Utilization of genetic analyses in human assisted reproduction process Mgr. Jan Smetana, Ph.D Ústav Experimentální Biologie, PřF MU Laboratoř molekulární cytogenetiky OLG FN Brno Assisted human reproduction (AHR) •Assisted reproduction is the name given to medical procedures and methods in which germ cells or embryos are manipulated, including their storage, for the purpose of treating infertility in women or men • •The complex process is now mostly based on in vitro fertilization techniques • •In addition to couples with a diagnosis, couples with normal fertility also benefit, due to the risk of transmitting genetic defects or pathological markers • •Specialized centers - clinics, sanatoriums • Goal - birth of healthy offspring = "infertility treatment“ Fertility Fertility - definition 1)The ability of an individual to reproduce sexually 2)A complex trait that results from the ability of males and females to produce healthy offspring in optimal numbers over time 3) 3)Demographic indicator expressing the average number of offspring per female Infertility Failure to achieve clinical pregnancy after 12 months or more of unprotected regular sexual intercourse (WHO) Infertility in Europe: statistical data - IVI Fertility The new epidemic of young India: 27.5 million couples suffer from infertility - The Economic Times Causes of female infertility Causes of female infertility ovarian factor - the ovary does not form or does not release a quality viable egg tubal factor - damage to the fallopian tubes, missing fallopian tubes, obstructed fallopian tubes endometriosis - presence of uterine lining outside the uterine cavity Genetic causes of female infertility Chromosome aberrations – structural ornumerical •Turner syndrome - 45, X •"Superwomen" - 47, XXX •Aneuploidy in gametes •Robertsonian translocations, centromeric fusion acrocentric chromosomes (13-15, 21, 22) Mutations - genes affecting blood clotting •MTHFR (1p36.3) •Leiden mutation (F5 - 1q23), •G20210A in the thrombin gene •CFTR Male factors poor sperm function – sperm incompetence to penetrate and fertilize a woman's egg •Oligozoospermia •(<15*106 in ejaculate) • •Asthenozoospermia •lack of motility • •Teratozoospermia •abnormal morphology • •Azoospermia •absence of sperm in the ejaculate Causes of male infertitlity Male Infertility Treatment Centre Punjab, India Genetic causes of male infertility Chromosomal aberrations •Klinefelter syndrome - 47, XXY •Males - 47, XYY •Structural abnormalities chr. Y •Deletion in (Yp)(11.3) - SRY - disorder of genitourinary development •Deletion Yq11 - AZF - azoosperima factor = disorder of sperm development •Autosomal translocations, Y/A, Robertsonian translocations - centromeric fusion of acrocentric chromosomes (13-15, 21, 22) •Aneuploidy in gametes (X,Y, 21, 13,18) Gene mutations Cystic fibrosis - F508 mutation in CFTR1, 97% of men infertile • History of IVF •17th century- van Graaf - Graaf follicles, van Leeuwenhoek - observation of mammalian sperm •19th century - first scientific papers on in vitro fertilization in animalsSchenk (1878), W. Heape - birth of 6 rabbit pups after vitro fertilization (1890). •1944 - Rock, Menkin - in vitro fertilization of human oocyte •1951 - Austin, Chang - the fertilizing ability of sperm is essential for its previous residence in the female genital tract (sperm capacitation) •2nd half of the 20th century - Cambridge University - R.G. Edwards •Description of oocyte maturation and in vitro fertilization, possibility of • embryo culture •1971 - Steptoe, Purdy: Nature - possibility of in vitro culture of human embryos • to the blastocyst stage •Late 70s - improvements in culture media, laparoscopic techniques, Cryopreservation •1978 - Lancet - clinical applications of in vitro fertilization •L. Brown 2010 - R.G. Edwards - Nobel Prize P.C. Steptoe R.G. Edwards A. van Leeuwenhoek Historie IVF Č(SS)R Prof. L. Pilka MUDr. J. Tesarik Prof. P. Trávník IVF centers in Brno IVF centers in Czech rep. •There are currently over 45 registered IVF centres in the Czech Republic (6x Brno, 8x Prague) • •Private IVF centers, (gynecology, obstetrics, reproductive medicine, genetics, biochemistry) • •Annually over 20,000 IVF cycles • •Over 50% covered by health insurance companies • •Specialization on foreign clientele - "reproductive tourism„ The Fertility Tourism Survey 2020 | FertilityClinicsAbroad.com IVF process Methods of AHR 1.Intrauterine insemination (IUI) = concentrated, purified sperm are introduced through a special catheter into the uterine cavity during ovulation 2.In vitro fertilization (IVF) = classical method of ectopic fertilization, in which sperm are cultured with oocytes in vitro. 