Can trophectoderm RNA analysis predict human blastocyst competency?

ABSTRACT

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 (HSD17B1) and Cytochrome P450 Family 11 Subfamily A Member 1 (CYP11A1), while the remaining 11 transcripts were significantly up-regulated, including BCL2 Antagonist/Killer 1 (BAK1) and KH Domain Containing 1 Pseudogene 1 (KHDC1P1) 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.

Abbreviations: 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

KEYWORDS:

• Transcriptome
• RNA sequencing
• differential gene expression
• trophectoderm
• implantation competence

Ethics approval and consent to participate

The study was approved by the National Health System, A’ Administration of the Health District of Attica, General Children Hospital ‘Aghia Sofia’ in Greece (Reference/Protocol Number: 19,964/04-09-2014).

Patients participating in the study provided signed informed consent to blastocyst biopsy before Day 5 transfer.

Acknowledgments

We would like to thank all the clinical embryologists, nurses, medical doctors, and other members of staff of Genesis Athens Clinic (Greece) for their assistance with recruitment and sampling.

Disclosure statement

No potential conflict of interest was reported by the authors.

Authors’ contributions

Contributed to the concept and design of the study: PN, GK, DM; Constructed the RNA sequencing libraries, QC, RT-qPCR, and prepared the original manuscript: PN; Carried out the bioinformatics analysis: DI, PN; Carried out the blastocyst biopsies and drafted the materials and methods section: GK. Reviewed the manuscript: GK, DI, HP, JH, MT. Undertook the clinical assessment of patients and sample collection: KP. Contributed to the supervision of the project, revised the final manuscript and gave the final approval: DM. All authors have read and approved the manuscript for submission.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Medical Research Council (MRC Grant number MR/K020501-1).