Can trophectoderm RNA analysis predict human blastocyst competency?

A systematic review of the literature showed that trophectoderm biopsy could assist in the selection of healthy embryos for uterine transfer without affecting implantation rates. However, previous studies attempting to establish the relationship between trophectoderm gene expression profiles and implantation competency using either microarrays or RNA sequencing strategies, were not sufficiently optimized to handle the exceptionally low RNA inputs available from biopsied material. In this pilot study, we report that differential gene expression in human trophectoderm biopsies assayed by an ultra-sensitive next generation RNA sequencing strategy could predict blastocyst implantation competence. RNA expression profiles from isolated human trophectoderm cells were analysed with established clinical pregnancy being the primary endpoint. Following RNA sequencing, a total of 47 transcripts were found to be significantly differentially expressed between the trophectoderm cells from successfully implanted (competent) versus unsuccessful (incompetent) blastocysts. Of these, 36 transcripts were significantly down-regulated in the incompetent blastocysts, including Hydroxysteroid 17-Beta Dehydrogenase 1 (<i>HSD17B1</i>) and Cytochrome P450 Family 11 Subfamily A Member 1 (<i>CYP11A1</i>), while the remaining 11 transcripts were significantly up-regulated, including <i>BCL2</i> Antagonist/Killer 1 (<i>BAK1</i>) and KH Domain Containing 1 Pseudogene 1 (<i>KHDC1P1</i>) of which the latter was always detected in the incompetent and absent in all competent blastocysts. Ontological analysis of differentially expressed RNAs revealed pathways involved in steroidogenic processes with high confidence. Novel differentially expressed transcripts were also noted by reference to a de novo sequence assembly. The selection of the blastocyst with the best potential to support full-term pregnancy following single embryo transfer could reduce the need for multiple treatment cycles and embryo transfers. The main limitation was the low sample size (N = 8). Despite this shortcoming, the pilot suggests that trophectoderm biopsy could assist with the selection of healthy embryos for embryo transfer. A larger cohort of samples is needed to confirm these findings. <b>Abbreviations:</b> AMA: advanced maternal age; ART: assisted reproductive technology; CP: clinical pregnancy; DE: differential expression; FDR: false discovery rate; IVF: in vitro fertilization; LD PCR: long distance PCR; qRT-PCR: quantitative real-time PCR; SET: single embryo transfer; TE: trophectoderm

现有文献的系统综述显示，滋养外胚层活检（trophectoderm biopsy）可辅助筛选可用于子宫内移植的健康胚胎，且不会影响着床率。然而，既往研究尝试通过微阵列（microarrays）或RNA测序（RNA sequencing）策略，建立滋养外胚层基因表达谱与着床能力之间的关联，但这些方法未得到充分优化，无法处理活检样本中极低的RNA起始量。
在本预实验中，我们报道：通过超灵敏下一代RNA测序（next generation RNA sequencing）技术检测人类滋养外胚层活检样本的基因差异表达，可预测囊胚的着床能力。本研究以确诊临床妊娠为主要终点，对分离得到的人类滋养外胚层细胞的RNA表达谱进行了分析。经RNA测序后，成功着床（具有着床能力）囊胚与失败（无着床能力）囊胚的滋养外胚层细胞间，共发现47个存在显著差异表达的转录本（transcripts）。其中，36个转录本在无着床能力的囊胚中显著下调，包括羟类固醇17-β脱氢酶1（Hydroxysteroid 17-Beta Dehydrogenase 1，HSD17B1）与细胞色素P450家族11亚家族A成员1（Cytochrome P450 Family 11 Subfamily A Member 1，CYP11A1）；剩余11个转录本则在无着床能力的囊胚中显著上调，包括BCL2拮抗剂/杀伤因子1（BCL2 Antagonist/Killer 1，BAK1）与含KH结构域1假基因1（KH Domain Containing 1 Pseudogene 1，KHDC1P1），其中后者仅在无着床能力的囊胚中被检测到，在所有具有着床能力的囊胚中均未出现。
对差异表达RNA的基因本体分析（Ontological analysis）显示，其显著富集于类固醇生成相关通路。通过从头序列组装（de novo sequence assembly）分析，还发现了新的差异表达转录本。
在单胚胎移植（single embryo transfer，SET）中选择最有望支持足月妊娠的囊胚，可减少多次治疗周期与胚胎移植的需求。本研究的主要局限性为样本量较小（N=8）。尽管存在这一不足，但本预实验仍表明，滋养外胚层活检可辅助筛选用于胚胎移植的健康胚胎。后续需开展更大样本量的队列研究以验证本研究结果。
缩写说明：AMA：高龄产妇（advanced maternal age）；ART：辅助生殖技术（assisted reproductive technology）；CP：临床妊娠（clinical pregnancy）；DE：差异表达（differential expression）；FDR：错误发现率（false discovery rate）；IVF：体外受精（in vitro fertilization）；LD PCR：长距离聚合酶链式反应（long distance PCR）；qRT-PCR：实时定量聚合酶链式反应（quantitative real-time PCR）；SET：单胚胎移植（single embryo transfer）；TE：滋养外胚层（trophectoderm）