Recurrent Pregnancy Loss and Its Relation to Combined Parental
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Transcript Recurrent Pregnancy Loss and Its Relation to Combined Parental
RECURRENT PREGNANCY LOSS
(RPL) AND ITS RELATION TO
COMBINED PARENTAL
THROMBOPHILIC GENE MUTATIONS
DOÇ. DR. A. GONCA İMİR YENİCESU
CUMHURİYET ÜNİVERSİTESİ
KADIN HASTALIKLARI VE DOĞUM AD.
2.4.2016
G. Yenicesu
UTD 2011
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RPL
RPL is classically defined as the occurrence of
three or more consecutive losses of clinically
recognized pregnancies prior to the 20th
week of gestation (ectopic and molar
pregnancies are not included).
The ASRM defines RPL as two or more failed
pregnancies (by ultrasound or
histopathological examination) and suggests
some assessment after each loss with a
thorough evaluation after three or more
losses.
Practice Committee of the American Society for Reproductive Medicine.
Definitions of infertility and recurrent pregnancy loss. Fertil Steril 2008; 89:1603.
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Approximately 15 % of pregnant women
experience sporadic loss of a clinically
recognized pregnancy.
Just 2 % of pregnant women experience
two consecutive pregnancy losses and
only 0.4 to 1% have three consecutive
pregnancy losses.
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Recurrent pregnancy loss (RPL) is a
significant clinical problem with many
etiologies.
In recent years there are many studies
that examined the incidence of specific
thrombophilic gene mutations in couples
with unexplained RPL.
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Thrombophilic mutations such as; FV L,
prothrombin G20210A, MTHFR lead to
enhanced blood coagulation and are
involved in folic acid metabolism which
influences DNA methylation.
An association with RPL has also been
reported for the Val34Leu polymorphism of
the FXIII gene by leading to fibrin
degradation.
Wang XP, 2004; 39:238–241, Wolf CE 2003, Coulam CB 2006, Muszbek L 2000.
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Apo E gene encodes three alternative
alleles –E2, E3 and E4 that play a crucial
role in cholesterol - triglycerides
metabolism(27).
Homozygous individuals harbouring the
E4 allele have a higher total cholesterol
level, hence cardiovascular risk (1,28,29).
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We have previously reported the high
frequency of ApoE4 gene polymorphisms
as a risk factor for RPL.
Goodman et al also reported the high
frequency of Apo E4 genotype in women
suffering from RPL.
Yenicesu GI, et al; A prospective case-control study analyzes 12 thrombophilic gene mutations in
Turkish couples with recurrent pregnancy loss. A J Reprod Immu 2010; 63(2):126-136.
Goodman C. Am J Reprod Immunol 2009; 61:34–38.
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In this study, we compare the prevalence
of 12 thrombophilic gene mutations in a
series of patients with two or more
consecutive, first-trimester losses and
fertile couples with no history of
miscarriages.
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The study was performed in gynecology
clinics and genetic departments at
Cumhuriyet University Hospital and
Canakkale Onsekiz Mart University Hospital.
The 543 women with RPL ( two or more
consecutive RPL at 5–12 weeks of gestation)
327 RPL male partners
106 couples with no history of RPL and at
least one normal pregnancy
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Chromosome analyses were performed in
all RPL and control couples.
Women with RPL were examined by US
or HSG , for immunologic risk factors
including APA, ANA, antithyroid
antibodies, and LA.
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Exclusion criteria:
anatomic abnormalities
endocrinologic dysfunction
autoimmune disease
urogenital infections
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Trombophilic genes
1. factor V G1691A (FV Leiden)
2. factor V H1299R
3. factor II prothrombin G20210A
4. factor XIII V34L
5. β-fibrinogen−455G>A
6. plasminogen activator inhibitor-1 (PAI-1)
7. GPIIIa L33P (HPA-1 a/b L33P)
8. MTHRF C677T
9. MTHFR A1298C
10. ACE I/D
11. Apo B R3500Q
12. Apo E (E2, E3, E4)
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Total genomic DNA was extracted by the
Magna Pure Compact (Roche) (Invitek).
Twelve thrombophilic genes were
simultaneously amplified in a biotinlabelled single multiplex amplification
reaction which is based on the reversehybridization principle and by Real Time
PCR, LightCycler 2.0 methods (Roche).
