Human Reproduction, Vol.26, No.5 pp.

1252 1258, 2011

Advanced Access publication on February 20, 2011 doi:10.1093/humrep/der017

ORIGINAL ARTICLE Reproductive genetics

The p53-HDM2 gene gene

polymorphism interaction is associated
with the development of missed
abortion
Yan Fang 1, Beihua Kong 1,*, Qifeng Yang 2, Daoxin Ma 3, and Xun Qu 4
1

Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, 107 Wenhua Xilu, Jinan, 250012 Shandong, China
Department of General Surgery, Qilu Hospital, Shandong University, 107 Wenhua Xilu, Jinan, 250012 Shandong, China 3Department of
Hematology, Qilu Hospital, Shandong University, 107 Wenhua Xilu, Jinan, 250012 Shandong, China 4Department of Basic Medicine,
Qilu Hospital, Shandong University, 107 Wenhua Xilu, Jinan, 250012 Shandong, China
2

Submitted on September 3, 2010; resubmitted on December 22, 2010; accepted on January 12, 2011

background: The interaction between p53 and human double minute 2 (HDM2) plays an important role in apoptosis; therefore, functional polymorphisms in these genes might have adverse effects in early pregnancy. In this study, we investigated whether p53 codon 72 and
HDM2 promoter (SNP309) polymorphisms were associated with the development of missed abortion.
methods: Women with missed abortions (n 60) and healthy controls (n 64) were included in the study. Genotyping of the p53
codon 72 and HDM2 SNP309 (T . G) polymorphisms was performed by PCR with sequence-specic primers and PCR-restriction fragment
length polymorphism analysis, respectively, using villous samples. The mRNA and protein levels for p53 and HDM2 were measured by realtime PCR and semi-quantitative immunohistochemistry, respectively.
results: For the p53 codon 72 polymorphism, no difference in genotype or allele frequencies was observed in women with missed abortion versus controls. However, for the HDM2 SNP309 (T . G) polymorphism, G/G genotype was associated with a higher risk of missed
abortion compared with the T/T + T/G genotypes (P 0.043). Women carrying the HDM2 G/G genotype or p53 Pro/Pro genotype had
higher HDM2 mRNA (P 0.04 and P 0.013, respectively) and protein (P 0.001 and P 0.037, respectively) levels than women
with other HDM2 SNP309 and p53 codon 72 genotypes.
conclusions: The genotypes HDM2 SNP309 G/G and p53 codon 72 Pro/Pro can induce high levels of HDM2, which may be associated with missed abortion.
Key words: missed abortion / p53 / human double minute 2 / polymorphism / pregnancy

Introduction
Missed abortion refers to a pregnancy in which there is a fetal
demise without outside intervention but the uterine activity is
absent to expel the products of conception before 20 weeks of gestation (Griebel et al., 2005). Appropriate apoptosis has been shown
to be critically important for the successful development of normal
pregnancy (Chatzaki et al., 2001; Jerzak et al., 2002; Savion et al.,
2002; Choi et al., 2003). Spontaneous pregnancy disorders are
associated with the excessive apoptosis of trophoblasts (Halperin
et al., 2000).
p53, as an important tumor suppressor gene, is called the gatekeeper
of cellular genome. p53 expression has been demonstrated in rst trimester placenta (Quenby et al., 1998). When a cell is exposed to DNA

damage, p53 can induce cell cycle arrest in G1 phase (DNA presynthetic
phase) and apoptosis. Increased human double minute 2 (HDM2;
human ortholog of murine double minute 2) levels were shown to
lead to attenuation of the p53 DNA damage response, and HDM2 is
a key negative regulator of p53 (Zauberman et al., 1995; Ries et al.,
2000). Enhanced p53 expression and activity subsequently leads to
induction of HDM2, which may act as an oncogene (Fakharzadeh
et at., 1991; Dubs-Poterszman et al., 1995; Jones et al., 1998) or to
inhibit growth (Brown et al., 1998). Specically, enhanced HDM2
levels have been shown to cause p53 degradation in surviving cells
and, consequently, attenuation of the p53-mediated DNA damage
response (Zauberman et al., 1995; Ries et al., 2000), thereby forming
a negative feedback loop (Michael et al., 2003).

& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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*Correspondence address. Tel: +86-531-82169008; Fax: +86-531-86927544; E-mail: kongbeihua@yahoo.com.cn

1253

p53/HDM2 gene polymorphisms and risk of missed abortion

Materials and Methods

Diagnosis
By ultrasound examination, the rst-trimester missed abortion was dened
as an intact gestational sac lacking any fetal cardiac activity [6 weeks after
last menstrual period (LMP)], intrauterine gestational sac with the largest
diameter exceeding 10 mm but devoid of yolk sac or an empty gestational
sac with a conrmed gestational age of no ,6 weeks (Griebel et al., 2005).

Patients
This prospective observational study involved 60 pregnant women diagnosed with rst-trimester missed abortion. All the pregnancies terminated
at the rst trimester ,10 weeks from the LMP. Prior to inclusion in the
study, all subjects underwent a standard diagnostic work-up to rule out
any veriable cause of missed abortion. The women were examined
using ultrasonography for uterine abnormalities, and blood was drawn
for testing for chromosomal abnormalities, immunologic factors (such as
positive anticardiolipin antibody and positive antinuclear antibody) and
infections, with these analyses resulting in an unexplained etiology.
Among the 60 women who suffered from missed abortion, 26 had at
least two prior miscarriages, 24 had one prior miscarriage and 10 experienced miscarriage for the rst time. None of these women had had a successful pregnancy. The control group consisted of 64 women in early
pregnancy with a healthy, viable intrauterine fetus and no prior

miscarriage. Fetal cardiac activity and gestational age were conrmed by

ultrasound. Written informed consent was obtained from all participating
subjects. The study design was approved by the Ethical Committee of
Shandong University.

Specimens
Villous samples (n 60, one per woman) from the missed-abortion group
were collected by curettage or manual vacuum aspiration. Villous samples
(n 64, one per woman) from the control group were obtained by
vacuum aspiration from women undergoing elective abortion at 7 10
weeks of gestation for social reasons. Each villous sample was divided
into two parts: one part was stored at 280oC before genomic DNA
and RNA isolation, the other part (only 30 from missed-abortion group
and 31 from control group, randomly selected) was stored in 4% formaldehyde at room temperature overnight for immunohistochemistry
analysis.

DNA preparation
DNA was extracted using a DNA isolation kit (Tiangen, Beijing, Peoples
Republic of China). The DNA content and purity of each sample were
analyzed by ultraviolet spectrophotometry (the E260/280 ratio ranging
between 1.6 and 1.8), and a 10 ng DNA aliquot of each sample was
used for PCR amplication. DNA samples were routinely stored at
2208C.

Genotyping of p53 codon 72 and HDM2

SNP309
PCR analysis of p53 codon 72 polymorphism
Analysis of p53 genotype at codon 72 was performed as described
(Pietrowski et al., 2005). Sequence-specic primers (SSPs) for the Pro
allele are F: 5 -GCC AGA GGC TGC TCC CCC-3 ; R: 5 -CGT GCA
AGT CAC AGA CTT-3 . Primers of Arg allele are F: 5 -TCC CCC TTG
CCG TCC CAA-3 ; R: 5 -CTG GTG CAG GGG CCA CGC-3 . In each
25-ml reaction, 10 ng genomic DNA was mixed with 1.25 U Taq Platinum
Polymerase (Tiangen), 250 mmol/l each dNTP, 25 mmol/l TrisHCl (pH
8.7), 10 mmol/l KCl, 2 mmol/l MgCl2 (Tiangen) and 10 mmol/l of each
primer (previously described). PCR conditions for the Pro allele were as
follows: 948C for 5 min, 5 cycles of 948C for 1 min, 618C for 50 s,
728C for 50 s and 35 cycles of 948C for 1 min, 528C for 50 s, 728C for
50 s and a nal extension of 728C for 7 min. PCR conditions for the Arg
allele were as follows: 948C for 5 min, 35 cycles of 948C for 1 min,
648C for 45 s, 728C for 50 s and a nal extension of 728C for 10 min.
PCR products (10 ml) were subjected to electrophoresis on 3% agarose
gels. The gels were stained with ethidium bromide and photographed
using an ultraviolet light transilluminator. The Pro product was 178 bp
and the Arg product was 136 bp.
The possible outcomes were: (i) if a PCR product was obtained only
with the Pro-specic primers, the patient was considered as Pro homozygote (Pro/Pro); (ii) if a PCR product was obtained only with the Arg-specic
primers, the patient was considered as Arg homozygote (Arg/Arg); (iii) if
the sample showed amplication with both two primers, the patient
was considered as heterozygote (Arg/Pro). Several PCR products were
sequenced to further validate the PCR results.

