(PGS) Is Compromised by Degree of Mosaicism of Human Embryos Norbert Gleicher1,2,3*, Andrea Vidali1,4, Jeffrey Braverman5, Vitaly A
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Gleicher et al. Reproductive Biology and Endocrinology (2016) 14:54 DOI 10.1186/s12958-016-0193-6 RESEARCH Open Access Accuracy of preimplantation genetic screening (PGS) is compromised by degree of mosaicism of human embryos Norbert Gleicher1,2,3*, Andrea Vidali1,4, Jeffrey Braverman5, Vitaly A. Kushnir1,6, David H. Barad1,2,7, Cynthia Hudson1, Yang-Guan Wu1, Qi Wang1, Lin Zhang1, David F. Albertini1,8 and the International PGS Consortium Study Group Abstract Background: To preclude transfer of aneuploid embryos, current preimplantation genetic screening (PGS) usually involves one trophectoderm biopsy at blastocyst stage, assumed to represent embryo ploidy. Whether one such biopsy can correctly assess embryo ploidy has recently, however, been questioned. Methods: This descriptive study investigated accuracy of PGS in two ways. Part I: Two infertile couples donated 11 embryos, previously diagnosed as aneuploid and, therefore, destined to be discarded. They were dissected into 37 anonymized specimens, and sent to another national laboratory for repeat analyses to assess (i) inter-laboratory congruity and (ii) intra-embryo congruity of multiple embryo biopsies in a single laboratory. Part II: Reports on human IVF cycle outcomes after transfer of allegedly aneuploid embryos into 8 infertile patients. Results: Only 2/11 (18.2 %) embryos were identically assessed at two PGS laboratories; 4/11 (36.4 %), on repeat analysis were chromosomally normal, 2 mosaic normal/abnormal, and 5/11 (45.5 %) completely differed in reported aneuploidies. In intra-embryo analyses, 5/10 (50 %) differed between biopsy sites. Eight transfers of previously reported aneuploid embryos resulted in 5 chromosomally normal pregnancies, 4 delivered and 1 ongoing. Three patients did not conceive, though 1 among them experienced a chemical pregnancy. Conclusions: Though populations of both study parts are too small to draw statistically adequately powered conclusions on specific degrees of inaccuracy of PGS, here presented results do raise concerns especially about false-positive diagnoses. While inter-laboratory variations may at least partially be explained by different diagnostic platforms utilized, they cannot explain observed intra-embryo variations, suggesting more frequent trophectoderm mosiaicsm than previously reported. Together with recentl published mouse studies of lineages-specific degrees of survival of aneuploid cells in early stage embryos, these results call into question the biological basis of PGS, based on the assumption that a single trophectoderm biopsy can reliably determine embryo ploidy. Keywords: Preimplantation genetic screening (PGS), In vitro fertilization (IVF), Embryos, Embryo mosaicism, Trophectoderm biopsy, Blastocyst Abbreviations: aCGH, Array comparative genomic hybridization; FOR, Functional ovarian reserve; IVF, In vitro fertilization; LFOR, Low functional ovarian reserve; PGS, Preimplantation genetic screening * Correspondence: [email protected] 1The Center for Human Reproduction, 21 East 69th Street, New York, NY 10021, USA 2The Foundation for Reproductive Medicine, New York, NY, USA Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gleicher et al. Reproductive Biology and Endocrinology (2016) 14:54 Page 2 of 7 Background Methods Human embryos are frequently aneuploid. The preva- Here reported study received Institutioanl Review Board lence further increases with advancing female age and (IRB) approval ER 10232015:01 at The Center for with low functional ovarian reserve (LFOR) [1]. Avoid- Human Reproduction. ing transfers of aneuploid embryos in association with in vitro fertilization (IVF) has been proposed since the PGS laboratories and PGS platforms early 1990s [2] under the assumption it would lead to The CHR received 11 donated embryos for Part 1 of better pregnancy rates and fewer miscarriages [3]. This here reported manuscript. These embryos had previ- effort was given the acronym preimplantation genetic ously at two national referral laboratories of inter- screening (PGS) but over almost two decades has national repute (both laboratories have extensively failed to demonstrate promised improvements of in contributed to the PGS literature) been reported as vitro fertilization (IVF) outcomes. aneuploid: Reprogenetics (Livingston, N.J., now a div- Initial criticisms of the procedure [4–7] were attrib- ision