3. ICSI - intracytoplasmic sperm injection through the zona pellucida into the egg 4. PICSI - enhanced ICSI allows only the sperm to be selected and injected into the oocyte mature sperm through the attachment to the oocyte complex (hyaluronan) www.gipom.com Chirurgical sperm aspiration Hormonal stimulation – harvesting of oocytes GnRH - gonadorelin, gonadotropin-releasing hormone CC - clomiphene citrate, synthetic estrogen, ovulation support IVF cycle •In the process of an IVF cycle, we usually obtain several embryos... •Ideally, a single embryo transfer is performed • x selection...which is „the best“ - morphology, genetics? Genetic aberrations and IVF a large proportion of embryos, regardless of the age of the mother, are aneuploid (54% under the age of 35, 82% aged 40 and over) Reason = disruption during meiosis Chromosomal aberrations in embryos •~ 90% of aneuploidies occur during meiosis I in women = gradual degradation of cohesin leads to violation of bivalent integrity Aberration of segregation during Meiosis I Preimplantation genetic analyses (PGA) PGT-M: Preimplantation genetic testing of monogenic diseases • •Previously PGD = monogenic diseases •Sex selection in X-linked diseases •Congenital structural abnormalities (Robertsonian translocations, balanced translocations PGT-A: Preimplantation genetic testing for aneuploidies •Screening of the most common congenital chromosomal aneuploidies PGA methods 1. Molecular cytogenetics (I-FISH, CGH) • aneuploidy, translocations, microdeletion syndromes, etc. 2.PCR - monogenic diseases •specific mutations - CF, thalassaemia, sickle cell anemia, hemophilia, DMD..... •QF PCR - +13,16,18,21, X,Y 3.Screening techniques - "PGD 2.0" - whole genome coverage •array-CGH (DNA chips) - numerical and structural CHA •SNP chips - KARYOMAPPING •NGS - comprehensive approach, PGD+PGS combination Výsledek obrázku pro NGS PGD PGA – biological material Oplození in vitro Biopsie embrya Genetický test Polární tělísko Blastomera (3. den) Blastocysta (5-6. den) Embryo biopsy difference between 3rd and 5th harvest day Day 3 embryo biopsy (blastomeres) •analysis of 1 - 2 cells •30 - 60% loss of implantation potential •higher risk of mosaicism •time constraint (24 hours) Embryo biopsy day 5-6 (blastocyst) •analysis of 5 - 10 cells •less risk of mosaicism •possibility of embryo vitrification = sufficient time for examination •not all embryos reach the stage blastocysts = slection Vitrification of embryos •Vitrification •modern method of effective cryopreservation of embryos, oocytes and sperm •Superfast freezing of biological material with a mixture of suitably selected cryoprotectants (sucrose, dimethylsulfoxide) at -196C •Viablity after thawing approx. 98% Chromosomal aberrtaions in embryos Multiple chromosome aberrations (aneuploidy) •are the most common genetic alteration in human embryos •aneuploidies often occur in morphologically normal developing embryos (A. Mertzanidou, 2013) • reduce the success rate of assisted reproductive techniques Structural chromosome aberrations •postzygotic mitotic disorders are very common in embryos •Chromosomal instability - duplication, amplification, deletion, UPD - has been demonstrated in up to 70% of embryos using SNP chips (Vanneste et al., 2009) SCREEINING OF MERE ANEUPLOIDIES IN EARLY EMBRYOS IS NOT ENOUGH! PGA with the use of I-FISH Screening - AneuVysion Vysis MultiVysion Probe Panel (13,18,21,X,Y,16,22) SpectrumGreen 21 SpectrumRed 13 SpectrumBlue X SpectrumGold Y SpectrumAqua 18 Multiple chromosomes on one cell - repeated FISH (FISH - evaluation, washout, new FISH - evaluation) PGA with I-FISH does not improve IVF success.....why? Problems of PGA I examination of single cell - possibility of diagnostic error ? Problems of PGA II normal (all diploid cells) Mosaic (diploid + aneuploid cells) abnormal (all cells abnormal) chaotic (each cell contains different number of chromosomes) EMBRYA: One cell does not have to represent the whole embryo !!! Problems of PGA I – structural CHAs •structural aberrations (deletions, duplications, UPD etc...) also occur in embryos ...post-zygotic mitotic disorders are more frequent than meiotic ones... • It's not enough to investigate aneuploidy! Whole genome testing! Use of whole genome screening techniques in PGA •Isolation of one - several cells + whole genome amplification • •Use of array-CGH microarray techniques, SNP chips, NGS • •Possibility to examine the whole genome - necessary in a short time interval (24 h) X frozen embryos (vitrification) • DNA amplification – key step in complex PGA Genomic methods – hunderts ng of DNA needed, = 106cells XX trophoectoderm aspirate 20 cells = pg DNA, DNA amplification required Single cell WGA principy – PCR •Advantages: •higher yield, simple protocol, less time consuming •XXX •creates artifacts, ADO •Applications: array-CGH, QF - PCR PCR based WGA amplification Výsledek obrázku pro mda single cell •Circular amplification using the thermostable phage mutant Phí29 •Advantages - lower ADO rate, no amplification products x more challenging, lower yield •Applications: NGS, methylation analysis (PWS/AS) Multiple displacement amplification BAC array CGH – PGS in 12 hours Aneuploidy and structural changes (deletions, duplications) in the whole genome ; Resolution ~ 5 Mbp Metodika screeningových technik u PGT Array-cgh workflow PGA using high-resolution array-CGH Material: cells from trophoectoderm of 5-day-old embryos Amplification protocol: PicoPLEX WGA Kit (Rubicon Genomics, USA) Microarrays: 8x15K - CytoSure™Single Cell Aneuploidy Array, OGT UK 8x60K - Agilent SurePrint G3 Oligo CGH Microarray Software: CytoSure Interpret Software, Genomic Workbench Comparison of chromosome 19 profile on Agilent and OGT platform Higher false positivity of the 15k platform, most commonly chr. 11, 16 and 19 Agilent 8x60k OGT 8x15 Porovnání profilu chromozomu 22 na platformě Agilent a OGT Agilent 8x60k OGT 8x15k Higher density of 60k microarrays gives more robust results compared to 15k Agilent 8x60k OGT 8x15 Mikulášová A. et al, SLG konference 2014, Praha •most common monosomy: chromosome 22 (7.7%; 5/65), 7, 8 and 18 (6.1% each; 4/65) •most common trisomy: chromosome 15, 21 and 22 (4.6% each; 3/65) • Results of PGS array-CGH screening Results of PGS array-CGH screening Visualization of 8.4 Mb segmental deletion in chromosome 13q21.32 - q22.2 affecting loci ofCDH9, KLHL1, ATXN8OS, DACH1, C13orf37, C13orf34, DIS3, PIBF1, KLF5, KLF1 gene. Karyomapping – PGA-M 23 Obtain genomic DNA from parents Obtain genomic DNA from reference (known genotype status e.g. affected child, siblings ... Karyomapping – PGA-M Karyomapping – PGA-M PGD condition Mode of inheritance Gene/Locus Phenotype MIM number Chr Region SNP coverage 5' Gene/ Locus 3' Crigler Najar ... Karyomapping – PGA-M 25 Karyomapping - Diagnostic Laboratory Process Process DNAs - Infinium HumanKaryomap-12 DNA analysis kit (20 hrs) Kit = 2... Karyomapping – PGA-M https://els-jbs-prod-cdn.jbs.elsevierhealth.com/cms/attachment/87c0990f-7465-402a-a562-20be1ed5580d /rbmo1376-fig-0001.jpg Kubeciek et al, Incidence and origin of meiotic whole and segmental chromosomal aneuploidies detected by karyomapping. 2018. https://doi.org/10.1016/j.rbmo.2018.11.023 • Karyomapping – PGA-M Karyomapping – PGA-M •Benefits •Fast and efficient method for complex PGA-M if we have a suitable reference •Detection of structural deletions, aneuploidies and monogenic diseases • •Disadvantages •Patented technology (Illumina), no competition for chemistry, costly, closed system •Potential for problems in the absence of reference DNA Next generation (massive parallel) sequnincg in PGS Massive parallel sequencing technology (MPS) in IVF •NGS technologies are starting to make their way into PGS • •Processing of a larger number of samples in one experiment compared to microchip techniques • •Currently used in large IVF clinics mainly for screening of aneuploidies x possibility of a comprehensive view (ploidy, structural changes, mutations) • •Most often a form of closed systems - Illumina, Ion Torrent, or a form of library preparation (e.g. Agilent, Roche, etc.) • VeriSeq PGS (Illumina) • •Sequenicng by synthesis •Anueploididy detection in 12 hours •Up to 24 samples, resolution 16 Mbp VeriSeq PGS (Illumina) 9 Massively parallel sequencing approach – 25 million reads per MiSeq run Multiplex up to 24 samples per run by using inde... VeriSeq PGS (Illumina) Výsledek obrázku pro veriseq illumina VeriSeq PGS (Illumina) BlueFuse analytický SW VeriSeq PGS (Illumina) Ion Torrent Semiconuctor Sequencing Související obrázek Ion Torrent Aneuploidy Analysis (Life Tech Inc.) Ion Torrent Aneuploidy Analysis •"Semiconductor" sequencing •Based on the detection of the pH change that occurs when H+ is released during base binding to deoxyribose • •Protocol within 24 hours •Resolution ~10 Mbp •Cost $70/embryo for 32 embryos analyzed together NGS in IVF • •Routine use is still hampered by cost and algorithm in laboratories (3 vs. 5 day embryos, vitrification technology, etc.) • •Advantages - more robust compared to array-CGH, higher capacity, • •Higher "dynamic interval" - detection of mosaicism • •Development - exome level detection - "all in" = CHA, mutations for monogenic diseases • NGS u PGD 11 Performance Comparison between array CGH & NGS on Day 5 Trophectoderm Biopsies NGS aCGH NGS u PGD 14 Improved dynamic range – Fiorentino (2014) ESHRE S07 NGS u PGD •Most accurate and informative •Sequence data from thousands of loci along chromosome •Multiple genomic loci and multiple ... NGS in IVF NGS in IVF – problems? 1)With more robust screening methods, the volume of data is increasing - interpretation? 2)Detection of mosaicism in embryos - transfer yes or no? 3)PGD 2.0 - does it really improve IVF outcomes? After PGD, the results should be consulted with a clinical geneticist + Follow-up with prenatal genetic diagnosis should be performed After PGA… Thank you for attention