PURPOSE: Preimplantation genetic testing for monogenic disorders (PGT-M) allows early diagnosis in embryos conceived in vitro. PGT-M helps to prevent known genetic disorders in affected families and ensures that pathogenic variants in the male or female partner are not passed on to offspring. The trend in genetic testing of embryos is to provide a comprehensive platform that enables robust and reliable testing for the causal pathogenic variant(s), as well as chromosomal abnormalities that commonly occur in embryos. In this study, we describe PGT protocol that allows direct mutation testing, haplotyping, and aneuploidy screening. METHODS: Described PGT protocol called OneGene PGT allows direct mutation testing, haplotyping, and aneuploidy screening using next-generation sequencing (NGS). Whole genome amplification product is combined with multiplex PCR used for SNP enrichment. Dedicated bioinformatic tool enables mapping, genotype calling, and haplotyping of informative SNP markers. A commercial software was used for aneuploidy calling. RESULTS: OneGenePGT has been implemented for seven of the most common monogenic disorders, representing approximately 30% of all PGT-M indications at our IVF centre. The technique has been thoroughly validated, focusing on direct pathogenic variant testing, haplotype identification, and chromosome abnormality detection. Validation results show full concordance with Sanger sequencing and karyomapping, which were used as reference methods. CONCLUSION: OneGene PGT is a comprehensive, robust, and cost-effective method that can be established for any gene of interest. The technique is particularly suitable for common monogenic diseases, which can be performed based on a universal laboratory protocol without the need for set-up or pre-testing.
- MeSH
- aneuploidie MeSH
- blastocysta patologie MeSH
- genetické testování metody MeSH
- lidé MeSH
- mutace genetika MeSH
- preimplantační diagnóza * metody MeSH
- těhotenství MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: To evaluate the decline in transferable embryos in preimplantation genetic testing for aneuploidy (PGT-A) cycles due to (a) non-biopsable blastocyst quality, (b) failure of genetic analysis, (c) diagnosis of uniform numerical or structural chromosomal aberrations, and/or (d) chromosomal aberrations in mosaic constitution. METHODS: This retrospective multicenter study comprised outcomes of 1562 blastocysts originating from 363 controlled ovarian stimulation cycles, respectively, 226 IVF couples in the period between January 2016 and December 2018. Inclusion criteria were PGT-A cycles with trophectoderm biopsy (TB) and next generation sequencing (NGS). RESULTS: Out of 1562 blastocysts, 25.8% were lost due to non-biopsable and/or non-freezable embryo quality. In 10.3% of all biopsied blastocysts, genetic analysis failed. After exclusion of embryos with uniform or chromosomal aberrations in mosaic, only 18.1% of those originally yielded remained as diagnosed euploid embryos suitable for transfer. This translates into 50.4% of patients and 57.6% of stimulated cycles with no euploid embryo left for transfer. The risk that no transfer can take place rose significantly with a lower number of oocytes and with increasing maternal age. The chance for at least one euploid blastocyst/cycle in advanced maternal age (AMA)-patients was 33.3% compared to 52.1% in recurrent miscarriage (RM), 59.8% in recurrent implantation failure (RIF), and 60.0% in severe male factor (SMF). CONCLUSIONS: The present study demonstrates that PGT-A is accompanied by high embryo drop-out rates. IVF-practitioners should be aware that their patients run a high risk of ending up without any embryo suitable for transfer after (several) stimulation cycles, especially in AMA patients. Patients should be informed in detail about the frequency of inconclusive or mosaic results, with the associated risk of not having an euploid embryo available for transfer after PGT-A, as well as the high cost involved in this type of testing.
