Kromosomscreening af humane præimplantationsembryoner ved hjælp af brugt dyrkningsmedium: prøveindsamling og kromosomal ploidianalyse
Summary
Denne undersøgelse rapporterer en protokol til kromosomscreening af menneskelige embryoner, der bruger brugt dyrkningsmedium, som undgår embryobiopsi og muliggør kromosomploidiidentifikation ved hjælp af NGS. Denne artikel præsenterer den detaljerede procedure, herunder forberedelse af dyrkningsmedium, helgenomamplifikation (WGA), næste generations sekventering (NGS) biblioteksforberedelse og dataanalyse.
Abstract
Ved klinisk in vitro-befrugtning (IVF) kræver den fremherskende metode til PGT-A biopsi af nogle få celler fra trophectoderm (TE). Dette er den slægt, der danner moderkagen. Denne metode kræver imidlertid specialiserede færdigheder, er invasiv og lider af falske positive og negative, fordi kromosomtallene i TE og den indre cellemasse (ICM), som udvikler sig til fosteret, ikke altid er de samme. NICS, en teknologi, der kræver sekventering af DNA, der frigives i kulturmediet fra både TE og ICM, kan tilbyde en vej ud til disse problemer, men har tidligere vist sig at have begrænset effektivitet. Denne undersøgelse rapporterer den fulde protokol for NICS, som inkluderer dyrkningsmediumprøveudtagningsmetoder, helgenomamplifikation (WGA) og biblioteksforberedelse og NGS-dataanalyse ved hjælp af analysesoftware. I betragtning af de forskellige kryopræserveringstider i forskellige embryolaboratorier har embryologer to metoder til indsamling af embryokulturmedium, der kan vælges i henhold til IVF-laboratoriets faktiske forhold.
Introduction
Assisteret reproduktionsteknologi (ART’er) er i stigende grad blevet brugt til behandling af infertilitet. Imidlertid har succesraten for ART, såsom IVF, været begrænset, og graviditetstabsraten er signifikant højere end for den normale befolkning1. Hovedårsagen til disse problemer er kromosomale abnormiteter, som almindeligvis findes i præimplantation menneskelige embryoner2. PGT-A er en effektiv metode til screening af embryoner for kromosombalance før implantation 3,4. Nogle undersøgelser har vist, at PGT-A kan reducere abortraten og forbedre graviditetsraten 5,6,7,8. PGT-A kræver imidlertid kompleks teknisk ekspertise, der kræver specifik træning og erfaring. Den invasive embryobiopsiprocedure kan også potentielt forårsage skade på embryonerne9. Undersøgelser har vist, at blastomerbiopsi kan hindre efterfølgende udvikling, og antallet af biopserede TE’er kan påvirke implantationshastighederne10. Selvom det langsigtede biosikkerhedsproblem ved embryobiopsi endnu ikke er blevet evalueret grundigt hos mennesker, har dyreforsøg vist dets negative indflydelse på embryoudvikling11,12,13.
Tidligere rapporter viste, at spormængder af DNA-materialer blev udskilt i dyrkningsmediet under embryoudvikling, og der er gjort en indsats for at udføre omfattende kromosomscreening (CCS) ved hjælp af brugt embryokulturmedium 14,15,16,17,18. Detektionsraterne og testenes nøjagtighed har imidlertid ikke opfyldt kravene til omfattende klinisk brug. Denne undersøgelse rapporterede en forbedring i NICS-analysen for at øge detektionsraterne samt nøjagtigheden af NICS-testen19. I de senere år er blastocoelevæske (BF) blevet undersøgt som en analytisk prøve af minimalt invasiv PGT-A. Imidlertid varierer andelen af vellykket genom-dækkende amplifikation og detekterbart DNA i blastocystvæskeprøver fra 34,8% til 82%20,21,22. Mængden af BF rapporteret i forskellige undersøgelser varierer fra 0,3 nL til 1 μL. I betragtning af den lave mængde DNA i BF er det muligt at øge mængden af cellefrit DNA ved at blande blastocystvæske og dyrkningsmedium for at forbedre succesraten og konsistensen af detektionen. Kuznyetsov et al.23 og Li et al.24 behandlede zona pellucida med en laser og frigav blastocystvæske i dyrkningsmediet for at forbedre den samlede mængde embryonalt DNA, og amplifikationshastigheden for de kombinerede medium / BF-prøver efter WGA var henholdsvis 100% og 97,5%. Jiao et al.25 opnåede også en 100% forstærkningssuccesrate ved at bruge den samme metode.
