Recurrent spontaneous abortion

재발성 자연 유산

1. Recurrent Spontaneous
Abortion (RSA)
- How to manage genetic cause?
관동대학교 의과대학 제일병원 산부인과
불임 생식내분비 분과
∙
양 광 문

2. Fates of embryo after transfer
Boomsma, C.M. et al., Hum Reprod 2009
RSA

3. Definition of RSA (I)
•Traditionally, ≥ 3 clinical pregnancy losses before 20
weeks from the last menstrual period
- occurs in about 1/300 pregnancies.
Novak 15
th
ed., WILCOX et al, 1988
•Risk of subsequent pregnancy loss
–24% after 2 clinical losses
–30% after 3 losses
–40~50% after 4 losses
Novak 15
th
ed., Regan
et al, 1989

4. Definition of RSA (II)
•In 2008, the American Society for
Reproductive Medicine (ASRM) defined
RPL by two or more failed pregnancies,
and pregnancy must be clinical
: documented by ultrasound or histopathologic
examination

5. Indications of clinical Indications of clinical
investigationinvestigation
•Clinical investigation may be initiated after
2 consecutive SA
especially,

• when fetal heart activity is identified
• when the women is older than 35 years old
• when the couple has had difficulty conceiving
-1% of pregnant women
Novak 15
th
ed., Alberman et al, 1988

6. Risk for subsequent pregnancy loss
Pregnancy loss risk Probability of live birth
24%
30%
40 -50%
76%
70%
50- 60%
3 previous losses
More than 4
previous losses
2 previous losses
Regan et al., 1989

7. Etiology of RSA
Genetic factor
2-5%
Anatomic factor
10-15%
Autoimmune
20%
Infections
0.5 -5%
Endocrine
factors
17-20%
Unexplained
Including
non-APA
thrombophilia
50%
Ford HB et al. Rev Obstet Gynecol 2009
Unexplained
Genetic
Translocation 60.3%
Anatomic
Synechia 64.3%
Ut. Septum 14.3%
Endocrine
Hyperthyroidism 71.4%
Infection
Ureaplasma 89.5%
N = 881
(2005. 1.1 - 2009. 12. 31)
50% of RSA classified as unexplained
Allo-immune etiology?

8. Diagnosis & management protocol of RSA
History taking
Ultrasonographic scanning / pelvic exam
Normal
Genetic evaluation
• Karyotyping of abortus
• Parental karyotyping
Immunologic evaluation
Routine lab
Genital infectionGenital infection
Cervical cultureCervical culture
ChlamydiaChlamydia
U. UrealyticumU. Urealyticum
MycoplasmaMycoplasma
HSG, MRI LH/FSH, E2, PRL, TSH, T3/ freeT4
Uterine anomaly (Septated uterus)
Hysteroscopy
or
Laparoscopy
Hormone therapy
Allo-immune study
NK number (CD
16,56)
NK cytolytic activity
Auto-immune study
ACA (IgG/IgM)
LAC
Antithyroid Ab
PGD
AntibioticsAntibiotics
Surgery
Prednisolone (PDS)
Low molecular weight heparin (LMWH)
IVIGIVIG
Uterine anomaly? Ovulatory dysfunction?

9. Genetic evaluation of RSA
•Fetal karyotyping


Chorionic villus sampling
•Parental karyotyping
: Parental lymphocytes

10. Trisomy
16
Trisomy
22

11. Karyotyping of the abortus in RSA
•41% of miscarriages are aneuploid in recurrent
miscarriage.
Ogasawara. et al., Fertil Steril 2000

12. •The prognosis is better after an aneuploid
abortion than a euploidy miscarriage.
•15% of patients will have repeat aneuploidy.
: can be offered pre-implantation genetic screening
(PGS).
Karyotyping of the abortus
- Howard et al., IMAJ 2008

13. •The value of parental karyotyping is limited in
recurrent miscarriage.
•Seeks balanced translocations and inversions rather
than the more common numerical aberrations such as
trisomy.
•Parental karyotypic aberrations have been found in
3–10% of couples with recurrent miscarriage.
: PGD is indicated
Parental karyotyping
- Howard et al., IMAJ 2008

14. Normal
Other RSA work up
Parental Karyotyping
Abnormal
PGD, PGS
Normal
Abnormal
Numerical abnormalities
• Trisomy
• Monosomy
• Polyploidy
1
st
numerical
abnormalities
Repeated numerical
abnormalities
Structural abnormalities
• Deletion
• Addition
• Inversion
• Translocation
• High dose folic acid
: 5mg/day
• Timed intercourse
Treatment options by the results of
karyotyping in RSA

