Recurrent Spontaneous Abortion (RSA)

- How to manage genetic cause?

관동대학교 의과대학 제일병원 산부인과불임 생식내분비 분과 ∙

양 광 문

Fates of embryo after transfer

Boomsma, C.M. et al., Hum Reprod 2009

RSA

Definition of RSA (I)• Traditionally, ≥ 3 clinical pregnancy losses

before 20 weeks from the last menstrual period

- occurs in about 1/300 pregnancies. Novak 15th 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 15th ed., Regan et al, 1989

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

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 15th ed., Alberman et al, 1988

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

Etiology of RSA

Genetic factor2-5%

Anatomic factor10-15%

Autoimmune 20%

Infections0.5 -5%

Endocrine factors17-20%

UnexplainedIncluding non-APA

thrombophilia50%

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?

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 orLaparoscopy

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?

Genetic evaluation of RSA

• Fetal karyotyping

Chorionic villus sampling

• Parental karyotyping : Parental lymphocytes

Trisomy 16

Trisomy 22

Karyotyping of the abortus in RSA

• 41% of miscarriages are aneuploid in recurrent miscarriage.

Ogasawara. et al., Fertil Steril 2000

• 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

• 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

Normal

Other RSA work up

Parental Karyotyping

Abnormal

PGD, PGS

Normal

Abnormal

Numerical abnormalities• Trisomy• Monosomy• Polyploidy

1st 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

1. Supplement of high dose folic acid2. Timed intercourse3. Prenatal Genetic Screening (PGS)

Prevention of repeated aneuploidy pregnancy

Folic acid supplement

Activated folic acid

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

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

Folic acid supplement

Folic acid supplement

Activated folic acid

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

OPU수정

IVF-PGD, PGS

PCR

FISH

배아 배양

26 일 16주

17141272 일생리

과배란 유도

초음파 배아이식

-hCG양수천자

PCR FISH

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

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

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%

Comparative genomic hybridization (CGH)

Normal DNANormal DNATest DNATest DNA

MonosomyMonosomyTrisomyTrisomyNormalNormal

Array CGH

27

Euploid embryo46, XY Aneuploid embryo

47, XY, +7

Euploid embryo46, XX

Aneuploid embryo45, XY, -16

arr CGH 22q11.1-q.ter x 1

arr 20q13.32-q.ter x 1

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).

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.

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

Early Diagnosis for Early Cure!

KFDA Certification

‘BAC Chip H1440’ was approved from the Korea Food and Drug Administration(KFDA) in March 2006

CC: centromere

Chromosome 21

Parental chromosomal abnormality Balanced translocation – most common Monosomy – X chromosome Inversions - not inv (9) Insertions Mosaicism Single gene defects

Genetic factor2-5%

Anatomic factor10-15%

Autoimmune 20%

Infections0.5 -5%

Endocrine factors17-20%

UnexplainedIncluding non-APA

thrombophilia50%

Genetic cause of RSA

Neither family history nor a history of prior term births is sufficient to rule

out a potential parental chromosomal abnormality

비정상적 감수 분열 (Adjacent 1)

비정상12

1

1

12

비정상1 1

12 12

유산기형아

Reciprocal translocation

50%

정상적 감수 분열 정상적 감수 분열 (Alternate)(Alternate)

균형 전좌정상1 1

12 12

121

1 12

정상 임신

25% 25%

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

  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

  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<0.005c Percent per embryo transfer

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

Thank you for your attention!