PCR-restriction fragment length

polymorphism analysis of HDM2 SNP309
polymorphism
Primers used in this study have been described: 5 -CGCGGGAGTT
CAGGGTAAAG-3 and 5 -AGCTGGAGACAAGTCAGGACTTAAC-3

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Single nucleotide polymorphisms (SNPs) in the p53 gene might have

functional relevance. At least two forms of wild-type p53 protein exist
among human populations; these forms are ascribed to amino acid
replacement at codon 72 of Arg (CGC) by Pro (CCC) in the transactivation domain of the p53 protein. The arginine and proline isoforms
have different biological and biochemical effects: the Arg-p53 could
induce apoptosis more efciently than the Pro-p53 (Thomas et al.,
1999; Dumont et al., 2003), while the Pro-p53 could induce a
higher level of G1 arrest than the Arg-p53 (Pim et al., 2004). The
p53 codon 72 polymorphism has been proposed as a candidate for
increasing the chance of miscarriage in otherwise healthy women (Pietrowski et al., 2005). HDM2 is vital in the regulation of p53 yet
sequence variations in the HDM2 promoter may result in altered
expression of the HDM2 protein. Recently, polymorphism SNP309
(a T to G change at nucleotide 309 in the rst intron) was found in
the promoter of HDM2. In our previous report, we found that the
HDM2 SNP309 G/G genotype was associated with a high risk of
missed abortion (Fang et al., 2009). Cells carrying the G/G genotype,
owing to an enhanced afnity for binding the stimulatory protein (Sp)
1, show a heightened HDM2 expression and signicant attenuation of
the p53 pathway compared with those carrying the T/T genotype.
Importantly, this HDM2 SNP309 G/G polymorphism enhanced promoter recognition by the transcription factor Sp1, which in turn
caused elevated HDM2 expression and attenuation of the
p53-mediated apoptotic response to cellular stresses, including
DNA damage (Bond et al., 2004; Hong et al., 2005). Polymorphisms
in the p53 and HDM2 genes have therefore been shown to be of functional signicance.
Based on the fact that the p53-HDM2 interaction plays an important role in apoptosis, we hypothesized that the functional polymorphisms in p53 codon 72 and HDM2 SNP309 might be associated with a
risk of missed abortion, and tested this hypothesis in this study

1254
(Ohmiya et al., 2006). The reagents were the same as for PCR-SSP above.
The PCR conditions for HDM2 SNP309 were as follows: 948C for 7 min,
35 cycles of 948C for 40 s, 608C for 40 s, 728C for 40 s and a nal extension of 728C for 7 min. The assay for the HDM2 polymorphism utilized the
enzyme MSPA1I (5 . . . CNGCKG . . . 3 ) (NEB, Beijing, Peoples
Republic of China). After DNA restriction, 10 ml of digested samples
were subjected to electrophoresis on 3% agarose gels. The gels were
stained with ethidium bromide and photographed using an ultraviolet
light transilluminator.
The possible outcomes were: (i) if only one DNA fragment of 237 bp
was observed, the patient was considered as T homozygous (T/T); (ii) if
only two DNA fragments of 189 and 48 bp were observed, the patient
was considered as G homozygous (G/G); (iii) if three DNA fragments of
237, 189 and 48 bp were observed, the patient was considered as heterozygous (T/G). Several PCR products were sequenced to further validate
the PCR results.

Total RNA was extracted from villous samples using the Trizol reagent
(Takara, Dalian, Peoples Republic of China). The quantity of RNA was
assessed spectrophotometrically. The OD260/280 of the RNA samples
ranged between 1.80 and 2.00. The total RNA was reverse transcribed
into cDNA using the Rever Tra Ace Kit (Takara) in a volume of 20 ml, including 4 ml of 5 reverse transcriptase buffer, 2 ml dNTP (10 mmol/l), 1 ml
RNAse inhibitor, 1 ml Rever Tra transcriptase, 1 ml Oligo (dT) 18
(0.5 mg/ml) and 5 ml ample RNA in a thermal cycler (room temperature
10 min, 428C for 1 h, and 708C for 10 min). The mRNA samples were
stored at 2808C before analysis.

