Topic: Novel Genetic Testing in ART. Matchuporn Sukprasert

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1 Topic: Novel Genetic Testing in ART Matchuporn Sukprasert

2 Topic: Novel Genetic Testing in ART Next-generation sequencing (NGS) The frequency of aneuploidy status of Day 5 and Day 6 blastocysts assessed by next-generation sequencing technology (NGS) application (Liss) First clinical applications of PGD for β-thalassemia combined with PGS on blastocysts from fresh and vitrified oocytes using Next Generation Sequencing (NGS) (Chamayou) ART, assisted reproductive technology; PGD, preimplantation genetic diagnosis; PGS, preimplantation genetic screening

3 Topic: Novel Genetic Testing in ART Improvement in outcomes and selection with genetic testing Preimplantation genetic screening using comprehensive chromosome screening technology improves embryo selection: a meta-analysis of randomized controlled trials (Dahdouh) High success rate of preimplantation genetic diagnosis in achieving healthy live-birth for rare monogenic disorders (Tac) Comparison of the live birth rate between PGD and natural conception in patients with recurrent pregnancy loss associated with translocation (Ikuma) Preimplantation genetic screening in IVF cycles with frozen embryo transfer: 2 year experience of one PGS center (Hrubá) IVF, in vitro fertilisation

4 Next-generation sequencing (NGS)

5 P-598 The frequency of aneuploidy status of Day 5 and Day 6 blastocysts assessed by next-generation sequencing technology (NGS) application J Liss Poster

6 Study background and aim Background Embryos with the highest morphological scores have a higher euploidy rate when compared with lower quality embryos During IVF cycles, the embryonic cohort is asynchronous in development and trophectoderm biopsy can equally be performed on Day 5 (D5) or Day 6 (D6) post-fertilisation and on blastocysts of different morphological quality It is still unknown whether blastocyst morphology and developmental rate relate to the embryo chromosomal constitution Aim To assess correlation between D5 and D6 blastocyst morphology and ploidy status. Liss J. Poster. P598

7 Study materials and methods A retrospective, single-centre study was performed in women with a median age of 38 years (range 34-40) between August 2013 and November blastocyst stage PGS cycles were studied Trophectoderm biopsy was performed on D5 (n=104) of development or, for slower growing embryos, on D6 (n=64) A total of 168 blastocysts were evaluated with the NGS protocol following whole genome amplification Liss J. Poster. P598

8 Aneuploidy rate was significantly higher in D6 blastocysts compared to D5 Day 5 biopsy Day 6 biopsy p Euploid, n 49 (47.1%) 18 (28.1%) Aneuploid, n 55 (52.9%) 46 (71.9%) 0.01 Good quality embryos, >3AA score, n 69 (80.2%) 46 (71.9%) NS >3AA score euploid/aneuploid <3AA score euploid/aneuploid p Day 5 biopsy 49.3%/50.7% 47.1%/52.9% NS Day 6 biopsy 28.3%/71.7% 27.8%/72.2% NS No significant difference in blastocyst morphology was observed between D5 and D6 blastocysts The euploidy/aneuploidy rates did not vary significantly between good and poor morphology embryos in either D5 or D6 blastocysts Liss J. Poster. P598

9 Transfer of D5 blastocysts resulted in higher CPR and IR n=18 D5 D6 30 n=3 % Clinical pregnancy rate Implantation rate Single FET of D5 (n=39) and D6 (n=11) euploid blastocysts was performed resulting in 18 and 3 clinical pregnancies, respectively CPR, clinical pregnancy rate; IR, implantation rate Liss J. Poster. P598

10 Conclusions and clinical implications Conclusions Slower developing blastocysts cryopreserved on D6 but at the same stage of development as those developing to the blastocyst stage on D5, do not have similar chromosomal status and provide a lower chance of achieving pregnancy Clinical implications Euploid embryos tend to show faster progression to the most advanced expansion stages when compared with aneuploid embryos. Liss J. Poster. P598

11 P-609 First clinical applications of PGD for β-thalassemia combined with PGS on blastocysts from fresh and vitrified oocytes using Next Generation Sequencing (NGS) S Chamayou Poster

