Next-Generation Sequencing Is More Efficient at Detecting Mosaic Embryos and Improving Pregnancy Outcomes than Single-Nucleotide Polymorphism Array Analysis
2021; Elsevier BV; Volume: 23; Issue: 6 Linguagem: Inglês
10.1016/j.jmoldx.2021.02.011
ISSN1943-7811
AutoresMin Xiao, Caixia Lei, Yanping Xi, Yulin Lu, Junping Wu, Xiaoyu Li, Shuo Zhang, Saijuan Zhu, Jing Zhou, Xiong Li, Yueping Zhang, Xiaoxi Sun,
Tópico(s)Assisted Reproductive Technology and Twin Pregnancy
ResumoWe compared chromosomal mosaicism, detected by next-generation sequencing (NGS), during preimplantation genetic testing (PGT) with that detected by single-nucleotide polymorphism (SNP) array–based PGT to assess the pregnancy outcomes associated with both platforms in a retrospective cohort study of patients undergoing in vitro fertilization in a single university-based assisted reproduction center. In total, 6427 blastocysts biopsied from 1513 patients who underwent 2833 oocyte retrievals from January 2017 to February 2019 were identified. The incidence of mosaicism was significantly higher in the NGS-based PGT group than in the SNP array–based PGT group. Furthermore, some aneuploid specimens were affected by mosaicism. The total mosaicism detection rate with NGS-based PGT (23.3%) was significantly higher than that with SNP array–based PGT (7.7%). Mosaicism rates were similar when stratified by maternal age or PGT type. The SNP array cohort showed a significantly higher spontaneous abortion rate than the NGS cohort (10.07% versus 6.33%; P = 0.0403). The ongoing pregnancy/live birth rate was higher in the NGS cohort (44.1%) than in the SNP array cohort (42.28%). Our results confirm that NGS-based PGT can detect mosaicism more frequently than SNP array–based PGT in trophectoderm specimens. Therefore, clinical application of NGS for PGT may improve pregnancy outcomes compared with that of SNP array–based PGT. More detailed blastocyst detection and classification is necessary to prioritize embryo transfers. We compared chromosomal mosaicism, detected by next-generation sequencing (NGS), during preimplantation genetic testing (PGT) with that detected by single-nucleotide polymorphism (SNP) array–based PGT to assess the pregnancy outcomes associated with both platforms in a retrospective cohort study of patients undergoing in vitro fertilization in a single university-based assisted reproduction center. In total, 6427 blastocysts biopsied from 1513 patients who underwent 2833 oocyte retrievals from January 2017 to February 2019 were identified. The incidence of mosaicism was significantly higher in the NGS-based PGT group than in the SNP array–based PGT group. Furthermore, some aneuploid specimens were affected by mosaicism. The total mosaicism detection rate with NGS-based PGT (23.3%) was significantly higher than that with SNP array–based PGT (7.7%). Mosaicism rates were similar when stratified by maternal age or PGT type. The SNP array cohort showed a significantly higher spontaneous abortion rate than the NGS cohort (10.07% versus 6.33%; P = 0.0403). The ongoing pregnancy/live birth rate was higher in the NGS cohort (44.1%) than in the SNP array cohort (42.28%). Our results confirm that NGS-based PGT can detect mosaicism more frequently than SNP array–based PGT in trophectoderm specimens. 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Next generation sequencing for preimplantation genetic screening improves pregnancy outcomes compared with array comparative genomic hybridization in single thawed euploid embryo transfer cycles.Fertil Steril. 2018; 109: 627-632Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Most recently, NGS has been utilized in PGT. This technique is based on massively parallel and deep sequencing and is more sensitive for mosaicism detection compared with other technologies.33Munne S. Wells D. Detection of mosaicism at blastocyst stage with the use of high-resolution next-generation sequencing.Fertil Steril. 2017; 107: 1085-1091Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,34Nakhuda G. Jing C. Butler R. Guimond C. Hitkari J. Taylor E. Tallon N. Yuzpe A. Frequencies of chromosome-specific mosaicisms in trophoectoderm biopsies detected by next-generation sequencing.Fertil Steril. 2018; 109: 857-865Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The PGT results of 1547 blastocysts from 378 patients were analyzed to examine the chromosome-specific frequencies of mosaicism with constitutional aneuploidy, and mosaicisms were detected in 17.5% of samples as the sole abnormality, with an overall incidence of mosaicism of 30.1%.34Nakhuda G. Jing C. Butler R. Guimond C. Hitkari J. Taylor E. Tallon N. Yuzpe A. Frequencies of chromosome-specific mosaicisms in trophoectoderm biopsies detected by next-generation sequencing.Fertil Steril. 2018; 109: 857-865Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar This result may be important for clinical treatment, as mosaic embryos can show impaired viability and a decreased potential to result in live births.31Zore T. Kroener L.L. Wang C. Liu L. Buyalos R. Hubert G. Shamonki M. Transfer of embryos with segmental mosaicism is associated with a significant reduction in live-birth rate.Fertil Steril. 