Genetics of rpl

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1. GENETICS OF RECURRENT PREGNANCY LOSS Dr Antima Rathore Fellow - Reproductive Medic Institute Of Human Reproduc Guwahati 2. 2 3. Definition 3 Pregnancy Loss…
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  • 1. GENETICS OF RECURRENT PREGNANCY LOSS Dr Antima Rathore Fellow - Reproductive Medic Institute Of Human Reproduc Guwahati
  • 2. 2
  • 3. Definition 3 Pregnancy Loss (Miscarriage) ↓ spontaneous demise of a pregnancy before the fetus reaches viability All pregnancy losses from the time of conception until 24 weeks of gestation 3 Green-top Guideline no 17, 2011
  • 4. Definition 4 “….involuntary termination of pregnancy before 20 weeks of gestation (dated from the last menstrual period) or below a fetal weight of 500 gm” Losses after 20 weeks are considered stillbirths or premature births Speroff, 8th Edition, 2011 4
  • 5. Definition 5 Recurrent miscarriage ↓ the loss of three or more consecutive pregnancies Speroff, 8th edition, 2011 : three or more pregnancy losses (not necessarily consecutive) 5 Green-top Guideline no 17, 2011
  • 6. Definition 6 ESHRE 2017 “A diagnosis of Recurrent Pregnancy Loss (RPL) could be considered after the loss of two or more pregnancies” ASRM 2012 “……2 or more failed clinical pregnancies” 6
  • 7. Definition 7 Pregnancy: confirmed at least by either serum or urine b-hCG includes non-visualized pregnancy losses : • biochemical pregnancy losses • resolved and treated pregnancies of unknown location • If identified as such, ectopic and molar pregnancies should be excluded from the definition 7 ASRM 2012, ESHRE 2017
  • 8. Definition 8 Primary RPL RPL without a previous ongoing pregnancy Secondary RPL RPL after one or more previous pregnancies 8
  • 9. 9
  • 10. 10 Miscarriage 15-25% 2 consecutive losses <5% 3 or more 1% ASRM 2012 10
  • 11. 11 11 ESHRE 2017 Sporadic 45% Subsequent 39%
  • 12. Incidence of chromosomal abnormalities 12 • 50-75 %First trimester • 15-30 %Second trimester • 3-5%Third trimester Speroff 8th edition, 2011 12
  • 13. Second Trimester 13 • Some abortuses recognized in the second trimester are actually missed abortions that were retained in utero after a first trimester demise • Chromosomal abnormalities - similar to those observed in liveborn infants: – trisomies 13, 18, and 21 – monosomy X – sex chromosome polysomies 13
  • 14. Incidence with maternal age 14 • 30 yrs →7–15% • 30 –34 yrs → 8–21% • 35–39 yrs → 17–28% • 40 yrs & older → 34–52% 14
  • 15. Increase Paternal Age 15 • Increased sperm DNA damage • Decreased fecundity • Adverse reproductive outcomes • exact mechanisms – not known Carrell et al. 2003 15
  • 16. Recurrence rate 16 SPEROFF 8TH EDITION, 2011 16
  • 17. The chance of subsequent live birth in untreated RPL patients with three, four, and five or more miscarriages has been found to be 42–86%, 41–72%, and 23– 51%,respectively Prognosis 17 17
  • 18. Tip of Iceberg 18 Speroff 8th edition, 2011 18
  • 19. Incidence : Underestimated 19 • Maternal cell contamination • Aneuploid cell less likely to grow in a culture • Newer techniques (FISH , CGH array) first trimester losses - upto 75% Speroff, 8th edition, 2011 19
  • 20. Causes of RPL 20 Speroff 8th edition, 2011 20
  • 21. Causes Of Rpl 21 ASRM 2012 2-5% 2-38% (avg-13%) 8-42% (avg 15%) 50-75% (avg 15%)
  • 22. GENETIC CAUSES OF RPL 22
  • 23. 1st report 23 (a triploidy in spontaneous abortion) It took several years before cytogenetic analysis of miscarriage became an option in laboratories ↓ the difficulties of culturing fetal tissue 23
  • 24. Genetic Abnormalities 24 • Numerical  Aneulpoidy  Polyploidy • Structural  Inversion  Insertion  Translocation • Point Mutation • Polygenic 24