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The frequencies of homozygous and
heterozygous thrombophilic gene
mutations,
the frequencies of allelic mutations in
female and male participants
a heterozygous mutation was considered
as one gene mutation
a homozygous mutation was considered as
two gene mutations.
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We have previously reported that
3/12 thrombophilic mutations (PAI-1 4G⁄
5G, FVL, and homozygous MTHFR C667T,
correlated significantly with RPL when
compared to fertile controls.
Yenicesu GI, et al; A prospective case-control study analyzes 12 thrombophilic gene mutations in
Turkish couples with recurrent pregnancy loss. A J Reprod Immu 2010; 63(2):126-136.
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Tablo 1. Demographic data
Group
Characteristics
Age(year), Median S. E.
(range)
Control (couple)
n:106
RPL
n:870
Female
n:543
Male
n:327
Female
n:53
Male
n:53
27.8 2.1(17-44)
29.1 3.2(21-36)
28.92.2
32.1 1.1
3.4 (2-10)
0
0
0
0
0
3.2(1-8)
0
No of RPL , Median (range)
No of live birth, Median (range)
P> 0.05; RPL, recurrent pregnancy loss
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SONUÇLAR
The frequencies of allelic mutations in
women for FVL, FVR2, MTHFR C677T,
MTHFR A1298C, PAI 1 5G/4G, ACE I/D and
APOE in the RPL women (20.02%, 13.3%,
57.4%, 63.0%, 83.2%, 86.9% and 35.0%)
respectively were significantly higher than in
the fertile control women respectively.
The rest of thrombophilic gene mutations
were the same as control women.
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FVL-FVR2
RPL
(n/%)
Gene/Genotype
Control
(n/%)
F
n:543
M
n:327
T
n:870
F
n:106
M
n: 106
T
n:212
Wild
433(79.8)
261(80.1)
694(79.8)
104(98.1)
103(97.1)
207(98.0)
Heterozygous
Mutated
109(20.0)
63(19.2)
172(19.7)
2 (1.9)
3(2.9)
5(2.0)
Homozygous
Mutated
1(0.002)
3(0.009)
4(0.005)
0 (0)
0 (0)
0 (0)
470(86.7)
269(82.2)
739(85.0)
103(97.2)
106(100)
209(98.6)
Heterozygous
Mutated
73(13.3)
58(17.8)
131(15.0)
3 (2.8)
0 (0)
3(1.4)
Homozygous
Mutated
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
FVL
FVR2
Wild
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FVL-FVR2
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MTHFR
RPL
(n/%)
Mutation/
Genotype
Control
(n/%)
Allele Frequency
(RPL/C)
F
n:543
M
n:327
T
n:870
F
n:106
M
n:106
T
n:212
C
T
C/C
231(42.6)
145(44.4)
376(43.3)
76(71.7)
79(74.5)
155(73.1)
0.64/0.87
0.36/0.13
C/T
239(44.0)
130(39.7)
369(42.4)
30(28.3)
27(25.5)
57(26.9)
T/T
73(13.4)
52(15.9)
125(14.3)
0(0)
0(0)
0(0)
C677T
P <0.0001
A1298C
A/A
201(37.0)
144(44.0)
345(39.7)
71(67.0)
69(65.0)
140(66.0)
A/C
257(47.4)
145(44.4)
402(46.2)
35(33.0)
37(35.0)
72(34.0)
C/C
85 (15.6)
38(11.6)
123(14.1)
0(0)
0(0)
0(0)
A
C
0.63/0.83
0.37/0.17
P <0.0001
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MTHFR
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The frequencies of allelic mutations in
men for FVL, FVR2, MTHFR C677T,
MTHFR A1298C, PAI 1 5G/4G, ACE I/D
and APOE in the RPL men (19.2%, 17.8%,
55.6%, 56.0%, 89.3%, 81.3% and 36.7%,
respectively) were statistically higher than
in the fertile control males.