15. 1. Supplement of high dose folic acid
2. Timed intercourse
3. Prenatal Genetic Screening (PGS)
Prevention of repeated
aneuploidy pregnancy

16. Folic acid supplement
Activated folic acid

17. Folic acid supplementation in
RSA patients
•Abnormal folate and methyl metabolism can lead to DNA
hypo-methylation, instability, abnormal segregation and
aneuploidy.
- Fenech M. Mutat Res 2001, Wang X, Mutat
Res. 2004
•Genomic instability is minimized when the plasma folate level
exceeds about 34 nmol/l and the Hcy level is less than 7.5
μmol/l.
•These levels can only be achieved when folic acid intake is
above 5mg per day.
- Fenech M. Mutat Res 2001

18. MTHFR gene mutation and folic
acid supplement
•MTHFR (methylenetetrahydrofoloate reductase)
gene
C677T
A1298C
•MTHFR gene mutation
heterozygous MTHFR carriers
- activate folate at 60-70%
homozygous carriers
- activate folate at 10%
•Activated folic acid or high dose folic acid supplement
are recommended

19. Folic acid supplement

20. Folic acid supplement
Activated folic acid

21. Timed intercourse for prevention of
repeated aneuploidy
•Prevention of delayed fertilization
 Secondary oocyte remains in MII metaphase in the
fallopian tube until it is fertilized.
 Ageing or over-ripeness of these cells could lead to a
higher incidence of spindle defects and so increase the chance
of non-disjunction.
Chromosomal errors increase with delayed fertilization,
although it is difficult to distinguish this from the maternal
age effect.
Ishikawa H et al., Hum Reprod 1995

22. OPU
수
정
IVF-PGD, PGS
PCR
FISH
배아
배양
26일 16
주
17141272 일생리
과배란 유
도
초음
파
배아이식
b-hCG
양수
천자
PCR
FISH

23. Embryo 7
Probe: 13, 16, 18, 21, 22
-> Monosomy 18, 21, 22
Probe: X, Y, 21
-> Trisomy X, Monosomy 21
A.Handyside, RBM Online 2011;23:686-91
Preimplantation Genetic Screening (PGS)
By FISH

24. The first techniques used for PGS were polar body biopsy or
cleavage-stage blastomere biopsy followed by fluorescence in
situ hybridization (FISH) analysis -> first-generation PGS
Initial studies with first-generation PGS suggested that
implantation rates increased and loss rates decreased.
 However, other studies, including several randomized
controlled trials (RCTs), showed no benefit or, worse, a
negative impact on implantation, pregnancy, or loss rates.
Preimplantation Genetic Screening (PGS)
By FISH

25. PGS in RSA
Aneuploidy Screening
Patients 87
Cycles 148
Age 36.9 ± 4.2 (26 ~ 46)
Biopsied embryos 1,413 (9.5 ± 4.0)
Diagnosed embryos 1,316 (8.9 ± 3.8)
(93.5%)
Normal or balanced 262 (2.4 ± 1.9)
(19.9%)
ET Cycles 129
(87.2%)
No. of transferred embryos 314
(2.1 ± 1.4)
Delivery rate/ET 15.5% (23.0% per patient)
Abortion rate 10.3%

26. Comparative genomic hybridization (CGH)
Normal DNANormal DNATest DNATest DNA
MonosomyMonosomyTrisomyTrisomyNormalNormal
Array CGH

27. 27
Euploid embryo
46, XY
Aneuploid embryo
47, XY, +7
Euploid embryo
46, XX
Aneuploid embryo
45, XY, -16
arr CGH 22q11.1-q.ter x 1
arr 20q13.32-q.ter x 1

28. An ideal technique would allow for the simultaneous analysis
of all 24 chromosomes (autosomes 1–22, X, and Y) and less
prone to technical issues that could lead to errors and
misdiagnosis than earlier FISH methods.
The first comprehensive analysis technique appearing after
FISH was comparative genomic hybridization (CGH), but it
was challenging to put into clinical practice because it needed
at least three full days for the analysis to be completed.
Finally, the advent of vitrification, which permitted the safe
cryopreservation of biopsied embryos, allowed all the
components for second-generation PGS to be assembled:
complete chromosome screening (via CGH); less damaging
embryo biopsy (at the blastocyst stage); and enough time to
carry out the test (afforded by vitrification).

29. CGH was later displaced by more automated techniques,
such as aCGH, single-nucleotide polymorphism (SNP) arrays,
and quantitative fluorescent polymerase chain reaction
(qPCR).
Of these techniques, aCGH and qPCR have been shown in
RCTs to improve pregnancy rates.