Real-time PCR analysis of p53 and HDM2

mRNA
The Quantitative SYBR-Green PCR mix kit (Toyobo, Shanghai, Peoples
Republic of China) was used for the quantication of p53 and HDM2
mRNA expression. Relative gene expression quantication for p53 and
HDM2, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
an internal reference gene, was carried out using the Roche sequence
detection system (Roche, Shanghai, Peoples Republic of China). The
primers used for p53 were: forward: 5 -ACTAAGCGAGCACTGCC
CAAC-3 , and reverse: 5 -CCTCATTCAGCTCTCGGAACATC-3 . The
primers used for HDM2 were: forward: 5 -GGACAAGAACTCTCAGAT
GAAGATG-3 , and reverse: 5 -ATCTGTTGCAATGTGATGAAG-3 . The
PCR mixture consisted of 10 mM of each primer, 1 ml, 10 ml Mix SYBR
Green I (Toyobo) and 500 ng cDNA to a nal volume of 20 ml. For negative controls, we used a complete DNA amplication mix in which the
target cDNA template was replaced by water. Cycling parameters were
the following: denaturation for one cycle at 958C for 10 s, 45 cycles (temperature transition of 208C/s) of 958C for 0 s, 588C for 10 s and 728C for
10 s and uorescence reading taken at 728C, and melting curve analysis
with continuous uorescence reading. Individual p53 and HDM2 measurements was calculated relative to expression of GAPDH using a modication of the method described by Lehmann et al. (2001).

Immunohistochemistry
Villous samples (61: 30 from women with missed abortion and 31 from
controls) were xed in 4% formaldehyde overnight at room temperature,
embedded in parafn wax and four-micron sections were cut (Zhongshan,
Guangzhou, Peoples Republic of China). Tissue sections were deparafnized and rehydrated through graded alcohol. Endogenous peroxidase
activity was quenched by incubation in 3% hydrogen peroxide for
10 min. After three rinses with phosphate-buffered saline, sections were

blocked with normal goat serum to suppress non-specic background

staining. Mouse monoclonal antibody for wild-type p53 (Zhongshan,
diluted 1:50) and HDM2 antibody (Zhongshan, diluted1:50) were
applied to the sections separately overnight at 48C. The sections were
incubated with biotinylated goat anti-rabbit immunoglobulin (Ig) G for
20 min at 378C and then processed according to the SP kit (Zhongshan)
protocol. In the negative control for staining, sections were incubated
with isotype-matched non-specic antibodies (MsIgG). Nuclear staining
of cells was regarded as a positive result. The percentages of p53- and
HDM2-positive cells were estimated by counting the number of immunoreactive cells in ve microscopic elds at high-power magnication (400)
in areas with the highest visually determined positive immunoreactivity.
The absence of any positive cells was scored as negative, ,5% immunopositive cells as +, 5 20% as ++, .20% as +++.

Statistical analysis
Chi-squared test was used to analyze the genotype distribution of p53
codon 72 and HDM2 SNP309 polymorphisms between women with
missed abortion and control groups. Chi-squared test was also used to
analyze the association of p53 and HDM2 polymorphisms with the immunohistochemistry results for p53 and HDM2. Students t-test was used to
compare the p53 and HDM2 mRNA levels in villous samples. All the
values were presented as mean + SD. The odds ratio (OR) was used
to measure the association between the allele frequencies and risk of
missed abortion. All P-values are two-tailed and 95% condence intervals
(CIs) were calculated. Signicant difference was dened as P , 0.05.

Results
The genotypes and allele frequencies of p53
codon 72 polymorphism
Arg/Pro (60%) was a dominant genotype, while the frequency of Pro/
Pro genotype was only 20%. No signicant difference in frequency
was observed between the Pro/Pro and the Arg/Arg + Arg/Pro genotypes in women with missed abortion versus controls (P 0.330;
OR, 1.750; 95% CI, 0.6614.636). For comparisons of the frequency
of Arg and Pro alleles, there was also no difference between women
with missed abortion and controls (P 0.373; OR, 0.778; 95% CI,
0.4721.282).

The genotypes and allele frequencies of

HDM2 SNP309 polymorphism
The G/G genotype was more in women with missed abortion
(28.33%) than controls (12.50%). In the comparison of frequency of
T/T + T/G and G/G genotypes, a signicant difference was seen
between women with missed abortion and controls (P 0.043;
OR, 2.767; 95% CI, 1.0927.011). A higher frequency of the G
allele was detected in women with missed abortion (48.33%) than
controls (35.94%) (P 0.054; OR, 1.668; 95% CI, 1.0032.773).