of Cooper Surgical, Trumbull, CT) served one uted to technical shortcomings of initial cleavage-stage couple utilizing array CGH [14], and the not-for-profit embryo biopsies and inadequate techniques of chromo- Foundation for Embryonic Competence (FEC), utilizing somal analyses [8]. Once embryo biopsy moved from an in-house developed test, called Select Comprehen- cleavage- to blastocyst-stage (trophectoderm), and sive Chromosome Screening (CCS) based on real-time selected chromosome investigation to full chromosomal polymerase chain reaction (qPCR) technology, developed complement analyses with highly accurate newly devel- by Reproductive Medicine Associates of New Jersey oped diagnostic platforms, the widely held assumption (Basking Ridge, N.J.) [15] served the other couple. was that PGS, finally, would show its clinical effective- ness. When this did not happen, the ability of different Specimen preparation and coding PGS platforms to accurately determine embryo ploidy All donated embryos investigated in this study were do- was questioned [9]. nated to The Center for Human Reproduction (CHR). Observing in clinical practice statistically improb- Most were donations of the center’s own patients but able high aneuploidy rates, especially in some younger some were donated by a couple that had their embryos women, The Center for Human Reproduction (CHR) stored at another Consortium member’s laboratory. decided in 2014 to offer women with only aneuploid CHR received these embryos cryopreserved and with full embryos embryo transfers with allegedly aneuploid documentation of PGS results from the original IVF cen- embryos in selected cases [10], an effort quickly joined ter. They were maintained in standard liquid nitrogen by other fertility centers, leading to the establishment tanks until dissection of all 11 embryos into 37 individ- of the International PGS Consortium, dedicated to ual specimens (1–5 per embryo, depending on technical investigations of effectiveness of PGS in association feasibility) by one of the authors (C.H.) Fragmentation of with IVF. embryos was achieved utilizing standard laser-assisted The Consortium since reported 3 chromosomally dissection instrumentation, as is routinely utilized for normal live births from 5 such transfers [11]. Concomi- trophectoderm biopsies during PGS. tantly, an Israeli member of the International PGS In mimickery routine, the 37 specimens were randomly Consortium completed a PGS study of embryos with coded as 4 patients (in Table 1, A1-A8, B1-B8, C1-C10 and single gene diseases, reporting significant discrepancies D1-D11) and sent to a third national PGS laboratory (IVI- between multiple trophectoderm and inner cell mass GEN, Miami, FL). This laboratory used for 24-chromosome biopsies (Prof. Raoul Orvieto, personal communication, assessment comparative genomic hybridization (aCGH, New York, October 2015). This study has since been BlueGnome, 24sure BAC-based arrays), in more detail de- published [12]. Shortly thereafter, Italian colleagues scribed at: http://www.cytochip.com. reported on 18 attempts of transfer of “mosaic” (i.e., The laboratory was aware that submitted specimens aneuploidy) embryos in women who had produced no involved a research project, but was uninformed about euploid embryos in IVF cycles, establishing 6 chromo- details and purpose of the project. Specimen codes were somally normal live births (33.3 %) [13]. broken upon receipt of results, at which time inter- This manuscript now raises additional doubts laboratory and intra-embryo/laboratory discrepancies about the ability of PGS to accurately determine were determined. embryos ploidy by reporting significant inter- laboratory and intra-embryo discrepancies for PGS Embryo transfers results as well as two additional normal pregnancies Transfers of supposedly chromosomally abnormal) established from transfer of embryos previously embryos were performed at three independent fertility designated as aneuploid. centers in New York City, CHR, Fertility Specialists in Gleicher et al. Reproductive Biology and Endocrinology (2016) 14:54 Page 3 of 7 Table 1 Comparison of embryo ploidy between two PGS 2.0 with appropriate and detailed informed consent allowed assessments transfer of selected supposedly aneuploid embryos if no Pat.# Emb# Biopsy # Original PGS analysis Repeat PGS analysisa or only inadequate numbers of euploid embryos were (all embryos reported (multiple biopsies) available for transfer. as abnormal) b 1 A1 1 45, XY, -18 Normal 46, XX Statistical considerations b 2 A2