Denne undersøgelse rapporterer en detaljeret protokol, der inkluderer forberedelse af brugte medier, NGS-forberedelse og dataanalyse. Ved omhyggeligt at fjerne cumulusceller fra oocytter udførte denne undersøgelse intracytoplasmatisk enkelt sædinjektion (ICSI) og blastocystkultur. Dag 4-dages 5/dag 6 brugt medium blev indsamlet til WGA og NGS bibliotek forberedelse. Ved hjælp af NICS-teknologi strømlinede denne undersøgelse forberedelsestrinnene til WGA- og NGS-biblioteket på ca. 3 timer og opnåede CCS-resultater noninvasivt på ca. 9 timer.
Protocol
Representative Results
Discussion
Ændringer og fejlfinding
Hvis NICS-resultaterne er forurenet med forældrenes genetiske materialer, skal du sørge for, at alle cumulus-corona radiata-celler fjernes, og sørg for, at ICSI udføres til befrugtning. Forkert medium opbevaring eller skabelonforberedelsesprocesser undgås, hvilket kan nedbryde DNA. Arbejdsområdet blev renset grundigt med DNase- og RNase-dekontamineringsreagenser. For at undgå kontaminering fra andre embryoner blev et embryo altid dyrket i en en…
Declarações
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke Shiping Bo og Shujie Ma for deres hjælp med NGS-dataanalyse. Finansiering: Dette arbejde blev støttet af National Key Research and Development Program (bevilling nr. 2018YFC1003100).
Materials
| 1.5 mL EP tube, 0.2 mL PCR tube | Axygen | MCT-150-C, PCR-02-C | DNase/RNase free, Low Binding PCR tubes and 1.5 mL micro-centrifuge tubes are recommended. |
| 10 µL, 200 µL, 1000 µL DNase /RNase Free Tips | Axygen | T-300-R-S, T-200-Y-R-S, T-1000-B-R-S | This can be replaced by other brand/For sample transfer |
| 100 % ethanol | Sinopharm Chemical | 10009218 | This can be replaced by other brand/For DNA library purification |
| Barcode Primer1-48 | Yikon Genomics | Reagent in NICSInst library preparation kit | For library amplificaton |
| BD Falcon Organ Culture Dish, Sterile | BD Bioscience | 363037 | This can be replaced by other brand/For embryo culture |
| BD Falcon Tissue culture Dishes (Easy Grip) , Sterile | BD Bioscience | 353001 | This can be replaced by other brand/For embryo culture |
| BD Falcon Tissue culture Dishes, Sterile | BD Bioscience | 353002 | This can be replaced by other brand/For embryo culture |
| Cell Lysis Buffer | Yikon Genomics | Reagent in NICSInst library preparation kit | For culture medium pre-treatment |
| Cell Lysis Enzyme | Yikon Genomics | Reagent in NICSInst library preparation kit | For culture medium pre-treatment |
| ChromGo software | Yikon Genomics | Data analysis | |
| CMPure Magbeads | Yikon Genomics | Reagent in NICSInst library preparation kit | For library purification |
| Cryotop open systerm | KITAZATO BioPharma | 81110 | This can be replaced by other brand/For embryo vitrification |
| Distill water | Yikon Genomics | Reagent in NICSInst library preparation kit | To dissolve DNA |
| ES (Vitrification kit) | KITAZATO BioPharma | Reagent inVitrification kit | This can be replaced by other brand/For embryo vitrification |
| HOLDNIG | ORIGIO | MPH-MED-35 | This can be replaced by other brand/For ICSI |
| Hyaluronidase solution, 80 U/mL | SAGE | ART4007-A | This can be replaced by other brand/Digest oocyte-corona-cumulus complex |