The mRNA levels of p53 and HDM2 in

villous samples
The relationship of the p53 codon 72 and HDM2 SNP309 polymorphisms to the levels of p53 and HDM2 mRNA were examined in villous
samples (n 124). In the comparison of the p53 codon 72
and HDM2 SNP309 polymorphisms, samples from women with the
Pro/Pro genotype and G/G genotype had a higher HDM2 mRNA

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RNA extraction and RT reaction

Fang et al.

1255

p53/HDM2 gene polymorphisms and risk of missed abortion

level than with the Arg/Arg genotype and T/T genotype (P 0.013 and
P 0.04) (Fig. 1b and a). No signicant difference can be seen in the
comparison of the Arg/Arg and T/T homozygote with the Pro/Pro and
G/G homozygote on p53 mRNA level separately (P 0.278 and P
0.367) (Fig. 1d and c).

Immunohistochemistry
Nuclear staining was scored as positive expression for p53 and
HDM2. The cytotrophoblast cells, syncytiotrophoblast cells and extravillous trophoblast cells showed positive nuclear staining in the villous
samples (Fig. 2)

p53 codon 72 genotype and the expression of p53 and HDM2

Sixty one villous samples were randomly selected, including 16 cases
of Arg/Arg genotype, 35 cases of Arg/Pro genotype and 10 cases of
Pro/Pro genotype (Table I). The Pro/Pro genotype had a higher percentage of HDM2-positive cells than the Arg/Arg + Arg/Pro genotypes
(P 0.037), but for p53 positive cells, no difference can be seen in
comparison of Arg/Arg genotype with the Arg/Pro + Pro/Pro genotypes (P 0.563).

HDM2 SNP309 genotype and the expression of p53 and HDM2

Discussion

Figure 1 p53-HDM2 gene gene polymorphisms and the mRNA.

levels for p53 and HDM2. (a) Comparison of mRNA levels
for HDM2 between genotypes HDM2 G/G and T/T (*P 0.04).
*a d, all Students t-test, using samples from women with missed
abortions and controls. (n 124). (b) Comparison of mRNA levels
for HDM2 between genotypes p53 Pro/Pro and Arg/Arg.
(P 0.013). (c) Comparison of mRNA levels for p53 between genotypes HDM2 G/G and T/T (P 0.367). (d) Comparison of mRNA
levels for p53 between genotypes p53 Pro/Pro and Arg/Arg
(P 0.278).

As described in our previous report, we found that the HDM2

SNP309 G/G genotype was associated with a high risk of missed abortion and concluded that the HDM2 SNP309 G/G genotype may be a
genetic risk factor for missed abortion (Fang et al., 2009).The present
study provides new insights, through molecular examination, into
whether the genetic polymorphism of p53 codon 72 and HDM2
SNP309 were associated with the risk of missed abortion.
In the present study, no signicant difference was observed in frequency of the Pro/Pro and the Arg/Arg + Arg/Pro genotypes between
women with missed abortion and controls (P 0.330). In the comparison of the Arg and Pro alleles, there was also no difference
between women with missed abortion and controls (P 0.373). Similarly, Coulam et al. (2006) found no signicant differences when comparing genotypic and allelic frequencies of p53 codon 72 among
patients with recurrent pregnancy loss and controls. However, Pietrowski et al. (2005) reported a signicant association between the
Pro allele and the occurrence of recurrent pregnancy loss. The difference may be related to the different populations used in the studies.
The patient population in our study included only Han people, while
Coulam et al. (2006) used an unselected population and Pietrowski
et al. (2005) included only Caucasian women whose parents were
of the same ethnicity. It has been found that there is signicant variation in p53 codon 72 polymorphisms among different ethnicities
(Inserra et al., 2003).
The Pro/Pro p53 codon 72 genotype can induce higher HDM2
mRNA levels than the Arg/Arg genotype (P 0.013); the HDM2
SNP309 G/G genotype can induce higher HDM2 mRNA levels than
the T/T genotype (P 0.04). Similarly, in villous samples, the Pro/Pro
genotype can induce higher HDM2 expression than the Arg/Arg +
Arg/Pro genotype (P 0.037); the G/G genotype can induce higher
HDM2 mRNA levels than the T/T + T/G genotype (P 0.001).
Missed abortion is a complicated problem; gene polymorphisms
have been proposed as susceptibility factors that increase the
chance of miscarriage in otherwise healthy women (Pietrowski et al.,
2003, 2005): furthermore, multiplicative interactions between p53
and HDM2 polymorphism genotypes had a higher risk of the occurrence of missed abortion. These ndings support our prior hypothesis