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PAI
Allele Frequency
(RPL/C)
RPL (n/%)
Control (n/%)
F
M
T
F
Genotyp
e
n:543
n:327
n:870
n:106
5G/5G
91(16.8)
35(10.7)
126(14.5)
34(32.0)
5G/4G
331(60.9
)
207(63.4
)
538(61.9)
4G/4G
121(22.3
)
85 (25.9)
206(23.6)
M
n:106
T
n:212
5G
4G
38(35.9)
72(34.0)
0.45/0.62
0.55/0.38
62(58.5)
59(55.7)
121(57.0)
10(9.5)
9(8.4)
19(9.0)
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PAI
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APOE E3/E2/E4
RPL (n/%)
Genotype
F
n:543
M
n:327
Control (n/%)
T
n:870
F
n:106
M
n:106
Allele Frequency (RPL/C)
T
n:212
E3/E3
353(65.0) 207(63.3) 560(64.4) 83(78.3) 84(79.2) 167(78.7)
E3/E2
116(21.4)
65(19.9)
181(20.9)
E3/E4
63(11.6)
49(15.0)
112(12.8) 15(14.2)
E2/E4
11(2.0)
6(1.8)
17(1.9)
8(7.5)
0(0)
15(14.2)
23(10.9)
7(6.6)
22(10.4)
0(0)
0(0)
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E3
E2
E4
0.81/0.89 0.11/0.06 0.08/0.05
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APOE E3/E2/E4
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The total frequencies of allelic mutations in RPL
subjects were due to FVL, 19.7% (176/870), FVR2, 15.0%
(131/870), MTHFR C677T, 56.7% (489/870), MTHFR
A1298C, 60.3% (525/870), PAI 1 4G/5G, 85.5%
(744/870), ACE I/D 84.8 (738/870), and APOE 35.6%
(310/870) respectively.
The total frequencies of allelic mutations in fertile
couple subjects were due to FVL, 2.0%(5/212), FVR2,
1.4%(3/212), MTHFR C677T, 26.9%(57/212), MTHFR
A1298C, 34.0%(72/212), PAI 1 4G/5G, 66.0%(140/212),
ACE I/D 68.3(145/212), and APOE 21.3% (45/212)
respectively.
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To further define the relationship
between some specific target genes with
RPL, we also evaluated the allelic
frequencies of 4G for PAI-1, D for ACE,
C677T and A1298C for MTHFR and E4
for Apo E in two different populations of
women experiencing RPL.
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The frequency of E3/E2, E3/E4 and E2/E4
profiles for Apo E were 20.9% ,12.8% and
1.9% respectively in the RPL couples,
and 10.9 %, 10.4 % and 0.0 % in fertile
couples.
while ApoE3 allele frequency was 0.81 in
RPL and 0.89 in fertile couples
the ApoE2 frequency was 0.11 for RPL and
0.06 for fertile couples.
ApoE4 allele frequency was detected as 0.08
for RPL and 0.05 for fertile couples.
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The frequencies of heterozygous
mutations for PAI-1, MTHFR C677T,
MTHFR A1298C, ACE I/D and Apo E
were significantly higher in RPL couples
when compared to fertile control couples.
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The frequency of homozygous mutation
for PAI-1 was significantly higher in RPL
men compared with control men.
The frequency of homozygous mutation
for MTHFR C677T was 15.9% in the RPL
men, but no homozygous mutation was
detected in the control men.
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The presented preliminary results
showed that ApoE2 allele profile may
contribute to the thrombophilic risk
factors for RPL.
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Increased T allele frequency of C677T
polymorphism in MTHFR gene was detected
in RPL patients (0.36) when compared to
fertile couples (0.13).
Also present results showed increased C
allele frequency of A1298C mutation in RPL
patients (0.37) when compared to the fertile
couples (0.17).
Difference in both common mutated alleles
in the MTHFR gene were statistically
significant.
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For the ACE I/D gene, the frequency of
heterozygous and homozygous mutations in
RPL couples were significantly higher than
the fertile couples.
D allele frequency for ACE was detected as
0.62 for RPL and 0.44 for fertile
couples(P<0.0001).
Current results also showed increased 4G
allele frequency for PAI in RPL (0.55) when
compared to healthy controls (0.38).
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CONCLUSION
Briefly, the homozygosity of 4G in PAI-1
and MTHFR C677T genes in women with
recurrent pregnancy loss, and
heterozygosity of FV Leiden, FVR2, ACE
and Apo E2 and ApoE4 genes in both
parents play crucial roles in RPL and
should be considered as risk factors in
RPL.
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CONCLUSION
Current results showed the maternal
mutated thrombophilic genes’ profiles
mainly have a potential risk for RPL but
paternal mutation profiles in specific
genes may also have a combined effect on
RPL phenomenon.
Study of mutations in a wide range of
thrombophilic genes associated with the
disease might be clinically useful as a
marker to assess couple’s risk for RPL.
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