Trophectoderm biopsy does not impact embryo viability after vitrification: results from a multicentre prospective study

Study question: Does trophectoderm biopsy technique increase blastocysts’ vulnerability to vitrification/warming injuries? Summary answer: Trophectoderm biopsy does not impact post-thawing behaviour of vitrified blastocysts. Only blastocyst re-expansion and degeneration grade after warming are predictive of blastocysts’ implantation potential. What is known already: The optimization of preimplantation genetic diagnosis for aneuploidies (PGD-A) cycles requires cryopreservation to obtain a reliable diagnosis, and the employment of an efficient vitrification program to preserve blastocyst developmental competence. Previous studies have shown that trophectoderm biopsy has no impact on embryo viability in fresh cycles, however, there is still a lack of knowledge about the impact of this procedure on blastocyst viability after warming. The developmental potential of warmed blastocysts has been previously evaluated in terms of re-expansion grade that has been reported as a predictive factor of clinical outcomes. Study design, size, duration: A multicenter prospective cohort study was performed between June 2016 and December 2016, including all the patients who underwent warming cycle with single blastocyst transfer (SET). A total of 383 warmed blastocysts were included in the study (258 from PGD-A cycles and 125 from standard ICSI cycles). 285 SETs had a conclusive clinical outcome when writing this abstract (197 and 88 in PGD-A and standard cycles, respectively). Participants/materials, setting, methods: The female age was 37.7 ± 3.5 and 34.6 ± 3.9 in PGD-A and standard group, respectively. As primary outcome, the post-warming blastocyst behaviour was evaluated 1.5hr after warming. Blastocysts were graded according to degeneration (absent/partial/full), re-expansion (1 = 80–100%/2 = 20–80%/3 = 0–20%) and morphology grades (excellent/good/average/poor). Secondary outcome was the ongoing implantation ( > 14 weeks of gestation). Logistic regression analysis corrected for confounding factors (female age,PGD-A/standard care group,BMI,stimulation protocol,total FSH dosage,main infertility factor,sperm factor,PGD-A indication, incubator,culture media,biopsy/vitrification/warming operator,blastocyst quality) was performed. Main results and the role of chance: The rate of degeneration after warming was similar in the PGD-A and in the standard groups (n = 2/258, 0.8% versus n = 3/125, 2.4%). Partial (n = 42/258, 16.3% versus n = 24/125, 19.2%) and absent degeneration (n = 214/258, 83.0% versus n = 98/125, 78.4%) rates were also similar. No difference was found also in the rate of no re-expansion (n = 24/258, 9.3% versus 9/125, 7.2%), partial expansion (n = 78/258, 30.2% versus n = 37/125, 29.6%) and full expansion (n = 156/258, 60.5% versus n = 79/ 125, 63.2%). The rates of blastocysts whose morphological quality worsened after warming was similar between PGD-A and standard cycles (n = 65/256, 25.4% versus n = 23/122, 18.9%). Even if either only ICM’s quality (n = 37/256, 14.4% versus n = 11/122, 9.0%) or the TE’s one was considered (n = 36/256, 14.1% versus n = 16/122, 13.1%). The overall clinical outcomes highlighted that not re-expanded blastocysts have a significantly lower ongoing implantation rate than fully expanded ones (n = 3/23, 13.0% versus n = 74/168, 44.0%; p = 0.006). Similarly, also partially-degenerated blastocysts have a significantly lower ongoing implantation rate than fully-viable ones (n = 13/58, 22.4% versus n = 94/227, 41.4%; p = 0.009). Logistic regression analysis identified the re-expansion grade 1.5hr after warming as the strongest predictor of ongoing implantation. Specifically, not reexpanded blastocysts showed an OR of 0.25 (P = 0.04). Limitations, reasons for caution: These findings are relative to the trophectoderm biopsy protocol that involves simultaneous zona opening and trophectoderm cells retrieval on fully-expanded blastocysts (no day3 hatching step involved). A higher sample size is advisable to confirm the current results. Wider implications of the findings: This study provides further evidences that trophectoderm biopsy does not impact embryo developmental and reproductive potential. Furthermore, interesting clues to increase predictive power upon implantation may arise from post-warming blastocysts evaluation which could influence the clinical practice (e.g. warming of an additional blastocyst). Trial registration number: none.