Balkan Journal of Medical Genetics

PREIMPLANTATION GENETIC TESTING WITHIN THE PUBLIC HEALTHCARE SYSTEM IN SLOVENIA
Volk M, Writzl K, Veble A, Jaklič H, Teran N, Prosenc B, Štimpfel M, Virant Klun I, Vrtačnik Bokal E, Ban Frangež H, Peterlin B
*Corresponding Author: Prof. Borut Peterlin, MD, PhD, Clinical institute of genomic medicine UMC Ljublja na, Šlajmerjeva 004, 1000 Ljubljana, Slovenia, Telephone: +3861 5226103, Fax: +3861 5401137, borut.peterlin@kclj.si
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MATERIALS AND METHODS

Subjects Two hundred and eleven (211) couples with a known genetic predisposition, 110 with monogenic disorder, 88 with chromosome structural rearrangement, and 13 with mosaic sex or numeric chromosome abnormality, were eligible for the PGT procedure. Ovarian stimulation and oocyte retrieval were performed according to standard protocol (5). All couples signed informed consent prior to the PGT procedure. Clinical operations have been con- ducted following the principles expressed in the Helsinki declaration. Methods Preimplantation genetic testing was implemented in 2004. Prior to enrolment, the couples underwent genetic counselling. Genetic counselling is organized stepwise to provide all the relevant education and information associ- ated with the procedure. Couples attending PGT cycles are informed about the benefits, limitations, and risks of the PGT procedure and the expected delivery rate per embryo transfer. The multidisciplinary approach manages coordi- nation between hormone stimulation, embryology part, and genetics. Since biopsy is performed only on good quality blastocysts, single embryo transfer is preferred. The con- firmatory prenatal diagnostic testing is still recommended following a PGT-M, and to a lesser extent of PGT-SR, due to difficulties of testing the limited number of cells obtained by embryo biopsy as well as recognition of the biological and human factors that may lead to misdiagnosis in a PGT cycle (6). A follow-up of pregnancies, deliveries, and postnatal development of born children, along with the cycle data, is maintained. For cycles from 2004 to the end of 2016, cleavage- stage embryo biopsy was performed on day three after fer- tilization, and two blastomeres, when possible, were with- drawn. Then according to the indication, either fluorescent in situ hybridization (FISH) analysis or polymerase chain reaction (PCR) based protocol were performed. The FISH based protocol consisted of set-up with probe selection and pre-cycle work-up on peripheral blood lymphocytes from both reproductive partners. FISH was carried out ac- cording to standard protocol using commercially available probes by Abbott Vysis, Cytocell, or Agilent SureFISH, and guidelines and recommendations by ESHRE (7). The turnaround time was 48 hours, which allowed for fresh embryo transfer on day five. PCR based protocol for single- gene disorders was performed according to guidelines and recommendations by ESHRE (8). PGT set-up included indirect analysis and direct genotyping, if appropriate (8). In 2017, blastocyst biopsy (trophectoderm biopsy- TE) on days 5 to 7 was introduced. This allowed for whole genome amplification and next-generation sequencing (NGS) based 24-chromosome screening for chromosome and segmental abnormalities with a resolution of 10-20 Mb. Another advantage of this approach is that a pre- diagnostic set up is usually not required. The NGS-based protocol was carried out according to the manufacturer’s recommendations (VeriSeq PGS, Illumina). In addition, genetic testing for single-gene disorders was carried out as mentioned above. We have reviewed the medical records from 2004 to 2019 at our institute to determine the proportion of couples with genetic indications that opted for preimplantation genetic testing and signed informed consent. Based on this data, we defined the proportion of Slovenian couples who would opt for PGT as a first genetic testing. Data analysis By first reviewing medical records, we estimated the proportion of couples that would opt for PGT. We retrospectively collected the referrals for all performed PGT-M, PGT-SR, and PGT-A cycles from 2004-2019. In addition, we calculated the average and median age of female partners enrolled in PGT. Then we analysed data of the PGT cycles regarding the referrals (PGT-M, PGT-SR, PGT-A or X-linked disorder), the type of embryo biopsy (blastomere biopsy in 2004-2016, blastocyst biopsy in 2017-2019) and genetic testing approach for chromosome rearrangements (FISH for chromosome rearrangements in 2004-2016, next-generation sequencing-based 24-chro- mosome screening in 2017-2019). Then, we compared the clinical outcomes in 2004-16 and 2017-19 using Chi- square statistics to test the clinical effectiveness of different PGT approaches.

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