- MeSH
- aneuploidie MeSH
- blastocysta patologie MeSH
- genetické testování metody MeSH
- lidé MeSH
- preimplantační diagnóza * metody MeSH
- retrospektivní studie MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
Autophagy is one of the basic cellular mechanism during preimplantation development of mammalian embryos, and it plays crucial role in several physiological processes. It is induced by interleukin (IL)-1β in mammalian cells. Our present study shows that IL-1β is important for autophagy activation in embryo development. Our in vitro culture system analysis shows effect of IL-1β in medium on the development of mouse embryos and it was found to be concentration dependent. A preimplantation embryo culture using medium containing IL-1β did not improve cleavage and blastocyst development rates of mouse embryos; however, blastocyst quality was significantly improved by increasing total cell number, especially in supplementary 20 ng/mL IL-1β. Furthermore, autophagy activation mainly occurs in 2 to 4 cell embryo and blastocyst, 20 ng/mL IL-1β into culture medium can effectively enhance levels of messenger RNA and protein of autophagy-related-factors in 2 to 4 cell embryos and blastocyst, while these factors reduce in VGX-1027 (IL-1β inhibitor) groups that also reduce the quality of blastocyst. Effects of IL-1β on the development of embryo reduced in 20 ng/mL IL-1β supplemented group when 5 mM 3-methyladenine (3-MA) was also added, which used to inhibit autophagy activation in endogenous PtdIns3Ks signal pathway. Our current results show that exogenous IL-1β can effectively induce autophagy in mouse embryos at stages of 2 to 8 cell and blastocyst, that also help to improve the quality of blastocyst.
- MeSH
- autofagie * MeSH
- blastocysta účinky léků patologie MeSH
- embryo savčí účinky léků patologie MeSH
- embryonální vývoj účinky léků MeSH
- interleukin-1beta farmakologie MeSH
- kultivace embrya MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cíl studie: Cílem této práce je shromáždit aktuální informace o možnostech preimplantačního genetického screeningu a diagnostiky. Typ studie: Souhrnný článek. Název a sídlo pracoviště: Gynekologicko-porodnické oddělení Nemocnice Šternberk, IVF Clinic Olomouc. Výsledky: Preimplantační genetické testování představuje soubor genetických a molekulárně cytogenetických vyšetření, s jejichž pomocí můžeme odhalit abnormality embrya ještě před jeho přenosem do dělohy matky. Toto specializované vyšetření vychází z nejnovějších poznatků genetiky a asistované reprodukce. Preimplantační genetické testování je nutně spojeno s metodou in vitro fertilizace a provádí se na izolovaných blastomerách třetí den kultivace embrya. V současnosti však bývá preferováno vyšetření buněk trofoektodermu z pětidenních blastocyst. Obecně lze konstatovat, že po provedení preimplantačního genetického testování lze k transferu do dělohy vybrat pouze embrya bez genetické zátěže. Závěr: Preimplantační genetické testování je v současné době důležitou součástí léčby neplodnosti. Komplexní diagnostika a léčba neplodných párů je stále více ovlivňována rozvojem a využitím moderních genomických technologií. Další rozvoj a aplikace těchto metod vyžaduje úzkou spolupráci mezi obory asistované reprodukce a klinické genetiky.
Objective: The aim of this work is to summarize the current knowledge about preimplantation genetic screening and diagnostics. Design: A review article. Setting: Department of Gynecology and Obstetrics, District Hospital Šternberk, IVF Clinic, Olomouc. Results: Preimplantation genetic testing is a complex of genetic and molecular cytogenetic examinations, which can help to detect abnormalities in embryos before transfer into the uterus of the mother. These specialized examinations are based on the latest findings in genetics and assisted reproduction. The preimplantation genetic testing is necessarily associated with a method of in vitro fertilization. It is performed on isolated blastomeres on the third day of embryo cultivation. Nowadays, it is preferred trophectoderm examination of cells from the five-day blastocysts. Generally speaking, after preimplantation genetic testing, we can select only embryos without genetic load to transfer into uterus. Conclusion: Preimplantation genetic testing is an important part of treatment of infertility. Complex diagnostics and treatment of infertile couples are increasingly influenced by the development and use of advanced genomic technologies. Further development and application of these modern methods require close cooperation between the field of assisted reproduction and clinical genetics.
- Klíčová slova
- aCGH, karyomapping, NGS,
- MeSH
- aneuploidie MeSH
- asistovaná reprodukce MeSH
- blastocysta cytologie patologie MeSH
- hybridizace in situ fluorescenční metody využití MeSH
- infertilita terapie MeSH
- lidé MeSH
- preimplantační diagnóza * metody MeSH
- srovnávací genomová hybridizace metody využití MeSH
- výzkum embrya MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- souhrny MeSH