12 Study background and aim Background Until now, the clinical application of PGD for single cell disease and PGS on embryos have been applied separately NGS has been applied and previously validated for diagnosis of aneuploidy in embryos Aim To use next-generation sequencing for preimplantation genetic diagnosis of β-thalassemia combined with preimplantation genetic screening. Chamayou S. Poster. P609

13 Study materials and methods This study involved 7 infertile couples carrying β-globin mutations (Cd39, HbS, IVS1nt6, IVS1nt110, IVS2nt745) Trophectoderm cells of blastocysts produced from fresh and vitrified/warmed oocytes were used for diagnosis of β- globinopathies and aneuploidies Trophectoderm cells were removed from day 5-6 blastocysts that had been fertilised by ICSI Whole genome amplification was performed on 39 samples Sample libraries were prepared with an in-house Ion AmpliSeq HBB panel and Ion plus fragment library kit. Enriched barcoded-sample libraries were sequenced on Ion PGM (Life Technology) ICSI, intracytoplasmic sperm injection; HBB, β-globin gene analysis Chamayou S. Poster. P609

14 After PGD and PGS, 10 embryos were diagnosed as euploid with 1 β-globin mutation 25 blastocysts from fresh oocytes 14 blastocysts from vitrified/warmed oocytes 39 blastocyst biopsies WGA on trophectoderm cells, Ampliseq with HBB Ion Ampliseq design primers, Ion Xpress barcoding, Library enrichment, NGS on PGM (Life Technology) HBB Analysis: 37 results - 9 affected embryos - 16 carrier embryos - 12 embryos w/o β-globin mutations Previous WGA, Fragment library kit, lon Xpress barcoding, Library enrichment, NGS PGS Results: 37 results - 22 aneuploid embryos - 15 euploid embryos 7 thawed and transferred embryos Final genetic diagnoses: - 10 euploid embryos with 1 β-globin mutation 4 clinical pregnancies and PND confirmations 2 from fresh oocytes 2 from vitrified/warmed oocytes Of these 10 euploid embryos, 7 were transferred resulting in 4 clinical pregnancies WGA, whole genome amplification; w/o, without; PND, prenatal diagnosis Chamayou S. Poster. P609

15 Conclusions and clinical implications Conclusions Using NGS technology, it is possible to diagnose aneuploidies and single cell diseases, such as haemoglobinopathies, simultaneously The use of fresh oocytes together with vitrified oocytes increases the number of available embryos to transfer Clinical implications In cases of limited ovarian response, it is possible to combine the diagnosis of embryos from fresh and vitrified/warmed oocytes. This option allows the couple to limit the cost of genetic analysis and increase the number of available embryos to transfer. Chamayou S. Poster. P609

16 Improvement in outcomes and selection with genetic testing

17 O-117 Preimplantation genetic screening using comprehensive chromosome screening technology improves embryo selection: a meta-analysis of randomized controlled trials E Dahdouh Oral presentation

18 Study background and aim Background Most RCTs that have examined the impact of preimplantation genetic screening using FISH show no increase in live birth rates Comprehensive chromosome screening, which analyses the whole chromosome complement, can be performed using various genetic platforms, and has been extensively tested and validated in PGS cycles (PGS-CCS) However, whether PGS-CCS improves embryo selection in IVF remains unclear Aim To determine whether the use of PGS-CCS technology improves embryo selection in IVF cycles. FISH, fluorescence in-situ hybridisation Dahdouh E. Session 33. Presentation O-117

19 Study materials and methods A meta-analysis of RCTs on PGS-CCS published before January 2015 was performed RCTs were eligible if they compared PGS-CCS to other methods of embryo selection Outcomes were clinical IR and sustained IR (probability that an embryo would implant and progress beyond 20 weeks gestation) 267 embryos transferred after PGS-CCS were compared to 383 embryos transferred after selection based on standard morphology criteria alone Dahdouh E. Session 33. Presentation O-117

