ProblemAlthough endometrial receptivity is a key factor in influencing implantation in both naturally conceived and assisted reproductive technology (ART) cycles, very little is known about the endometrium milieu around the time of implantation. Previous studies have demonstrated the presence of several cytokines in the endometrium that affect implantation. However, there is lacking data about the presence of immune cell subtypes within the endometrium and in the uterine cavity at the time of implantation.Method of studyThis study was approved by the Institutional Review Board (# 225589). The study was designed as a prospective observational cohort study between May 2021 and December 2022 at a single academic‐based fertility center. All patients underwent at least one In Vitro Fertilization (IVF) cycle and have frozen embryos. Twenty‐four participants were recruited for this study which was conducted during the frozen embryo transfer (FET) cycle regardless of the outcome of previous cycles. Two samples were acquired from each subject, denoted as lower and upper. A trial transfer catheter was introduced under ultrasound guidance into the lower uterine segment. Upon removal, the tip was rinsed in IMDM medium containing 10% FBS (lower uterus). A transfer catheter was then loaded with the embryo that was placed in the upper uterus under ultrasound guidance. The tip of the transfer catheter was rinsed in separate aliquot of the above media (upper uterus). After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD66b, CD14, CD16, CD56 and acquired on Sony SP6800 Spectral Analyzer.ResultsUpon staining the pelleted cells, we were able to identify viable leukocytes from samples obtained from both, upper and lower uterus (0.125 × 106 cells ± SD 0.32), (0.123 × 106 cells ± SD 0.12), respectively. Among total viable cells, there was no significant difference in both percent and number of CD45+ cells between the upper and lower uterus (9.88% ± 6.98 SD, 13.67% ± 9.79 SD, p = .198) respectively. However, there was significantly higher expression of CD3+ (p = .006), CD19+ (p = .032) and CD14+ (p = .019) cells in samples collected from upper compared to lower uterus. Within all CD3+ cells, we found that gamma delta T cells (GDT) were the major population of T cells in both upper and lower uterus. In contrast, CD8+ T cells were significantly higher in the lower uterus when compared to the upper uterus (p = .009). There was no statistically significant difference in the expression of CD4+ T cells, T regulatory cells (CD4+CD25+CD127‐), NK cells (CD56+), neutrophils (CD66b+) and FcγRIII+ cells (CD16+) between upper and lower uterus.ConclusionsWe believe the immune milieu at the time of embryo transfer will affect implantation. Understanding the composition of immune cells will guide further research in identifying optimal immune milieus that favor implantation. Comprehensive analysis of endometrium is expected to lead to new diagnostic and therapeutic approaches to improve IVF outcomes.

中文翻译：

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胚胎移植时子宫液中免疫细胞的评估

问题尽管子宫内膜容受性是影响自然受孕和辅助生殖技术 (ART) 周期中着床的关键因素，但人们对着床时的子宫内膜环境知之甚少。先前的研究表明，子宫内膜中存在多种影响着床的细胞因子。然而，缺乏有关子宫内膜内和植入时子宫腔内免疫细胞亚型存在的数据。研究方法本研究得到了机构审查委员会的批准（#225589）。该研究被设计为 2021 年 5 月至 2022 年 12 月在单一学术生育中心进行的一项前瞻性观察队列研究。所有患者均接受过至少一个体外受精 (IVF) 周期并拥有冷冻胚胎。这项研究招募了 24 名参与者，该研究是在冷冻胚胎移植 (FET) 周期内进行的，无论之前周期的结果如何。从每个受试者处获取两个样本，表示为下样本和上样本。在超声引导下将试验转移导管引入子宫下段。取出后，将尖端在含有 10% FBS（子宫下部）的 IMDM 培养基中冲洗。然后将胚胎装入转移导管，并在超声引导下放置在子宫上部。将转移导管的尖端用上述培养基的单独等分试样（子宫上部）冲洗。离心后，对沉淀细胞进行以下表面标记物染色：CD45、CD3、CD19、CD4、CD8、gamma delta TCR、CD25、CD127、CD66b、CD14、CD16、CD56，并在 Sony SP6800 光谱分析仪上获取。 结果细胞，我们能够从上子宫和下子宫（0.125 × 106细胞 ± SD 0.32), (0.123 × 106细胞±SD 0.12），分别。在总活细胞中，上下子宫之间 CD45+ 细胞的百分比和数量没有显着差异（9.88% ± 6.98 SD、13.67% ± 9.79 SD、p= .198) 分别。然而，CD3+ 的表达显着升高（p= .006), CD19+ (p= .032) 和 CD14+ (p= .019) 与下子宫相比，从上子宫收集的样本中的细胞。在所有 CD3+ 细胞中，我们发现 γδT 细胞 (GDT) 是上子宫和下子宫中 T 细胞的主要群体。相比之下，与上子宫相比，下子宫的 CD8+ T 细胞明显更高（p=.009）。上下子宫间CD4+ T细胞、调节性T细胞（CD4+CD25+CD127-）、NK细胞（CD56+）、中性粒细胞（CD66b+）和FcγRIII+细胞（CD16+）的表达量无统计学差异。结论我们认为胚胎移植时的免疫环境会影响着床。了解免疫细胞的组成将指导进一步的研究，以确定有利于植入的最佳免疫环境。对子宫内膜的综合分析有望带来新的诊断和治疗方法，以改善体外受精的结果。