POCS FIVAP 1
Description
Chromosomal analysis of products of conception using targeted direct embryo biopsy by operative hysteroscopy
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Inclusion criteria were gestational age between 6 and 12 weeks at the time of abortion and patient´s willingness to have chromosomal analysis of POCs. Operative hysteroscopies for uterine evacuation were performed from September 2015 to January 2019 in a single private infertility clinic. Specimens were obtained within 72 hours of bleeding or loss of fetal heartbeat by ultrasound. All surgeries were performed under anesthetic and transabdominal ultrasound guidance. A 5 mm compact hysteroscope with an operative channel was inserted trans cervically. After entry to the endometrial cavity, the implantation site and other characteristics were identified by direct visualization plus transabdominal ultrasound. Opening the gestational sac with hysteroscopic scissors gave access to the extra-coelomic cavity, after which the yolk sac and embryo were evaluated. Embryo and chorionic villi sampling were performed with hysteroscopic forceps. After removal of the embryo, resection of the gestational sac was accomplished with hysteroscopic forceps and scissors. In some cases, evacuation of the uterine cavity was completed using soft vacuum aspiration under ultrasound guidance. DNA was extracted from POC samples using Wizard SV Genomic DNA Purification System extraction kit (Promega, Madison, USA). After DNA extraction and amplification, the presence of maternal contamination was assessed by qPCR techniques, comparing the pattern obtained in the fetal and maternal DNA samples. These techniques also allow determination of the genotypic sex of the fetal sample. Chromosomal analysis of the samples was performed using aCGH (Genoma, Italy). This analysis used a comparative genomic hybridization oligonucleotide microarray of approximately 60,000 probes (qChip PdC). Samples were hybridized against an internal reference DNA of the same sex. The data were analyzed with the Genomic Workbench software and interpreted following the published parameters (Agilent Technologies Platform). The same samples were analyzed by NGS (CooperSugical Inc, USA), using PGTai 2.0Plus protocol. Samples were amplified using PicoPex DNA whole amplification system (Takara Bio, US). DNA samples were prepared for NGS using the Illumina (Illumina Inc. US) DNA Prep library protocol and sequenced on a NextSeqTM (Illumina Inc), using a paired end 2x36 base pair read strategy (4M paired end, 8M total reads, 288M base pairs total). Sequencing analysis was performed using the CooperSurgical artificial intelligence (PGTai Plus) module as described previously .