Prevalence of genetic abnormalities in cases of recurrent pregnancy losses and different diagnostics techniques to identify them.

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