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Sixty one villous samples were randomly selected, including 21cases of

T/T genotype, 24 cases of T/G genotype and 16 cases of G/G genotype (Table II). The G/G genotype had a higher HDM2 level than
T/T + T/G genotypes (P 0.001) but there was no difference in
protein levels for p53 in comparison of T/T genotype with the T/
G + G/G genotypes (P 0.289).

1256

Fang et al.

Figure 2 Immunohistochemical staining of p53 and HDM2 (200) in villous samples of control group. Mouse monoclonal antibodies of wild-type
p53 (diluted 1:50) and HDM2 (diluted 1:50) were applied to the sections separately (A and B). In the negative control for staining, sections were
incubated with isotype-matched non-specic antibodies (MsIgG) (C). A nuclear yellow brown stain indicated a positive result.

HDM2

................................................................
61b

2 or 1

Pa

p53

.............................................

11 or 1 11

2 or 1

1 1 or 1 11

.............................................................................................................................................................................................
Arg/Arg

16

11

Arg/Pro

35

17

18

Pro/Pro

10

0.155c
0.037d

21

14

0.563e
1.000f

All by x 2 test.
Thirty randomly selected from women with missed abortions and 31 from control group.
c
HDM2 immunostaining in Arg/Arg versus Arg/Pro and Pro/Pro genotypes.
d
HDM2 immunostaining in Pro/Pro versus Arg/Arg and Arg/Pro genotypes.
e
p53 immunostaining in Arg/Arg versus Arg/Pro and Pro/Pro genotypes.
f
p53 immunostaining in Pro/Pro versus Arg/Arg and Arg/Pro genotypes.
b

Table II HDM2 SNP309 genotype and the levels of p53 and HDM2 protein, as assessed by immunohistochemistry in
human villous samples.
HDM2

.................................................................
61b

2 or 1

Pa

p53

..............................................

11 or 111

2 or 1

11 or 111

.............................................................................................................................................................................................
T/T

21

14

T/G

24

14

10

G/G

16

14

0.062c
d

0.001

10

11

13

11

12

0.289e
0.142f

All by x 2 test.
Thirty randomly selected from women with missed abortions and 31 from control group.
c
HDM2 immunostaining in T/T versus T/G and G/G genotypes.
d
HDM2 immunostaining in G/G versus T/T and T/G genotypes.
e
p53 immunostaining in T/T versus T/G and G/G genotypes.
f
p53 immunostaining in G/G versus T/T and T/G genotypes.
b

that the genetic polymorphisms of p53/HDM2 and their interactions

were associated with missed abortion.
The trophoblast cells are similar to malignant cancer cells in terms
of their invasiveness, high cellular proliferation rate, lack of cell contact
inhibition and immune privilege (Quenby et al., 1998). Placental trophoblast invasion and placental reorganization is an ongoing process

during pregnancy. Apoptosis and cell proliferation are coordinately

regulated during pregnancy (Pietrowski et al., 2005). One key regulator may be p53, a potent transcription factor that controls the
expression of multiple target genes involved in cell-cycle progression
and apoptosis (Sivaraman et al., 2001; Carvajal et al., 2005). Meanwhile, p53 is also a potential mediator for pregnancy (Sivaraman

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Table I p53 codon 72 genotype and the levels of p53 and HDM2 protein, as assessed by immunohistochemistry in human
villous samples.

1257

p53/HDM2 gene polymorphisms and risk of missed abortion

epidemiologic study investigated whether the p53 and HDM2 polymorphisms were associated with a risk of missed abortion. The investigated polymorphisms of p53 and HDM2 have been shown to be of
functional signicance. In our study, we observed that the woman who
carry the G/G or Pro/Pro genotype had signicantly higher expression
levels of HDM2. Missed abortion may be related to trophoblast cells
undergoing severe DNA damage. The high expression of HDM2 can
attenuate the apoptosis reaction following DNA damage, possibly
then resulting in the death of the embryo. This study supports our
prior hypothesis that polymorphism variations in p53 and HDM2
might have a relationship with missed abortion.
In conclusion, our ndings support the hypothesis that the polymorphisms of p53 codon 72 and HDM2 SNP309 and their multiplicative gene gene interaction are associated with an increased risk of
missed abortion. Further study will be necessary to determine the
mechanisms of missed abortion.