2019; 111: 69-76Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar A previous study compared pregnancy outcomes using NGS-based PGT with aCGH-based PGT and found that clinical implementation of NGS resulted in a significantly better OP/LBR than that of aCGH.16Friedenthal J. Maxwell S.M. Munne S. Kramer Y. McCulloh D.H. McCaffrey C. Grifo J.A. Next generation sequencing for preimplantation genetic screening improves pregnancy outcomes compared with array comparative genomic hybridization in single thawed euploid embryo transfer cycles.Fertil Steril. 2018; 109: 627-632Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Few studies have focused on pregnancy outcomes based on PGT with clinical implementation of NGS compared with those of PGT using SNP arrays. These two PGT platforms each have advantages with respect to UPD, polyploidy, and mosaicism detection. Herein, we performed clinical evaluation to determine whether PGT using the NGS platform improves pregnancy outcomes compared with that of PGT using the SNP array platform. Given the superior ability of the NGS platform to detect embryonic mosaicism compared with that of the SNP array platform, we hypothesized that patients undergoing IVF who had been evaluated by PGT with NGS would have better pregnancy outcomes than those of patients undergoing IVF who had been tested by PGT with SNP array testing. This was a retrospective cohort study of all SNP array and NGS results of patients presenting for PGT from January 2017 to February 2019 and single frozen-thawed euploid embryo transfer cycle from January 2017 to June 2018 at a single large university-based fertility center. All 1513 patients with various conditions,35Fesahat F. Montazeri F. Hoseini S.M. Preimplantation genetic testing in assisted reproduction technology.J Gynecol Obstet Hum Reprod. 2020; 49: 101723Crossref PubMed Scopus (9) Google Scholar including advanced maternal age, severe oligospermia, recurrent miscarriage, and recurrent implantation failure, which belong to the PGT type for aneuploidies (PGT-A), and Robertsonian translocation, reciprocal translocation, inversion, or chromosomal abnormalities and mosaicism, which belong to the PGT type for structural rearrangements (PGT-SR), were recruited in the PGT program at the ShangHai JIAI Genetics and IVF Institute of the Obstetrics and Gynecology Hospital of Fudan University in China. Both PGT-A and PGT-SR were included in this retrospective cohort study. In detail, 744 patients underwent SNP array–based PGT cycles, whereas 769 patients underwent NGS-based PGT cycles. Demographic and baseline clinical data were collected on the patients from each platform group, including the age, proportion of patients with advanced maternal age, number of embryos, and infertility diagnosis (Table 1). Exclusion criteria included patients who underwent mosaic frozen-thawed embryo transfers (FET), double FET, or transfer of an embryo with a second biopsy, or those with incomplete clinical pregnancy outcome data. In our clinical report, trophectoderm biopsies containing ≤20% abnormal cells were deemed euploid, and corresponding blastocysts are recommended to priority transfer. Trophectoderm biopsies containing >20% and ≤50% abnormal cells were deemed mosaic with low proportion of abnormal cells, and corresponding blastocysts are recommended to retain and undergo genetic counseling on mosaic embryo transfer.Table 1Demographic and Baseline Clinical Data of Patients Undergoing Euploid Frozen-Thawed Embryo Transfer with Either NGS-Based PGT or SNP Array–Based PGTCharacteristicsSNP array (n = 744)NGS (n = 769)P valueAge, mean ± SD, years31.96 ± 4.5433.11 ± 4.68<0.001Patients (aged ≥35 years), % (n/total)28.8 (214/744)38.8 (301/769)<0.001Embryos, mean ± SD, n4.21 ± 2.824.29 ± 2.850.5939Embryos remaining, mean ± SD, n2.25 ± 2.002.42 ± 2.060.0985Embryos remaining (excluding mosaic embryos), mean ± SD, n2.04 ± 1.881.78 ± 1.680.0047Patients with embryos remaining in storage, % (n/total)56.0 (417/744)70.0 (538/769) 100%, because some patients have a combination of multiple factors leading to infertility.E2, estrogen; E2 peak, peak value of E2; IVF, in vitro fertilization; NGS, next-generation sequencing; PGT, preimplantation genetic testing; SNP, single-nucleotide polymorphism. Open table in a new tab The numbers in the Infertility Diagnosis section add up to >100%, because some patients have a combination of multiple factors leading to infertility. E2, estrogen; E2 peak, peak value of E2; IVF, in vitro fertilization; NGS, next-generation sequencing; PGT, preimplantation genetic testing; SNP, single-nucleotide polymorphism. The PGT and human embryo research was approved by the Ethics Committee of JIAI Genetics and IVF Institute, and all protocols followed the ethical guidelines of this committee. Informed written consent was provided by all patients before the commencement of IVF and PGT. The primary outcomes are positive human chorionic gonadotropin (hCG), implantation rate, fetal heartbeat, OP/LBR, BPR, and SAB rate. Conception based on positive hCG was defined by a serum β-hCG level ≥10 mIU/mL at 14 days after embryo transfer. Implantation rate was defined as the presence of gestational sacs observed by transvaginal ultrasound at 6 weeks of pregnancy. Fetal heartbeat was defined as an ultrasound detection of a fetal heartbeat 9 weeks after embryo transfer. Biochemical pregnancies implied that an embryo had implanted. In such cases, secretion of the pregnancy hormone (ie, a positive β-hCG level ≥10 mIU/mL on cycle days 28 to 30) was detected in the