  • 25. PARENTAL GENETIC ABNORMALITIES 25 25 Balanced translocations (reciprocal, Robertsonian) - most common 4-8 % sex chromosome mosaicism chromosome inversions other
  • 26. PARENTAL GENETIC ABNORMALITIES 26 • Carrier Couples – miscarriage rate was higher – live birth rate was lower – cumulative live birth rate was 64% • Ongoing pregnancies with unbalanced translocations are however very rare - less than 1% “More pregnancy losses in carriers of reciprocal translocations and inversions” Sugiura-Ogasawara et al., 2004, Franssen et al., 2006, Stephenson and Sierra, 2006 26
  • 27. PARENTAL GENETIC ABNORMALITIES 27 (Sugiura-Ogasawara et al., 2008) 63% 78.70% 0% 20% 40% 60% 80% 100% Carriers Normal live birth rate 27
  • 28. History 28 • Normal Child + Early Pregnancy Losses → 6–7% • Only Spontaneous Miscarriages Or 4-5% Combinations of malformed children, stillbirths, and abortions • evaluated in the same fashion Speroff 8th edition, 2011 28
  • 29. History 29 Family History of recurrent pregnancy loss, stillbirths, or birth defects  possibility of an occult chromosomal abnormality even after ONE SPONTANEOUS MISCARRIAGE 29
  • 30. 25% and 60% 30 structural Mosaicism Numerical 80-90% 5% 90% 5% Monosomy X Autosomal Trisomy Polyploidy Chromosomal Abnormalities Among Abortuses 30
  • 31. Incidence of genetic abnormality in abortus 31 54.10% 7.70% 2.60% 18.60% 1.50% 0.20% 0.10% 22.30% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 31 Simpson et al, 1987
  • 32. Frequency of Autosomal trisomies 32 Chrom. 1 0 Chrom. 2 1.11 Chrom. 3 0.25 Chrom. 4 0.64 Chrom. 5 0.04 Chrom. 6 0.14 Chrom. 7 0.89 Chrom. 8 0.79 Chrom. 9 0.72 Chrom. 10 0.36 Chrom. 11 0.04 Chrom. 12 0.18 Chrom. 13 1.07 Chrom. 14 0.82 Chrom. 15 1.68 Chrom. 16 7.27 Chrom. 17 0.18 Chrom. 18 0.15 Chrom. 19 0.01 Chrom. 20 0.61 Chrom. 21 2.11 Chrom. 22 2.26 Double trisomy 0.7 Mosaic trisomy 1.3 Other/ not specified 0.9 Simpson et al, 1987 32
  • 33. Ageing of gametes 33 Aneuploidy rates in aging female • Aneuploidy rates in aging male - 1–2% < 35 yrs 10% 40 yrs 30% 43 50% Speroff 8th edition,2011; CARP 2nd edition, 2015 33
  • 34. 34 34 Majority of chromosome abnormalities in spontaneous miscarriages –  De Novo  Random Errors - during gametogenesis and embryonic development
  • 35. Errors of Meiosis & Mitosis 35 35
  • 36. Undiagnosed genetic abnormalities 36 • Not be detected by standard cytogenetic techniques • Isolated gonadal or germline mosaicism (including a trisomic cell line) • Single Gene Defects 36
  • 37. Parental Gonadal Mosaicism 37 • Only germ cells are affected • Risk of transmission 37
  • 38. Normal Variants 38 • t(11;22)(q23;q11) - the most common reciprocal translocation in humans • Pericentric inversions - often have no clinical consequences, e.g. – chromosome 9, inv(9)(p11q13), (1–1.5% in the general population) Speroff 8th edition, 2011 38
  • 39. Genetic Evaluation 39
  • 40. Parental 40 4-8 % KARYOTYPE 40
  • 41. ESHRE 2017 41 • Parental karyotyping is not routinely recommended in couples with RPL. It could be carried out after individual assessment of risk Strong • In case of established carrier status, couples should be advised that the long-term prognosis of a live birth is good in carriers of a structural chromosome abnormality (LBR of 71% in 2 years). Strong • Assessing sperm DNA fragmentation in couples with RPL can be considered based on indirect evidence 41
  • 42. Karyotyping The Products Of Conception 42 Without karyotyping, women who repeatedly miscarry generally are assumed to be losing normal pregnancies; when, in fact, most are not 42