| ICSI | ORIGIO | MPH-35-35 | This can be replaced by other brand/For ICSI |
| Illumina MiSeq® System | Illumina | SY-410-1001 | For library sequencing |
| Incubator | Labotect | Inkubator C16 | This can be replaced by other brand/For embryo culture |
| Library buffer | Yikon Genomics | Reagent in NICSInst library preparation kit | For library amplificaton |
| Library Enzyme Mix | Yikon Genomics | Reagent in NICSInst library preparation kit | For library amplificaton |
| Magnetic Stand | DynaMagTM-2 | 12321D | For library purification |
| Microscope | OLYMPUS | 1X71 | This can be replaced by other brand/For embryo observation |
| Mini-centrifuge | ESSENSCIEN | ELF6 | For separation |
| MT Enzyme Mix | Yikon Genomics | Reagent in NICSInst library preparation kit | For culture medium pre-treatment |
| NICSInst library preparation kit | Yikon Genomics | KT1000800324 | Whole genome amplification and library construction |
| NICSInst Sample Prep Station | Yikon Genomics | ME1001003 | Amplificate DNA |
| Nunc IVF 4-Well Dish | Thermo Scientific | 144444 | This can be replaced by other brand/For embryo washing and blastocyst culture |
| Pasteur Pipette | Oirgio | MXL3-IND-135 | This can be replaced by other brand/For embryo tansfer |
| Pasteur pipettes | ORIGIO | PP-9-1000 | This can be replaced by other brand/For IVF laboratory |
| Pre-Lib Buffer | Yikon Genomics | Reagent in NICSInst library preparation kit | Pre-library preparation |
| Pre-Lib Enzyme | Yikon Genomics | Reagent in NICSInst library preparation kit | Pre-library preparation |
| Qubit® 3.0 Fluorometer | Thermo Scientific | Q33216 | For library quantification |
| Quinn's Advantage Blastocyst Medium | SAGE | ART-1029 | For embryo blastocyst stage culture |
| Quinn's Advantage Cleavage Medium | SAGE | ART-1026 | This can be replaced by other brand/For embryo cleavage stage culture |
| Quinn's Advantage Fertilization Medium | SAGE | ART-1020 | This can be replaced by other brand/For oocyte and sperm fertilization |
| Quinn's Advantage m-HTF Medium with HEPES | SAGE | ART-1023 | This can be replaced by other brand/For embryo clutrure |
| Quinn's Advantage SPS Serum protein Substitute Kit | SAGE | ART-3010 | This can be replaced by other brand/To denude the oocyte |
| Quinn's Advantage Tissue culture mineral oil | SAGE | ART-4008P | This can be replaced by other brand/To cover the culture medium |
| STRIPPER TIPS | ORIGIO | MXL3-IND-135 | This can be replaced by other brand/For denudating granulosa cells |
| Vitrification Cryotop Open systerm | KIZTAZATO | 81111 | This can be replaced by other brand/For embryo vitrification |
| Vitrification kit | KITAZATO BioPharma | VT101 | This can be replaced by other brand/For embryo vitrification |
| Vortexer | Qilinbeier | DNYS8 | Sample mix |
| VS (Vitrification kit) | KITAZATO BioPharma | Reagent inVitrification kit | This can be replaced by other brand/For embryo vitrification |
| ZILOS-tk Laser System | Hamilton Thorne | CLASS 1 laser | This can be replaced by other brand/For artificial blastocoele collapse |
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