20 PGS-CCS was associated with significantly higher clinical IR compared to morphology-based embryo selection Clinical implantation rate PGS-CCS Control Risk ratio Risk ratio Study or subgroup Events Total Events Total Weight M-H, Fixed, 95% Cl M-H, Fixed, 95% Cl Yang et al % 1.55 [1.09, 2.20] Forman et al % 1.22 [0.98, 1.52] Scott et al % 1.26 [1.09, 1.46] Total (95% Cl) % 1.29 [1.15, 1.45] Total events Heterogeneity: Chi 2 =1.34, df=2 (p=0.51); l 2 =0% Test for overall effect: Z=4.27 (p<0.0001) Favours control Favours PGS-CCS Of 750 articles identified, only 3 RCTs met full inclusion criteria, allowing direct comparison of PGS-CCS versus IVF with morphology-based embryo selection One of the three RCTs used array comparative genomic hybridisation; the other two used qpcr qpcr, quantitative polymerase chain reaction Dahdouh E. Session 33. Presentation O-117

21 PGS-CCS was associated with significantly higher sustained IR compared to morphologybased embryo selection Sustained implantation rate (> 20 weeks) PGS-CCS Control Risk ratio Risk ratio Study or subgroup Events Total Events Total Weight M-H, Fixed, 95%Cl M-H,Fixed,95% Cl Yang et al % 1.66 [1.14, 2.42] Forman et al % 1.29 [1.03, 1.61] Scott et al % 1.39 [1.14, 1.70] Total (95% Cl) % 1.39 [1.21, 1.60] Total events Heterogeneity:Chi 2 =1.29, df=2 (p=0.53); l 2 =0% Test for overall effect: Z=4.61 (p<0.0001) Favours control Favours PGS-CCS Use of PGS-CCS was also associated with a significantly higher sustained IR compared to embryo selection based on morphology alone CI, confidence interval Dahdouh E. Session 33. Presentation O-117

22 Conclusions and clinical implications Conclusions In patients with normal ovarian reserve, PGS-CCS improves embryo selection compared to use of morphology alone PGS-CCS is associated with significantly higher clinical IR and sustained IR However, PGS-CCS is invasive and carries financial burdens Further evidence is required to determine whether the advantage extends to other patient groups Clinical implications PGS-CCS improves embryo selection compared to use of embryo morphology alone. PGS-CCS might be helpful when used in the setting of elective single embryo transfer practice. Dahdouh E. Session 33. Presentation O-117

23 P-602 High success rate of preimplantation genetic diagnosis in achieving healthy live-birth for rare monogenic disorders H Tac Poster

24 Study background and aim Background Since its first application, PGD for monogenic disorders has become an effective alternative to prenatal diagnosis Since then, it is estimated that PGD has been performed for over 350 monogenic conditions, resulting in the birth of thousands of healthy children There are few reports on the application of this technique for rare monogenic disorders, such as Coenzyme Q deficiency and Leber congenital amaurosis Aim To determine the clinical outcomes of PGD cycles performed for very rare monogenic disorders, and to evaluate whether the method has been successful in terms of both diagnostic and clinical efficiency. Tac H. Poster. P602

25 Study materials and methods A retrospective analysis was conducted of 17 PGD cycles for 9 couples referred for PGD due to 9 different rare monogenic disorders between 2003 and 2013 PGD set-up procedure was performed initially on the parents' peripheral blood DNA samples in order to establish informative STR markers PGD was performed at the cleavage or blastocyst stage External and nested PCR amplifications were performed in all cases Mutation analysis was performed using the PCR-RFLP method and polymorphic STR markers were used for both mutation analysis and HLA typing DNA, deoxyribose nucleic acid; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; STR, short tandem repeat; HLA, human leukocyte antigen Tac H. Poster. P602

26 Pregnancy was achieved in 62.5% of PGD cycles with an implantation rate of 48.1% Disease Name Female age Biopsied embryos Type of biopsy Transferable Embryos Transferred Embryos Prenatal confirmation Pregnancy outcome Ehler Danlos Syndrome Type VllC Coenzyme Q deficiency 31 8 Trophectoderm 2 1 Yes 34 5 Blastomere 4 2 Yes Delivered (singleton) Delivered (singleton) Lafora disease Blastomere 10 2 Miscarriage 38 Frozen embryo transfer 2 Yes Delivered (singleton) Leber congenital amaurosis 30 6 Trophectoderm 5 2 Yes Blastomere 10 2 No Delivered (twin) Delivered (triplet) Fraser syndrome 31 Frozen embryo transfer 1 No 32 4 Blastomere 1 1 No Delivered (singleton) 200 oocytes were retrieved, 172 mature oocytes injected and 151 oocytes fertilised in the 17 cycles, resulting in 126 diagnosed embryos out of 142 biopsied embryos Tac H. Poster. P602