  • 43. Evaluation 43 Hogge et al. 2003, Bernardi et al. 2012, Foyouzi et al. 2012 43 Genetic evaluation of abortus Genetic evaluation of parents & Proceed Think about other causes & proceed Abnormal Normal
  • 44. Evaluation 44 • Decreased financial expenditure • Provide valuable information regarding the current miscarriage and subsequent reproductive potential • To give closure - easier to overcome the grief • Allows patients to make a more educated decision in pursuing future pregnancies 44
  • 45. Evaluation of abortus 45 Karyotype FISH aCGH NGS 45
  • 46. Cell Cycle 46 46
  • 47. Cell Division 47 47
  • 48. Karyotype 48 48 Collect the sample Culture the cells Stain them Visualise under microscope
  • 49. FISH (Fluorescent In Situ Hybridization) 49 49
  • 50. aCGH array – Comparative Genomic Hybridization 50 50
  • 51. Next Generation Sequencing 51 51 LIBRARY PREPARATION AMPLIFICATION SEQUENCING
  • 52. Karyolite 52 52
  • 53. Methods of evaluation 53 Conventional Karyotyping FISH aCGH NGS Method G-banding Fluorescently labelled probes WGA, Fluorescently labelled DNA probes WGA , tagging with specific bar code, mixing of samples, parallel sequencing detection threshold (Resolution) 5-10 million bases (Mb) 3-5 Mb 5-6Mb ~ 3 Mb Type of cells needed Active dividing cells Both dividing/nondividi ng cells Non dividing cells Approx. time 7-14 days 10-15 days (Interphase – 24 hr) 8 hr 14 hr 53
  • 54. Methods of evaluation 54 Detection capability Conventional Karyotyping FISH aCGH NGS No of chromosomes detected 24 5-14 (12) 24 3,000 probes 24 1,50,000 probes Haploidy and polyploidy + limited limited limited Segmental aneuploidies - ? + + familial balanced chromosome rearrangement s + - - Uniparental disomy - - - - 54
  • 55. Methods of evaluation 55 Conventional Karyotyping FISH aCGH NGS Deletion/ Duplication + + + Microdeletio ns (<5 Million Base Pair) - -/+ + + Mosaicism + (>2%) + (<20%) + inversion + ? + Single gene mutation - - - + False positive rate Moderate Low Very low 55
  • 56. Eshre 2017 56 • Genetic analysis of pregnancy tissue is not routinely recommended but it could be performed for explanatory purposes Conditional • For genetic analysis of the pregnancy tissue, array- CGH is recommended based on a reduced maternal contamination effect Strong 56
  • 57. Sampling 57 • Collect 30mg of abortus tissue in the tube containing sterile transport media • If sterile media is not available, use glucose saline and add two drops of crystalline geramycin or gentamycin • DO NOT USE FORMALIN FOR ANY GENETIC TESTING • Blood sample from the mother be submitted along with the POC sample - STR markers to determine maternal/fetal origin of the tissue 57
  • 58. 58
  • 59. Genetic Testing As Therapy in RPL 59
  • 60. Role Of Genetics 60 • Preimplantation Genetic Diagnosis • Preimplantation Genetic Screening 60
  • 61. ESHRE 2011 (PGD CONSORTIUM) 61 • AMA - 36 completed years • Recurrent Implantation Failure e.g. ≥3 embryo transfers with high-quality embryos or the transfer of ≥10 embryos in multiple transfers—exact numbers to be determined by each centre Implantation failure is defined as the absence of a gestational sac on ultrasound at 5 or more weeks postembryo transfer • Recurrent miscarriages 61
  • 62. PGD/PGS 62 ESHRE PGD/PGS Consortium 2011 “The current guidelines from this consortium do not give a Recommendation in favor or against PGS for RM couples” 62
  • 63. PGS In Women With Unexplained RPL 63 ESHRE PGD/PGS Consortium 2011 • No reported higher chances of live birth rate after PGS compared to natural conception • Relatively good pregnancy outcomes after natural conception in women with unexplained recurrent miscarriage • Requirement of invasive and expensive techniques (IVF/ICSI) • 63
