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46,XX male? Reality, PGT-A misdiagnosis or laboratory quality management error?

Authors: Schiewe MC, NL Nugent, CM Rios, S Zozula, S Shahbazian, JB Whitney, RE Anderson

Study question: Does a mid-term ultrasound of a confirmed male fetus derived from a single non-mosaic euploid 46,XX blastocyst transfer definitely imply a laboratory error occurred?

Summary answer: Sex determination errors from non-mosaic embryos are rare, but not when random meiotic X translocations of a SRY gene causes an uncommon XX male syndrome.

What is known already: Blastocyst biopsy (BL-Bx)/preimplantation genetic aneuploidy testing (PGT-A) have been shown to be subject to false-negative diagnosis due to mosaicism.  We have previously shown that the BL-Bx technique can contribute to mosaic diagnoses.  Wrong sex determinations are extremely rare based on the precision of NGS tests.  Errors in the sex of offspring are typically associated with a laboratory handling/identification error.  Conversely, viable aneuploidy of sex chromosomes is not unusual as it pertains to Klinefelter(<1:1000) or Turner’s syndrome(<1:5000). Less common(1:20,000) is the random meiotic translocation of the SRY gene onto the sperms’ X chromosome, causing a female to be born biologically male.

Study design, size, duration: Following a “wrong sex” ultrasound confirmation at 18-20 weeks, a retrospective case study analysis was performed. All aspects of embryo handling in the patients IVF/ICSI cycle and subsequent thaw/biopsy (“thawopsy”) procedure were evaluated for potential error and confirmation of witness identification.  Furthermore, the timing and procedural order/paperwork of other same day, BL-Bx/vitrification/FET cases was scrutinized for possible embryo mix-up.  In addition, the patients’ NGS tests were reviewed and rerun to assess accuracy of outcome.

Participants/materials, setting, methods: An infertility couple performed an ICSI cycle with blastocyst vitrification in May 2013.  A successful term male birth followed and in May 2018 the couple chose to perform a thawopsy of their residual embryos.  In June 2018 they performed a single euploid ET using a 6AA Day 6 46,XX blastocyst with a non-mosaic NGS profile.   During the second trimester of pregnancy, a perinatologist performed ultrasound exams of the fetus and additional genetic testing (NIPT, amniocentesis).

Main results and the role of chance:  A 35 year old patient entered her second trimester with a predetermined female pregnancy, only to be told and shown at 20 weeks, that the fetus had a penis.  The distraught patient contacted the REI physician, whom informed the IVF Lab looking for answers to how such an error could occur. A detailed investigation of patient records revealed that the initial cycle produced 25 mature oocytes, 21 zygotes and 13 blastocysts (1 transferred and 12 vitrified) and that all embryo handling events had been witnessed, as were the subsequent thawopsy procedures.  Of the 7 embryos re-biopsied/re-vitrified, 5 euploid (2-46,XY and 3-46,XX) blastocysts were confirmed.  In retrospect, the only fact not fully detailed to alleviate any doubt in potentially mishandling another patients’ male embryo post-biopsy was the exact time of vitrification.  As we typically vitrify within 45 min post-biopsy, we had confidence that the prospects of a mix-up 90 min later was highly doubtful.  Post-repeated ultrasound, the NIPT outcome was “inconclusive”, which we assumed meant it did not support the ultrasound scan findings.  Finally, in October 2018, the amniocentesis finding was conclusive revealing the rare XX male syndrome condition.

Limitations, reasons for caution: It is imperative that laboratories implement strict quality management practices involving witness verification and time/date details for all embryo handling/movement events to confidently assess and troubleshoot potential lab errors.  As we integrate routine genetic testing, we must inform patients about the risks of potential rare events, besides issues of mosaicism.

Wider implications of the findings: When faced with a potential misdiagnosis, it is comforting to everyone when quality management efforts effectively confirm the elimination of lab error.  Furthermore, we must anticipate incorrect mosaic profile interpretations, as well as unusual/rare developmental events to best inform and support our patients.