27 Pregnancy was achieved in 62.5% of PGD cycles with an implantation rate of 48.1% (con t.) Disease Name Female age Biopsied embryos Type of biopsy Transferable Embryos Transferred Embryos Prenatal confirmation Pregnancy outcome Leukocyte Adhesin Syndrome type lll Arthrogryposis- Renal Dysfunction- Cholestasis syndrome 29 5 Blastomere No embryo for transfer 30 5 Trophectoderm 2 2 Yes Ongoing 32 2 Blastomere 2 1 No 32 2 Blastomere 1 1 No Blastomere 7 3 No Barter s syndrome with sensorineural deafness 35 Frozen embryo transfer 2 No Blastomere 12 2 Miscarriage Glazmann thrombasthenia Blastomere 1 1 No Blastomere 2 2 Yes Delivered (twin) Live birth rate per cycle was 41.2%, and per patient was 77.7% Eleven babies were born from 7 full-term deliveries (1 ongoing pregnancy), including 4 singletons, 2 sets of twins and 1 set of triplets Tac H. Poster. P602

28 Conclusions and clinical implications Conclusions Live birth rate per cycle, per transfer and per patient were high using this diagnostic procedure Live birth rate per patient was 77.7% Clinical implications The high efficiency of the diagnostic procedures and the high livebirth rate reported in this study indicate that PGD is highly successful when performed for couples at risk of transmitting very rare monogenic disorders. Lindeman M. Poster. P601

29 P-613 Comparison of the live birth rate between PGD and natural conception in patients with recurrent pregnancy loss associated with translocation S Ikuma Poster

30 Study background and aim Background Established causes of recurrent pregnancy loss (RPL) include parental chromosomal abnormalities, particularly translocations PGD can be used to prevent miscarriage in patients with RPL associated with translocations To date, there has been no cohort study conducted to compare the live birth rates in patients matched for age and number of previous miscarriages undergoing PGD or conceiving naturally Aim To determine whether PGD improves the live birth rate, compared to natural conception, in patients with RPL associated with translocation. Ikuma S. Poster. P613

31 Study materials and methods A cohort study was conducted between August 2003 and November 2013; subsequent pregnancies were followed up until July Japanese patients with RPL associated with translocation were included; 52 patients chose to attempt natural conception and 74 patients opted to undergo PGD after genetic counselling Live birth rate, cumulative live birth rate and miscarriage rate were compared between 37 patients undergoing PGD who were matched for age with 52 patients who selected natural conception and did not undergo PGD PGD was performed by FISH analysis on blastomeres obtained from day 3 embryos at about 8-cell stage Ikuma S. Poster. P613

32 PGD significantly decreased miscarriage rate 37 matched patients who underwent PGD 52 patients who conceived naturally OR (95%CI) p-value Live birth rate at the first trial 37.8% (14/37) 53.8% (28/52) 0.52 ( ) 0.10 Cumulative live birth rate 67.6% (25/37) 65.4% (34/52) 1.10 ( ) 0.83 Total (range) and mean number of further miscarriages till live birth Biochemical pregnancy* Ectopic pregnancy* 9 (0 1) 0.24±0.40 (1) (2) 30 (0 3) 0.58±0.78 (1) (1) 0.02 Infertility 18.9% (7/37) 3.8% (2/52) 1.19 ( ) 0.03 Congenital anomaly 1 1 Twin pregnancy/live birth 29.0% (9/31) 5.1% (2/39) 7.57 ( ) Cost/patient $7,956 - No difference in birth rate was found between groups The prevalence of twin pregnancy was significantly higher in the PGD group Ikuma S. Poster. P613

33 Conclusions and clinical implications Conclusions While PGD significantly prevented further miscarriages, there was no difference in the live birth rate Single embryo transfer should be selected to prevent a higher risk of multiple pregnancies and the risk of preterm delivery Clinical implications During genetic counselling, couples should be fully informed of the advantages and disadvantages of PGD, and the advantages of natural pregnancy. Ikuma S. Poster. P613

