Kromosomscreening av humane preimplantasjonsembryoer ved bruk av brukt kulturmedium: prøveinnsamling og kromosomal ploidianalyse
Summary
Denne studien rapporterer en protokoll for kromosomscreening av humane embryoer som bruker brukt kulturmedium, som unngår embryobiopsi og muliggjør kromosomploidiidentifikasjon ved hjelp av NGS. Denne artikkelen presenterer den detaljerte prosedyren, inkludert utarbeidelse av kulturmedium, hele genomamplifikasjon (WGA), neste generasjons sekvensering (NGS) bibliotekforberedelse og dataanalyse.
Abstract
Ved klinisk in vitro-fertilisering (IVF) krever den rådende metoden for PGT-A biopsi av noen få celler fra trophektoderm (TE). Dette er linjen som danner morkaken. Denne metoden krever imidlertid spesialiserte ferdigheter, er invasiv og lider av falske positive og negative fordi kromosomtallene i TE og den indre cellemassen (ICM), som utvikler seg til fosteret, ikke alltid er de samme. NICS, en teknologi som krever sekvensering av DNA som slippes ut i kulturmediet fra både TE og ICM, kan tilby en vei ut til disse problemene, men har tidligere vist seg å ha begrenset effekt. Den nåværende studien rapporterer hele protokollen til NICS, som inkluderer prøvetakingsmetoder for kulturmedium, helgenomamplifisering (WGA) og bibliotekforberedelse, og NGS-dataanalyse ved analyseprogramvare. Tatt i betraktning de forskjellige kryokonserveringstidene i forskjellige embryolaboratorier, har embryologer to metoder for å samle embryokulturmedium som kan velges i henhold til de faktiske forholdene i IVF-laboratoriet.
Introduction
Assistert befruktning (ART) har i økende grad blitt brukt til behandling av infertilitet. Imidlertid har suksessraten for ART, som IVF, vært begrenset, og graviditetstapsraten er betydelig høyere enn for den normale befolkningen1. Hovedårsaken til disse problemene er kromosomale abnormiteter, som vanligvis eksisterer i preimplantasjon menneskelige embryoer2. PGT-A er en effektiv metode for screening av embryoer for kromosombalanse før implantasjon 3,4. Noen studier har vist at PGT-A kan redusere abortfrekvensen og forbedre graviditetsraten 5,6,7,8. PGT-A krever imidlertid kompleks teknisk ekspertise som krever spesifikk trening og erfaring. Den invasive embryobiopsiprosedyren kan også potensielt forårsake skade på embryoene9. Studier har vist at blastomerbiopsi kan hindre senere utvikling, og antall biopsierte TEs kan påvirke implantasjonsratene10. Selv om det langsiktige biosikkerhetsproblemet med embryobiopsi ennå ikke har blitt evaluert grundig hos mennesker, har dyreforsøk vist sin negative innflytelse på embryoutvikling11,12,13.
Tidligere rapporter indikerte at spormengder av DNA-materialer ble utskilt i kulturmediet under embryoutvikling, og det har blitt gjort forsøk på å utføre omfattende kromosomscreening (CCS) ved bruk av brukt embryokulturmedium 14,15,16,17,18. Deteksjonsratene og nøyaktigheten av testene har imidlertid ikke oppfylt kravene til omfattende klinisk bruk. Denne studien rapporterte en forbedring i NICS-analysen for å øke deteksjonsraten samt nøyaktigheten av NICS-testen19. De siste årene har blastocoelevæske (BF) blitt studert som en analytisk prøve av minimalt invasiv PGT-A. Imidlertid varierer andelen vellykket genom-bred amplifisering og detekterbart DNA i blastocystvæskeprøver fra 34,8% til 82%20,21,22. Volumet av BF rapportert i ulike studier varierer fra 0,3 nL til 1 μL. I lys av den lave mengden DNA i BF, er det mulig å øke mengden cellefritt DNA ved å blande blastocystvæske og kulturmedium for å forbedre suksessraten og konsistensen av deteksjon. Kuznyetsov et al.23 og Li et al.24 behandlet zona pellucida med en laser og frigjorde blastocystvæske i kulturmediet for å forbedre den totale mengden embryonalt DNA, og amplifikasjonshastigheten til de kombinerte medium / BF-prøvene etter WGA var henholdsvis 100% og 97,5%. Jiao et al.25 oppnådde også en suksessrate på 100% forsterkning ved å bruke samme metode.
Den foreliggende studien rapporterer en detaljert protokoll som inkluderer forberedelse av brukte medieprøver, NGS-forberedelse og dataanalyse. Ved forsiktig å fjerne cumulusceller fra oocytter, utførte denne studien intracytoplasmatisk enkeltspermieinjeksjon (ICSI) og blastocystkultur. Dag 4-dagers 5/dag 6 brukt medium ble samlet inn for WGA og NGS bibliotek forberedelse. Ved å bruke NICS-teknologi strømlinjeformet denne studien WGA- og NGS-bibliotekforberedelsestrinnene på omtrent 3 timer og oppnådde CCS-resultater ikke-invasivt på omtrent 9 timer.
Protocol
Representative Results
Discussion
Endringer og feilsøking
Hvis NICS-resultatene er forurenset med foreldrenes genetiske materiale, må du sørge for at alle cumulus-corona radiata-celler fjernes og sørge for at ICSI utføres for befruktning. Feil medium lagring eller malforberedelsesprosesser unngås, noe som kan forringe DNA. Arbeidsområdet ble renset grundig med DNase og RNase dekontamineringsreagenser. For å unngå forurensning fra andre embryoer ble ett embryo alltid dyrket i en enkelt dråpe medium fo…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gjerne takke Shiping Bo og Shujie Ma for deres hjelp i NGS dataanalyse. Finansiering: dette arbeidet ble støttet av National Key Research and Development Program (Grant No. 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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