31. 575 couples: 169 (SNP-PGD; 2011.10.-2012.8.) + 406 (2005.1.-2011.10.)

33. Early Diagnosis for Early Cure!
KFDA Certification
‘BAC Chip H1440’ was approved from the Korea Food and Drug
Administration(KFDA) in March 2006

34. CC: centromere
Chromosome 21

35. Parental chromosomal
abnormality
Balanced translocation – most common
Monosomy – X chromosome
Inversions - not inv (9)
Insertions
Mosaicism
Single gene defects
Genetic factor
2-5%
Anatomic factor
10-15%
Autoimmune
20%
Infections
0.5 -5%
Endocrine
factors
17-20%
Unexplained
Including
non-APA
thrombophilia
50%
Genetic cause of RSA
Neither family history nor a history of prior term births is sufficient to rule
out a potential parental chromosomal abnormality

36. 비정상적 감수 분열 (Adjacent 1)
비정상
12
1
1
12
비정상
1 1
12 12
유산
기형아
Reciprocal translocation
50%

37. 정상적 감수 분열 정상적 감수 분열 (Alternate)(Alternate)
균형 전좌
정상
11
12 12
12
1
112
정상 임신
25% 25%

38. Robertsonian translocation
(13, 14, 15, 21, 22 chromosome)
14 21
21
1421
21
14
21
14
21
14
정상 (1/6)
로벗슨
전좌
(1/6)
비정상 비정상
14
21
14
비정상
(2/3)
비정상
21

39.   Female carrier Male carrier Total
Patients 35 30 65
Cycles 77 56 133
Mean female age 33.0 ± 3.9 30.8 ± 2.7 32.1 ± 3.6
Retrieved oocytes 1,298 1,038 2,336
Injected oocytes 1,132 877 2,009
Fertilized oocytes (%) 871 (77.0) 704 (80.3) 1,575 (78.4)
Frozen zygotes 98 116 214
Thawed zygotes/embryos 133 120 253
Biopsyed embryos 898 700 1598
Diagnosed embryos 843 665 1508
Transferable embryos (%) 158 (18.7) 127 (19.1) 282 (18.7)
Unbalanced embryos (%) 685 (81.3) 538 (80.9) 1,226 (81.3)
ET cycles 66 50 116
Transferred embryos 144 (2.1 ± 1.1) 105 (1.8 ± 1.2) 249 (1.9 ± 1.1)
Frozen embryos 7 7 14
Positive β-hCG (%) 25 (37.9) 25 (50.0) 50 (43.1)
Biochemical pregnancy (%) 7 (10.6) 10 (20.0) 17 (14.6)
On-going preg. or delivery (%) 11 (18.2) 14 (28.0) 25 (21.6)
Miscarriage (%) 7 (10.6) 1 (2.0) 8 (4.3)
Clinical outcome of PGD for reciprocal translocation

40.   Female carrier Male carrier Total
Patients 46 16 62
Cycles 94 26 120
Mean female age 31.2 ± 2.9 32.8 ± 5.4 31.5 ± 2.6
Retrieved oocytes 1,067 400 2,007
Injected oocytes 1,310 346 1,656
Fertilized oocytes (%) 1,035 (79.0) 254 (73.4) 1,289 (77.8)
Frozen zygotes 98 21 119
Thawed zygotes/embryos 161 45 206
Biopsied embryos 1,048 263 1,311
Diagnosed embryos 995 252 1,247
Transferrable embryos (%) 246 (24.7)
a
85 (33.7)
a
331 (26.5)
Unbalanced embryos (%) 749
b
167
b
916
ET cycles 89 24 113
Transferred embryos 221 (2.4 ± 1.2) 63 (2.4 ± 1.3) 284 (2.4 ± 1.2)
Positive β-hCG (%)
c
37 (41.1) 12 (50.0) 49 (43.4)
Biochemical pregnancies (%)
a
10 (11.2) 2 (8.3) 12 (10.6)
Deliveries (%)
a
14 (15.7) 8 (33.3) 22 (19.5)
Miscarriages (%)
a
8 (9.0) 0 8 (7.1)
Follow-up loss (%)
a
3 (3.3) 2 (8.3) 5 (4.4)
Termination of pregnancy 1 0 1
Stillbirth 1 0 1
Overall pregnancy outcomes of PGD for Robertsonian translocation carriers
a
P<0.001;
b
P<0.005
c
Percent per embryo transfer

41. Summary
•Benefits of high dose folic acid
•Efficacy of timed intercourse
•MTHFR gene mutation needs activated or high
dose folic acid
•CGH in patients with repeated aneuploidy
•PGD in patients with inheritable chromosome
abnormalities

42. Thank you for your attention!