VEGF165 n = 24: P < 0.001) and outgrowth (n = 15/treatment: 66 h, P < 0.001; 74, 90, 98 and 114 h, P < 0.01) on fibronectin compared with control. Furthermore, rhVEGF improved implantation rates and enhanced fetal limb development (P < 0.05).
LIMITATIONS, REASONS FOR CAUTION: Due to the nature of this work, embryo development and implantation was only examined in the mouse.
The absence or reduction in levels of VEGF during the preimplantation period likely affects key events during embryo development, implantation and placentation. The potential for improvement of clinical IVF outcomes by the addition of VEGF to human embryo culture media needs further investigation.
STUDY FUNDING/COMPETING INTERESTS: This study was supported by a University of Melbourne Early Career Researcher Grant #601040, the NHMRC (L.A.S., Program grant #494802; Fellowship #1002028; N.J.H., Fellowship # 628927; J.E.; project grant #1047756) and L.A.S., Monash IVF Research and Education Foundation. N.K.B. was supported by an Australian Postgraduate Award. Work at PHI-MIMR Institute was also supported by the Victorian Government's Operational Infrastructure Support Program. There are no conflicts of interest to declare.
血管内皮生长因子（VEGF）在早期胚胎发育和着床过程中是否发挥重要作用？
VEGF在小鼠植入前胚胎发育过程中发挥关键作用，对空泡化时间、囊胚细胞数量和生长、着床率以及胎儿肢体发育均有有益影响。
胚胎着床需要母体细胞与孕体之间的同步对话。排卵后，子宫内膜腺体的分泌物增加并积聚在子宫腔内。这些分泌物含有在植入前期支持孕体的重要介质。此前，我们证明不明原因不孕症女性子宫腔内的VEGFA显著减少。功能研究表明，VEGF显著增强子宫内膜上皮细胞的黏附特性和胚胎生长。
研究设计、规模、持续时间：从有生育能力的女性获取人子宫内膜灌洗液（n = 6）。对4周龄的瑞士小鼠进行超排卵处理，并与瑞士雄性小鼠交配以获取胚胎，用于体外VEGF处理。在胚胎移植前评估植入前胚胎发育情况（每个处理组/输出n = 19 - 30）。将受体F1雌性小鼠（8 - 12周龄）与输精管结扎的雄性小鼠交配以诱导假孕，然后进行胚胎移植。在妊娠第14.5天，收集子宫角以分析着床率以及胎盘和胎儿发育情况（每个处理n = 14 - 19）。
参与者/材料、环境、方法：通过蛋白质免疫印迹分析评估灌洗液，以确定存在的VEGF异构体。用重组人（rh）VEGF或VEGF异构体121和165处理小鼠胚胎。使用延时显微镜对植入前胚胎发育进行定量分析。囊胚（i）进行细胞数量染色，（ii）转移到包被纤连蛋白的孔中以检查滋养层生长，或（iii）转移到假孕受体中以分析着床率、胎盘和胎儿发育情况。
蛋白质免疫印迹分析显示人子宫液中存在VEGF121和165异构体。延时显微镜分析显示，VEGF（n = 22）和VEGF121（n = 23）处理显著缩短了植入前小鼠胚胎的空泡化时间（P < 0.05）。与对照组相比，VEGF和VEGF165增加了囊胚细胞数量（VEGF n = 27；VEGF165 n = 24：P < 0.001）和在纤连蛋白上的生长（每个处理n = 15：66小时，P < 0.001；74、90、98和114小时，P < 0.01）。此外，rhVEGF提高了着床率并促进了胎儿肢体发育（P < 0.05）。
局限性、谨慎的原因：由于这项研究的性质，仅在小鼠中研究了胚胎发育和着床情况。
植入前期VEGF水平的缺失或降低可能会影响胚胎发育、着床和胎盘形成过程中的关键事件。向人胚胎培养基中添加VEGF以改善临床体外受精结果的潜力需要进一步研究。
研究资金/利益冲突：本研究得到墨尔本大学早期职业研究员资助#601040、澳大利亚国家卫生与医学研究委员会（L.A.S.，项目资助#494802；奖学金#1002028；N.J.H.，奖学金#628927；J.E.，项目资助#1047756）以及L.A.S.、莫纳什体外受精研究与教育基金会的支持。N.K.B.获得澳大利亚研究生奖学金支持。PHI - MIMR研究所的工作也得到维多利亚州政府运营基础设施支持计划的支持。无利益冲突声明。