Acknowledgements
The authors thank Qingshen Li and Qiuling Zhu from the Maternal and
Child Health hospital of Jinan City for help with collecting the blood
and villous samples from the pregnancy women. The authors thank
Prof. Shukang Wang from the School of Public Health, Shandong University for help with statistical analysis.

Funding
This work was supported partly by National Natural Science Foundation of China (No. 30872738).

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et al., 2001). Investigations have shown that a large amount of p53

protein is produced by the human placenta in abnormal pregnancies;
thus, p53 is suspected as an important factor for the pathogenesis of
placental disorders through the induction of the trophoblastic apoptosis (Fulop et al., 1998; Qiao et al., 1998; Levy et al., 2002; Hu et al.,
2006). The major regulator of p53 activity is the HDM2 protein,
which also feeds back to promote p53 degradation (Alarcon-Vargas
et al., 2002; Michael et al., 2002). Elevated HDM2 protein binds to
p53 to inactivate its transcriptional activity and facilitate the destabilization of p53. This interplay of p53 and HDM2 has been characterized
as the p53-HDM2 autoregulatory feedback loop (Wu et al., 1993).
Post-translational modications of HDM2 play a dynamic role in regulating the ability of HDM2 to destabilize p53 (Khosravi et al., 1999;
Mayo et al., 2001; Goldberg et al., 2002).
At least two forms of wild-type p53 protein exist among major
human population groups, such as Caucasian and Han Chinese, and
a number of studies have revealed the functional differences of the
two polymorphisms (Hong et al., 2005; Pietrowski et al., 2005). The
Pro/Pro genotype is believed to induce higher levels of G1 arrest
than the Arg/Arg genotype. The Arg/Arg genotype seems to induce
apoptosis with faster kinetics and to suppress transformation more
efciently than the Pro/Pro genotype (Thomas et al., 1999; Pim
et al., 2004). In our study, the homozygous Pro/Pro genotype was
found more often among women with missed abortion compared
with controls. Allele frequencies may differ slightly when analyzing
specic ethnicities, and there were signicant variants of p53 codon
72 polymorphisms among different ethnicities (Inserra et al., 2003).
For the HDM2 polymorphism, Bond et al. (2004) showed that after
treatment with etoposide to induce DNA damage, which activates the
p53 pathway leading to DNA repair, cell cycle arrest and apoptosis,
signicant death was observed in cells with the HDM2 T/T genotype
but not in cells with the HDM2 G/G genotype. In our study, we
found that the G/G genotype can induce higher mRNA and protein
levels of HDM2 (P 0.04 and P 0.001, respectively).
The maintenance of placenta is essential for the exchange between
the fetus and its mother and apoptosis and cell proliferation are major
processes contributing to a successful pregnancy. One explanation for
the observed increase of missed abortion for p53 Pro and HDM2 G/G
carriers might be their higher potential to resist apoptosis. p53 is activated by stressful stimuli, including DNA damage and hypoxia. We
suggest that p53 Pro carriers might be at a disadvantage, with a
later onset of response to stress or a lower level of the required physiological apoptosis. The lower level of apoptosis might lead to misguided growth of cells and high expression of HDM2. The HDM2
G/G carriers might more easily respond to stress, leading to the
high expression of HDM2. Specically, enhanced HDM2 levels
have been shown to cause p53 degradation in surviving cells and
consequently attenuate the p53-mediated DNA damage response
(Ries et al., 2000; Zauberman et al., 1995), which forms a negative
feedback loop.
Researchers found that both p53 codon 72 polymorphism and
HDM2 SNP309 were associated with the age at onset of oral
carcinoma (Yoon et al., 2008), early development of hepatocellular
carcinomar in patients with chronic hepatitis B virus infection (Han
et al., 2008), predicting the clinical outcome of advanced non-small
cell lung cancer and inuencing the clinical outcome of transitional
cell carcinoma of the bladder (Horikawa et al., 2008).This

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