Multiple vitrification-warming and biopsy procedures on human embryos: clinical outcome and neonatal follow-up of children

Abstract

STUDY QUESTION Does double vitrification and warming of human blastocysts having undergone biopsy once or twice have an impact on the clinical outcome? SUMMARY ANSWER The clinical pregnancy rate obtained with double vitrification single biopsy blastocysts was comparable to that obtained with single vitrification single biopsy blastocysts in our center in the same time period (46%; 2016–2018), whereas that obtained with double-vitrified double-biopsied blastocysts seemed lower and will need further study. WHAT IS KNOWN ALREADY Genetic testing on cryopreserved unbiopsied embryos involves two cryopreservation procedures. Retesting of failed/inconclusive-diagnosed blastocysts inevitably involves a second round of biopsy and a second round of vitrification as well. To what extent this practice impacts on the developmental potential of blastocysts has been studied to a limited extent so far and holds controversy. Additionally, the obstetrical/perinatal outcome after the transfer of double-vitrified/single or double-biopsied blastocysts is poorly documented. STUDY DESIGN, SIZE, DURATION This retrospective observational study included 97 cycles of trophectoderm biopsy and preimplantation genetic testing (PGT) on vitrified-warmed embryos followed by a second round of vitrification between March 2015 and December 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS In 36 warming cycles, no biopsy was performed on the embryos before the first vitrification (single biopsy group). In 61 warming cycles, the embryos had been biopsied on Day 3 (n = 4) or on Day 5/6 (n = 57) before the first vitrification (double biopsy group). A second biopsy was mostly indicated in cycles of failed or inconclusive diagnosis at the first biopsy. Two cycles involved a more specific mutation test for X-linked diseases on male embryos and one cycle involved testing for a second monogenic indication supplementary to a previously tested reciprocal translocation. Post-warming suitability for biopsy, availability of genetically transferable embryos and clinical outcome of subsequent frozen-thawed embryo transfer (FET) cycles were reported. Neonatal follow-up of the children was included. MAIN RESULTS AND THE ROLE OF CHANCE In total, 91 cleavage-stage embryos and 154 blastocysts were warmed, of which 34 (37.4%) and 126 (81.8%), respectively, were of sufficient quality to undergo trophectoderm biopsy and were subsequently vitrified for a second time. Out of these, 92 underwent biopsy for the first time (single biopsy), whereas 68 underwent a second biopsy (double biopsy). After diagnosis, 77 blastocysts (48.1%) were revealed to be genetically transferable (44 in the single biopsy group and 33 in the double biopsy group). In 46 warming cycles, 51 blastocysts were warmed and 49 survived this second warming procedure (96.0%). Subsequently, there were 45 FET cycles resulting in 27 biochemical pregnancies and 18 clinical pregnancies with fetal heartbeat (40.0% per FET cycle: 44.0% in the single biopsy group and 35.0% in the double biopsy group, P = 0.54). Thirteen singletons were born (eight in the single biopsy group and five in the double biopsy group), while three pregnancies were ongoing. A total of 26 embryos (13 in each group) remain vitrified and have the potential to increase the final clinical pregnancy rate. The neonatal follow-up of the children born so far is reassuring. LIMITATIONS, REASONS FOR CAUTION This is a small retrospective cohort, thus, the implantation potential of double vitrification double biopsy blastocysts, as compared to double vitrification single biopsy blastocysts and standard PGT (single vitrification, single biopsy), certainly needs further investigation. Although one could speculate on birthweight being affected by the number of biopsies performed, the numbers in this study are too small to compare birthweight standard deviation scores in singletons born after single or double biopsy. WIDER IMPLICATIONS OF THE FINDINGS PGT on vitrified-warmed embryos, including a second vitrification-warming step, results in healthy live birth deliveries, for both single- and double-biopsied embryos. The neonatal follow-up of the 13 children born so far did not indicate any adverse effect. The present study is important in order to provide proper counseling to couples on their chance of a live birth per initial warming cycle planned and concerning the safety issue of rebiopsy and double vitrification. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER N/A.

Pre-Vitrification and Post-Warming Variables of Vitrified-Warmed Blastocysts That Are Predictable for Implantation

“…The warming of a second embryo is usually only performed if the first embryo does not survive or shows severe signs of degeneration. Warming a second embryo due to a potential prognostic parameter that turns out to be not optimal-like size after warming-may be less likely in clinical practice, despite the option of re-vitrification of one of the two embryos [41]. Still, the preferred option is to have parameters at hand that can be used to decide which embryo to warm to avoid any risk of potential embryo wastage.…”

Embryo and fetal gene editing: Technical challenges and progress toward clinical applications

“…PGT is subject to the limitations of embryo culture the diagnostic accuracy of one- or two-cell biopsy, potentially reduced viability following one or more freeze/thaw and biopsy cycles, and the probability of mosaicism. 13 , 14 , 15 Embryo biopsy is indicated to diagnose specific monogenic (PGT-M) or chromosomal anomalies (PGT-A), or in certain cases structural rearrangements (PGT-SRs) in couples with balanced translocations, using comparative genomic hybridization (aCGH) or next-generation sequencing (NGS). 12 As these technologies improve and become more widely accessible and cost-effective, the uptake of PGT is expected to increase and may expand to include non-congenital genetic conditions such as Huntington’s disease, a lethal neurodegenerative disorder caused by expansion of a CAG repeat within the Huntingtin ( HTT ) gene, or hereditary cancer syndromes (e.g., BRCA 1/2).…”

“…Repeated manipulation of the embryo may also affect embryo survival and pregnancy health, although repeat freeze-thawed embryos appear to function normally post transplantation. 14 …”

Clinical Outcome, Socioeconomic Status and Psychological Constrains of Patients Undergoing Preimplantation Genetic Testing (PGT) in Northern Greece

“…Preimplantation genetic testing for aneuploidy (PGT-A) has therefore been proposed as a solution that allows the diagnosis of chromosomal abnormalities in embryos before implantation to the uterus [ 10 , 11 ]. PGT-A is offered to patients of advanced maternal age (AMA) ≥ 35 years, repeated implantation failure (RIF) > 3, repeated miscarriages (RM) > 2, previous pregnancy with a chromosomally abnormal fetus irrespective of maternal age, and in cases of severe male factor infertility including non-obstructive azoospermia (NOA) and oligoasthenoteratozoospermia (OAT) [ 9 , 10 , 11 , 12 , 13 , 14 , 15 ].…”