Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies

Antonio Capalbo1,2, Filippo Maria Ubaldi1,3,4, Danilo Cimadomo1,2, Roberta Maggiulli1, Cristina Patassini2, Ludovica Dusi3, Federica Sanges4, Laura Buffo3, Roberta Venturella4, and Laura Rienzi1,3,4

1 GENERA Roma, Clinica Valle Giulia, Roma, Italy

2 GENETYX, Molecular Genetics Laboratory, Marostica, Vicenza, Italy

3 GENERA Marostica, Poliambulatorio SALUS, Marostica, Italy

4 GENERA Napoli, Clinica Ruesch, Napoli, Italy

Submitted on May 1, 2015; resubmitted on October 10, 2015; accepted on October 30, 2015

Study question: Is blastocyst biopsy and quantitative real-time PCR based comprehensive chromosome screening a consistent and re- producible approach across different biopsy practitioners?

Summary answer: The blastocyst biopsy approach provides highly consistent and reproducible laboratory and clinical outcomes across multiple practitioners from different IVF centres when all of the embryologists received identical training and use similar equipment.

What is known already: Recently there has been a trend towards trophectoderm (TE) biopsy in preimplantation genetic screening (PGS)/preimplantation genetic diagnosis (PGD) programmes. However, there is still a lack of knowledge about the reproducibility that can be obtained from multiple biopsy practitioners in different IVF centres in relation also to blastocysts of different morphology. Although it has been demonstrated that biopsy at the blastocyst stage has no impact on embryo viability, it remains a possibility that less experienced individual biopsy practitioners or laboratories performing TE biopsy may affect certain outcomes. We investigated whether TE biopsy practitioners can have an impact on the quality of the genetic test and the subsequent clinical outcomes.

Study design, size, duration: This longitudinal cohort study, between April 2013 and December 2014, involved 2586 consecutive blastocyst biopsies performed at three different IVF centres and the analysis of 494 single frozen euploid embryo transfer cycles (FEET).

Participants/materials, setting, methods: Seven biopsy practitioners performed the blastocyst biopsies in the study period and quantitative PCR was used for comprehensive chromosome screening (CCS). The same practitioner performed both the biopsy and tubing procedures for each blastocyst they biopsied. To investigate the quality of the biopsied samples, the diagnostic rate, sample-specific concurrence and the cell number retrieved in the biopsy were evaluated for each biopsy operator. Clinical outcomes following FEET cycles were stratified by biopsy operator and compared. Cellularity of the biopsy sample was also correlated with clinical outcomes.

Main results and the role of chance: The seven practitioners performed 2586 biopsies, five in centre IVF-1 and one in each of the other two IVF centres (IVF-2 and IVF-3). Overall, 2437 out of 2586 (94.2%) blastocyst biopsies resulted in a conclusive diagnosis, 119 (4.6%) showed a nonconcurrent result and 30 (1.2%) failed to amplify, suggesting the absence of TE cells in the test tube or presence of degenerated/lysed cells only. Among the samples producing a conclusive diagnosis, a mean concurrence value of 0.253 (95% CI 1⁄4 0.250–0.257) was observed. Logistic regression analysis adjusted for confounding factors showed no differences in the diagnosis rate and in the concurrence of the genetic analysis between different biopsy practitioners. An overall mean number of 7.32 cells (95% CI 1⁄4 6.82–7.81; range 2–15) were predicted from all biopsies. Higher cellularity was significantly associated with a better quality of the CCS diagnosis (P , 0.01) and with the conclusive diagnosis rate, with nonconcurrent samples showing significantly lower numbers of cells (2.1;95%CI1⁄41.52.7) compared with samples resulting in a conclusive diagnosis (mean cells number 7.5; 95% CI 1⁄4 7.1 – 7.9, P , 0.01). However, no differences were recorded between different biopsy practitioners regarding cellularity of the biopsy. Finally, logistic analysis showed no impact of the biopsy practitioners on the observed ongoing rates of implantation, biochemical pregnancy loss and miscarriage after the FEET cycles.

Limitations, reasons for caution: These data come from a restricted set of laboratories where all of the embryologists received identical training and use identical equipment. A single TE biopsy method and CCS technology was used and these data particularly apply to PGS programmes using blastocyst biopsy without zona opening at the cleavage stage and using qPCR-based CCS. To make firm conclusions on the potential impact of biopsy on biochemical pregnancy loss and miscarriages according to practitioner and biopsy cellularity, a larger sample size is needed.

Wider implications of the findings: We reported a very high consistency and reproducibility of the blastocyst biopsy approach coupled with qPCR-based CSS for both genetic and clinical outcomes across different practitioners working in different IVF centres when appropriate training is provided and when the same laboratory setting is used. These data are important considering the trend towards the use of blasto- cyst biopsy worldwide for PGD/PGS applications.

Study funding/competing interest(s): None.

Key words: blastocyst biopsy / preimplantation genetic screening / aneuploidies / embryo selection / blastocyst evaluation