Reproductive Medical Center of Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, 197 Ruijin 2nd Road, Shanghai 200025, China.
Department of Obstetrics and Gynaecology, BC Children's Hospital Research Institute, University of British Columbia, Room 317, 950 West 28 Avenue, Vancouver, British Columbia, Canada V5Z 4H4.
Hum Reprod. 2018 May 1;33(5):832-843. doi: 10.1093/humrep/dey022.
Is recurrent implantation failure (RIF) associated with decreased expression of platelet and endothelial cell adhesion molecule 1 (PECAM1) and transforming growth factor β1 (TGF-β1) in the endometrium during the implantation window?
The present study demonstrates that the expression of PECAM1 and TGF-β1 is significantly decreased in the mid-secretory endometrium in women with RIF, which may account for embryo implantation failure.
RIF has become a bottleneck issue that hampers the improvement of pregnancy rates in IVF-embryo transfer (IVF-ET). The causes of RIF are complex and may involve the dysregulation of various growth factors, metabolites, and inflammatory cytokines. At present, the precise pathogenesis of RIF has not been elucidated.
STUDY DESIGN, SIZE, DURATION: This was a prospective case-control study. Endometrial tissue samples were obtained from January 2014 to December 2016 from two groups of women who had undergone IVF (RIF group, 22 women who underwent ≥3 ETs including a total of ≥4 good-quality embryos without pregnancy, control group, 18 women who conceived in their first treatment cycle). At the same time, samples were obtained from 18 women with infertility secondary to tubal factor in the early proliferative, late proliferative and mid-secretory phases of the menstrual cycle (n = 6 per group). Samples used for isolation of primary human endometrial epithelial cells and stromal cells (HEECs and HESCs) were collected in December 2017 from six women with infertility secondary to tubal factor.
PARTICIPANTS/MATERIALS, SETTING, METHODS: We investigated gene expression using integrative whole genome expression microarray analysis, including differentially expressed gene screening, principal component analysis, and functional enrichment analysis. RT-qPCR, western blotting, immunohistochemistry, immunofluorescence co-localization analysis and short hairpin RNA (shRNA) plasmid transfection in Ishikawa cell line, HEECs and HESCs were used to investigate the expression of PECAM1 and TGF-β1.
Integrative data mining of whole-genome expression profiles identified cell adhesion as a key regulator in RIF. Database retrieval and literature review screened several novel cell adhesion-related genes that might participate in embryo implantation, which include PECAM1, intercellular adhesion molecule 2 (ICAM2), integrin subunit β2 (ITGB2), selectin P (SELP) and TEK receptor tyrosine kinase (TEK). Among these targets, the mRNA and protein levels of PECAM1 were significantly lower in the RIF group than those in the control group. During the menstrual cycles of women with secondary infertility, the protein expression level of PECAM1 was the lowest in early proliferative phase, slightly increased in late proliferative phase and was the highest in mid-secretory phase. While the expression level of HOXA10, an endometrial receptivity marker, kept at a low level in early proliferative phase and increased in late proliferative phase, then maintained at a high level in the mid-secretory phase. Furthermore, TGF-β1, mediated by PECAM1, was also decreased significantly in the RIF group. Using shRNA-based approach, we demonstrated that the depletion of PECAM1 significantly decreased the expression of TGF-β1 in Ishikawa cells, as well as in primary HEECs and HESCs. These results indicated that PECAM1 and TGF-β1 might play a pivotal role in modulating endometrial receptivity.
Although we have shown that PECAM1 and TGF-β1 were down-regulated in the women with RIF, the molecular mechanism of the effect of the factors on the endometrial receptivity remain unclear.
Our findings provide insight into the contribution of PECAM1 and TGF-β1 in regulating implantation, which could be used to develop potential therapeutic methods for RIF.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the National Natural Science Foundation of China (Nos. 81771656 and 81370763), Special fund for clinical research of the Chinese Medical Association (No. 16020480664), and the Merck Serono China Research Fund for Fertility Agreement. The authors have no competing interests.
复发性种植失败（RIF）是否与子宫内膜种植窗期血小板内皮细胞黏附分子 1（PECAM1）和转化生长因子-β1（TGF-β1）表达降低有关？
本研究表明，RIF 患者的中分泌期子宫内膜中 PECAM1 和 TGF-β1 的表达显著降低，这可能是胚胎着床失败的原因。
RIF 已成为阻碍 IVF-胚胎移植（IVF-ET）妊娠率提高的瓶颈问题。RIF 的病因复杂，可能涉及各种生长因子、代谢物和炎症细胞因子的失调。目前，RIF 的精确发病机制尚未阐明。
研究设计、规模、持续时间：这是一项前瞻性病例对照研究。2014 年 1 月至 2016 年 12 月，从两组接受 IVF 的妇女中获得子宫内膜组织样本，一组是经历≥3 次 ET 包括≥4 个优质胚胎但未妊娠的妇女（RIF 组，22 例），另一组是在第一次治疗周期中怀孕的妇女（对照组，18 例）。同时，在 2017 年 12 月，从 18 例因输卵管因素导致不孕的妇女的早期增殖期、晚期增殖期和中分泌期获得用于分离原发性人子宫内膜上皮细胞和基质细胞（HEECs 和 HESCs）的样本。
参与者/材料、设置、方法：我们使用整合全基因组表达微阵列分析方法进行基因表达研究，包括差异表达基因筛选、主成分分析和功能富集分析。采用 RT-qPCR、western blot、免疫组织化学、免疫荧光共定位分析和 Ishikawa 细胞系、HEECs 和 HESCs 的短发夹 RNA（shRNA）质粒转染来研究 PECAM1 和 TGF-β1 的表达。
全基因组表达谱的综合数据挖掘确定细胞黏附作为 RIF 的关键调节剂。数据库检索和文献综述筛选出几种可能参与胚胎着床的新型细胞黏附相关基因，包括 PECAM1、细胞间黏附分子 2（ICAM2）、整合素亚基β2（ITGB2）、选择素 P（SELP）和 TEK 受体酪氨酸激酶（TEK）。在这些靶标中，RIF 组的 PECAM1 mRNA 和蛋白水平明显低于对照组。在继发性不孕妇女的月经周期中，PECAM1 的蛋白表达水平在早期增殖期最低，在晚期增殖期略有增加，在中分泌期最高。而子宫内膜容受性标志物 HOXA10 的表达水平在早期增殖期保持在较低水平，并在晚期增殖期增加，然后在中分泌期保持在较高水平。此外，TGF-β1 也通过 PECAM1 介导，在 RIF 组中显著降低。通过 shRNA 方法，我们证明 PECAM1 的耗竭显著降低了 Ishikawa 细胞以及原代 HEECs 和 HESCs 中 TGF-β1 的表达。这些结果表明 PECAM1 和 TGF-β1 可能在调节子宫内膜容受性方面发挥关键作用。
局限性/谨慎原因：尽管我们已经表明 RIF 患者的 PECAM1 和 TGF-β1 表达下调，但这些因素对子宫内膜容受性的影响的分子机制仍不清楚。
我们的研究结果为 PECAM1 和 TGF-β1 在调节着床中的作用提供了新的认识，这可能有助于开发治疗 RIF 的潜在治疗方法。
研究资助/利益冲突：本研究得到国家自然科学基金（No. 81771656 和 81370763）、中华医学会临床研究专项基金（No. 16020480664）和默克雪兰诺中国生育基金的资助。作者没有利益冲突。