34 P-630 Preimplantation genetic screening in IVF cycles with frozen embryo transfer: 2 year experience of one PGS center M Hrubá Poster

35 Study background and aim Background The benefit of comprehensive chromosomal screening is still controversially discussed Recently, a significant increase in pregnancy rates was reported in young, good-prognosis-patients when performing PGS on trophectoderm samples by acgh followed by fresh embryo transfer (Yang et al. Mol Cytogenet 2012) Further, a benefit of PGS was shown in patients with advanced maternal age (AMA) (Harton et al. Fertil Steril 2013) Aim To ascertain whether PGS improves pregnancy rates (PR) in IVF cycles with ET of frozen embryos, and whether PGS results and clinical outcomes differ in relation to AMA. acgh, array-based comparative genomic hybridisation Hrubá M. Poster. P630

36 Study materials and methods PGS data from IVF cycles with FET from a 2 year period ( ) were retrospectively analysed in correlation to maternal age Clinical outcome (PR per FET) was compared with age-matched outcome of FETs without PGS (nopgs, 2014 only) (A) PGS/FET Group I: 32 years 192 blastocysts biopsied from 27 egg donors, 24 FETs Group II: 38 years 162 blastocysts biopsies from 30 patients (own oocytes), 19 FETs Group III: >38 years 261 blastocysts biopsied from 61 patients (own oocytes), 28 FETs (B) nopgs/fet 372 FETs 200 FETs 95 FETs PGS analysis was performed on trophectoderm samples by acgh FET, frozen embryo transfer Hrubá M. Poster. P630

37 The proportion of aneuploid embryos increased with maternal age p< % of abnormal embryos % 58% 75% 0 Group I A ( 32y) Group II A ( 38y) Group III A (>38y) The proportion of aneuploid embryos (including both whole chromosomal and segmental unbalanced changes) increased with AMA (p< ) Hrubá M. Poster. P630

38 However, in the PGS group PR did not significantly differ between the age groups Pregnancy rate per FET (%) % P=0.451, NS 59% 68% P= % 57% P=0.077, NS 38% PGS/FET (A) NoPGS/FET (B) 0 Group I ( 32y) Group II ( 38y) Group III (>38y) PRs per FET did not significantly differ among PGS groups (p=0.674) Overall PR per FET after PGS (i.e. without selection for maternal age) was significantly improved in comparison with FETs without PGS (63% vs. 49%, p= 0.025) NS, not significant Hrubá M. Poster. P630

39 Conclusions and clinical implications Conclusions A statistically significant overall increase in PR after PGS/FET was observed when compared with patients not undergoing PGS/FET The higher increase in PRs was seen predominantly in the older maternal age groups PGS can overcome the adverse effect of AMA. Although the proportion of aneuploid embryos strongly increased with AMA, PRs did not significantly differ from younger patients when PGS was applied Clinical implications PGS is a useful method for choosing viable embryos for FET, particularly in patients with AMA. Hrubá M. Poster. P630

40 Summary

41 Summary: Novel Genetic Testing in ART next-generation sequencing (NGS) Liss et al: Euploid embryos tend to show faster progression to the most advanced expansion stages when compared with aneuploid embryos Chamayou et al: In cases of limited ovarian response, it is possible to combine the diagnosis of embryos from fresh and vitrified/warmed oocytes. This option allows the couple to limit the cost of genetic analysis and increase the number of available embryos to transfer

42 Summary: Novel Genetic Testing in ART improvement in outcomes and selection with genetic testing Dahdouh et al: PGS-CCS improves embryo selection compared to use of embryo morphology alone Tac et al: The high efficiency of the diagnostic procedures and the high live-birth rate reported in this study indicate that PGD is highly successful when performed for couples at risk of transmitting very rare monogenic disorders Ikuma et al: While PGD significantly prevented further miscarriages, there was no difference in the live birth rate Hrubá et al: A statistically significant overall increase in PR after PGS/FET was observed when compared with patients not undergoing PGS/FET. The higher increase in PRs was seen predominantly in the older maternal age groups

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