  • 64. PGD/PGS 64 ASRM 2012 “………..the available evidence does not support the use of PGS as currently performed to improve live birth rates in patients with recurrent pregnancy loss, because RCTs are not available.” 64
  • 65. ESHRE 2017 65 PGD versus expectant management (Translocations Carrier) • Live Birth Rate PGD = 37.8% first natural pregnancy = 53.8% • Miscarriage rate – reduced • Cumulative live birth rate – almost same • Time to pregnancy - similar Ikuma et al., 2015 65
  • 66. SOGC 2015 66 • Preimplantation genetic screening using Fluorescence In Situ Hybridization technology on day-3 embryo biopsy is associated with decreased live birth rates and therefore should not be performed with in vitro fertilization. (I-E) • Preimplantation genetic screening using comprehensive chromosome screening technology on blastocyst biopsy, increases implantation rates and improves embryo selection in IVF cycles in patients with a good prognosis (I-B) 66
  • 67. PGD/PGS 67 limited evidence for preimplantation genetic testing in couples with RPL ↓ no clear benefit of treatment *overall quality of the evidence is very low 67
  • 68. Variations Of Unknown Significance (VOUS) 68 At present VOUS embryos are not replaced ↓ certain number of normal embryos may be discarded 68
  • 69. Gamete Donation 69 • ASRM 2012 • Remohi et al 1996 – Oocyte Donation 69
  • 70. Sperm FISH 70
  • 71. Genetics of Sperm 71 • Male partners of patients suffering from unexplained RPL are at high risk for sperm chromosomal abnormalities at least numerically • Specifically for sex chromosomes and also autosomes such as 1, 17, 8, 18, or 21 71
  • 72. Genetics of Sperm 72 Percentage of sperm DNA fragmentation is significantly higher in male partners of patients who had unexplained RPL Hwang K et al, 2010 72
  • 73. Genetics of Sperm 73 • Nuclear chromatin decondensation of spermatozoa & subsequent male pronucleus formation → essential for fertilization and normal embryonic development • Chromatin damage → the loss of fertilization potential and poor embryo quality, resulting in pregnancy loss • Idiopathic RPL - a higher incidence of aberration in sperm chromatin packaging 73
  • 74. Genetics of Sperm 74 • Paternal Genome – Embryonic Activation “spermatozoa with defective DNA can fertilize an oocyte and produce high-quality early-stage embryos, but then, as the extent of the DNA damage increases, the likelihood of a successful term pregnancy decreases” Hwang et al 2010 74
  • 75. Genetics of Sperm 75 • DNA integrity decreases during epididymal transition of spermatozoa • Testicular sperm from men with nonobstructive azoospermia (NOA) displays higher rates of aneuploidy in spermatozoa and blastomeres in IVF/ICSI-derived embryos (Gianaroli et al. 2005) • Testicular sperm from men with NOA had a higher rate of aneuploidy than epididymal sperm from men with obstructive azoospermia (OA) (Palermo et al.2002) 75
  • 76. Abnormal Sperm morphology & RPL 76 Kobayashi et al. 1991 • Increase in morphologically abnormal sperm was associated with delayed fertilization and cleavage rates and a greater risk for miscarriages Sbracia 1996, Bhattacharya 2010 • No Correlation 76
  • 77. Genetics Of Sperm 77 Normospermic men who are partners in couples with unexplained recurrent miscarriages and repeated IVF failure have a higher rate of sperm aneuploidy (Petit et al. 2005; Bernardini et al. 2004) 77
  • 78. Sperm FISH 78 To determine the proportion of aneuploidy present in sex chromosomes and autosomes in sperm 78
  • 79. Sperm FISH 79 79
  • 80. Conclusion 80 “Keep Your Eyes And Mind Open” 80
  • 81. Be Thankful For Your Existence 81
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