目的:为了研究冷冻的效果,体外培养(IVC)和移植到绒毛尿囊膜(CAM)对人卵巢组织卵泡结局的影响。
方法:实验研究。
方法:大学研究实验室。
方法:10例患者的新鲜和冷冻保存的卵巢组织在获得同意和机构审查委员会批准的情况下捐赠给研究。
方法:将新鲜和冻融的卵巢皮质块进行体外培养和比较(新鲜IVC与FT-IVC)。然后针对接枝到CAM(FT-CAM)的片段检查FT-IVC片段。IVC和CAM移植后,在第0、1和6天分析卵巢皮质块(4×2×1mm3)。
方法:卵泡分析包括组织学(计数和分类)和免疫组织化学(Ki67[增殖],caspase-3[凋亡],1A和1B轻链3B[自噬],p-Akt,FOXO1和p-rpS6[PI3K激活])。液滴数字聚合酶链反应进一步探索PI3K通路和卵母细胞相关基因在组织切片中的表达。
结果:在任何进行的分析中,新鲜IVC和FT-IVC组织之间均未检测到主要差异。尽管在新鲜IVC和FT-IVC组中观察到原始卵泡(PF)比例显着下降(d0vs.d6,P<.002),他们在FT-CAM组中保持稳定(d0与d6,P>.05)。在d6时,FT-CAM组的PF率也明显高于FT-IVC组(P=0.02)。重要的是,鸟类红细胞已经存在于d1植入物的30%中。在IVC期间,凋亡和自噬卵泡率增加(P<.008),但FT-CAM组仍然显著较低(P<0.01),确认CAM移植组织中的卵泡保存良好。在IVC组中建立了PI3K/FOXO途径的上调,证明PF激活,而在FT-CAM组检测到显著的通路下调(P<.03)。液滴数字聚合酶链反应测试证实了所有组的IVC和卵泡自噬期间卵母细胞的生长;然而,PI3K通路在组织和卵泡中似乎受到不同程度的调节.
结论:体外培养诱导PF耗竭,而没有额外的冷冻影响。移植到CAM通过抑制卵泡激活来保留PF池,凋亡,和自噬,可能是由于快速的移植物血运重建和/或循环的胚胎抗米勒激素。这些发现强调了在卵巢移植物中增强新血管生成的重要性,并研究了使用抗米勒激素预防PF倦怠的潜在益处。
To study the effect of freezing, in vitro culture (IVC) and grafting to chorioallantoic membrane (CAM) on follicle outcomes in human ovarian tissue.
An experimental study.
University-based research laboratory.
Fresh and cryopreserved ovarian tissue from 10 patients was donated to research with their consent and institutional review board approval.
Fresh and frozen-thawed ovarian cortical pieces were in vitro-cultured and compared (fresh-IVC vs FT-IVC). The FT-IVC fragments were then examined against fragments grafted to CAM (FT-CAM). After both IVC and CAM grafting, ovarian cortical pieces (4×2×1 mm3) were analyzed on days 0, 1, and 6.
Follicle analyses included histology (count and classification) and immunohistochemistry (Ki67 [proliferation], caspase-3 [apoptosis], 1A and 1B light chain 3B [autophagy], p-Akt, FOXO1, and p-rpS6 [PI3K activation]). Droplet digital polymerase chain reaction further explored expression of PI3K pathway- and oocyte-related genes in tissue sections.
No major differences were detected between fresh-IVC and FT-IVC tissues in any conducted analyses. Although a significant drop was observed in primordial follicle (PF) proportions in the fresh-IVC and FT-IVC groups (d0 vs. d6, P<.002), they held steady in the FT-CAM group (d0 vs. d6, P>.05). The PF rates were also significantly higher in the FT-CAM group than the FT-IVC group on d6 (P=.02). Importantly, avian erythrocytes were already present in 30% of implants from d1. Apoptotic and autophagic follicle rates increased during IVC (P<.008), but remained significantly lower in the FT-CAM group (P<.01), confirming superior follicle preservation in CAM-grafted tissue. Upregulation of the PI3K/FOXO pathway was established in the IVC groups, demonstrating PF activation, whereas significant pathway downregulation was detected in the FT-CAM group (P<.03). The droplet digital polymerase chain reaction tests confirmed oocyte growth during IVC and follicle autophagy in all groups; however, the PI3K pathway appeared to be differentially modulated in tissues and follicles.
In vitro culture induces PF depletion with no additional impact of freezing. Grafting to CAM preserves the PF pool by curbing follicle activation, apoptosis, and autophagy, probably thanks to rapid graft revascularization and/or the circulating embryonic antimüllerian hormone. These findings highlight the importance of enhancing neoangiogenesis in ovarian grafts and investigating the potential benefits of administering antimüllerian hormone to prevent PF burnout.