blood of the mother coupled with declining hCG levels before the development of a gestational sac was detected with transvaginal ultrasound. The BPR was calculated as the number of biochemical pregnancies per FET with a subsequent positive hCG level. The SAB rate was defined as clinically recognized pregnancies lost up to 22 weeks of pregnancy after a previously documented gestational sac was detected by transvaginal ultrasound divided by the total number of clinical pregnancies. The OP/LBR was defined as the number of ongoing pregnancies after the presence of either a gestational sac or fetal heartbeat was detected by ultrasound or live births divided by the total number of embryos transferred. Monozygotic twins resulting from the transfer of a single embryo were counted as one implantation and one ongoing pregnancy or live birth. Either a gonadotropin-releasing hormone antagonist protocol or a long gonadotropin-releasing hormone agonist protocol was used for standard ovarian stimulation with gonadotrophins. At 34 to 36 hours after triggering with hCG or an agonist, the oocyte was retrieved under transvaginal ultrasound guidance. Standard IVF techniques were used in the ShangHai JIAI Genetics and IVF Institute. Briefly, oocytes were fertilized by intracytoplasmic sperm injection and incubated in fertilization media (Vitrolife, Goteborg, Sweden). Normal fertilization was assessed and confirmed by the presence of two pronuclei and a second polar body at 16 to 18 hours after insemination. The embryos were washed and cultured for 5 to 6 days in sequential media (G1 and G2; Vitrolife) to develop to the blastocyst stage. On day 4 after fertilization, an 18-μm hole was made in the zona pellucida of all embryos. On day 5 or day 6 after fertilization, blastocysts with TE cells herniating out of the zona pellucida were chosen for biopsy. Approximately three to eight biopsied TE cells were washed in 3-(4-Morpholino) propanesulphonic acid medium (Vitrolife) and placed into PCR tubes containing phosphate-buffered saline. They were either used directly for whole genome amplification (WGA) or stored at −20°C to await detection with amplification protocols that varied depending on the detection platform. All experiments were performed in and the data analyzed at the JIAI local laboratory. Mosaicism calls were made when 20% to 80% of the cells were aneuploid. Under ultrasound guidance, a single euploid blastocyst was transferred to the patient using a soft embryo-transfer catheter and, as part of routine clinical practice, the remaining embryos of the cohort were frozen. Serum hCG levels were checked after FET for 14 days. If the serum hCG level was positive and had doubled as expected, transvaginal ultrasound was performed 4 weeks later to locate the pregnancy and confirm fetal viability. If the serum hCG level was negative, all hormone therapy stopped. The pregnant women continued hormonal therapy until reaching 12 weeks of gestation. Subsequent management was the same as that in any other natural early pregnancy. In summary, a total of three serum hCG and three ultrasound follow-up visits were included. The patients were referred for antenatal care and a routine prenatal examination in the maternity hospital when the ongoing pregnancy had reached 12 weeks. The patients were followed up by telephone to obtain clinical pregnancy information. The outcomes of the pregnancy (delivery or miscarriage), delivery mode (natural labor or caesarean section), gestation week, number of babies born, birth weight, and obstetric complications were recorded. The multiple displacement amplification method with phi 29 DNA polymerase was performed using an REPLI-g Single Cell Kit (Qiagen, Hilden, Germany) for WGA, according to the manufacturer's protocol. The TE cell sample was lysed, and the DNA was denatured, which was stopped by adding neutralization buffer, and a master mix containing buffer and DNA polymerase was added. The isothermal amplification reaction was performed for 12 hours at 30°C; the reaction was stopped by incubation at 65°C for 3 minutes. The SNP genotype and intensity of the WGA products were determined with an Illumina Human Cyto-12 microarray for PGT-A and PGT-SR (Illumina, Inc., San Diego, CA). Each bead chip contained approximately 300,000 SNPs (Illumina, Inc.). In accordance with the Infinium chip protocol, the TE cell amplification product was amplified in a genome-wide manner in an overnight isothermal reaction. The DNA was fragmented, with the fragments ranging from approximately 300 to 500 bp. After precipitating and resuspending the fragments, the samples were hybridized on a BeadChip overnight for approximately 20 hours, extended, and stained. After staining, the BeadChips were imaged on an iScan System (Illumina, Inc.). The CNVs or cytogenetics were analyzed with GenomeStudio version 2-0-4-5, KaryoStudio version 1.4.3_64, and BlueFuse-Multi software version 4.4 (Illumina, Inc.). Call genotypes were determined to identify the CNVs, including aneuploidy, CNVs, and UPD. The copy numbers >2.2 and <1.8 were marked as duplications and deletions, respectively. The size threshold for calling CNVs was ≥3 Mb and that of mosaic CNVs was ≥5 Mb. Biopsied TE cells were subjected to WGA with a Pre-implantation Genetic Screening for Aneuploidy Kit (Berry Genomics Corp., Beijing, Ch
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