Engineering, 2013, 5, 371-375
http://dx.doi.org/10.4236/eng.2013.510B075 Published Online October 2013 (http://www.scirp.org/journal/eng)
The Expressions of P53, MDM2 in Trophoblasts of
Spontaneous Abortion Mouse Model and the Relevant
Researches
Chunlan Zhou, Qian Wang, Menghuan Zeng, Liping Cheng, Xiaoyun Xie*
Jiaxing Univer sity , Jia xing City, China
Received 2013
ABSTRACT
Objective: To explore the mRNA expression of the related genes of p53, MDM2, vascular endothelial growth factor
(VEGF) and hypoxia inducible transcription factors-1 a (HIF-la) in villous samples of spontaneous abortion mouse
models and normal pregnancy models, and to discuss the effect of p53, MDM2 on the growth of villous trophoblast
cells. Methods: The abortion-prone CBAXDBA/2 matings were established as the model of spontaneous abortion and
the non-abortion-prone CBAXBALB/c matings as the model of normal pregnancy. Applied q Real-time PCR method to
detect the mRNA expression levels of p53, MDM2, VEGF and HIF-la in villous samples of spontaneous abortion
mouse models and normal pregnancy models. Results: The relationship of the mRNA expression level of p53, MDM2,
VEGF and HIF-la in vinous samples of spontaneous abortion mouse models: in the villous samples of spontaneous
abortion mouse models, the expression of p53 was po sitively correlated with the expression of MD M2, HIF-la (r = 0.35;
r = 0.63), and the relationship was significant (P = 0.01; P < 0.001); but negatively correlated to the expression of
VEGF (r = −0.30), and the relationship was significant (P = 0.03). The expression of MDM2 was positiv ely correlated
with the expr ession of HIF-la (r = 0.28), and the relationship was significant (P = 0. 04); and negatively correlated with
the expression of VEGF (r = −0.08), but the relationship was not significant (P = 0.57). The expression of HIF-la was
negatively correlated with the expression of VEGF (r = −0.37), and the relationship was significant (P = 0.007). The
relationship of the mRNA expression level of p53, MDM2, VEGF and HIF-1 a in vinous samples of normal pregnancy
models: in the vinous samples of normal pregnancy models, the expression of p53 was positively correlated with the
expression of MDM2, VEGF and HIF-la (r = 0. 31; r = 0. 48; r = 0. 67), and the relationship was significant (P = 0.03; P
= 0.003; P < 0.001). The expression of MDM2 was positively correlated with the expression of VEGF (r = 0. 23), but
the relationship was not significant (P = 0.11); and negatively correlated with the expression of HIF-la (r = −0. 03), but
the relationship was not significant (P = 0.84). The expression of HIF-la was positively correlated with the expression
of VEGF ( r = 0. 35), and the relationship was significant (P = 0.01). Conclusion: angiogenesis reduces in villous sam-
ples of spontaneous abortion mouse model, P53 and MDM2 involve in angiogenesis in villous samples, unlikely p53,
and MDM2 have effects on normal early pregnancy villous angiogenesis and when the cell DNA damages or hypoxia
exacerbates, it can induce high expression of p53, MDM2, inhibit angiogenesis in villous samples in early pregnancy.
P53, MDM2 generegulate villous trophoblast cell growth by adjusting expression of HIF-1a and VEGF gene, finally
influences pregnancy.
Keywords: p53; MDM2; VEGF; HIF-la; Spontaneous Abortion
1. Introduction
Spontaneous abortion is one of the common obstetric
pathological pregnancies, its etiology is complex. In ad-
dition to anatomy, endocrine, infection, chromosome, ge-
netic factors, of which 40% - 80% unexplained sponta-
neous abortion may be associated with immune factors.
Although studies have confirmed the occurrence of spon-
taneous abortion may be associated with excessive cell
apoptosis, at present its specific regulation mechanism
lack systematic and thorough research. The related genes
of p53, MDM2 play an important role in regulating apop-
tosis, occurrence mechanism of spontaneous abortion.
The spontaneous abortion mouse models were chosen for
the study. We applied q Real-time PCR method to detect
genes expression levels in villous samples of spontane-
ous abortion mouse models and normal pregnancy mod-
els, discussed the effect of p53, MDM2 on pregnancy,
studied the mechanism of spontaneous abortion.
*Corresponding a uthor.

C. L. ZHOU ET AL.
372
2. Material and Method
1) Experim ent animals and reagent
Female CBA/J, male DBA/2, BALB/C three mouse,
clean 8 - 10 weeks of age, were bought in Shanghai
Science Development Company.p53, MDM 2 Specific
primer, p53, MDM2 VEGF and HIF-1a antibody, DNA
color-developing agent all related reagents were purchased
at Nanjing University.
2) Model preparation and grouping
Establish a mouse model, the abortion-prone CBAXD-
BA/2 were established as the model of spontaneous abor-
tion and the non-abortion-prone CBAXBALB/c prone as
the model of normal pregnancy, and detected Vaginal
suppository as pregnanc y 0 d.
3) Specimen handling
At the 13th day, two groups of pregnant mouse models
were put to death. We collected a partial villous samples
(samples came from the normal survival embryonic) and
stored them at −70˚C. Another small part fixed with 4%
formalin in room temperature was immobilized for 24
hours. Serial sections, the thickness about 4 μm, put in
the 60 degre e s oven, dried after 72 h for backup.
4) Applied q Real-time PCR method to detect the mRNA
expression levels of p53, MDM2, VEGF and HIF-la in
villous samples of spontaneous abortion mouse models
and normal pregnancy mode ls
Did strictly as agent instructions said. Reaction proce-
dure: pre degenerated at 95˚C for 10 min, degenerated at
95˚C for 15 sec. Annealed at 58˚C for 20 sec (p53,
MDM2, HIF-1a), Annealed at 61˚C for 20 sec (VEGF),
extended at 72˚C for 10 sec, circulated a total of 45 times,
at each end of circulation amplification detected fluores-
cence signal. Subsequently doing melting curve analysis,
a slow heating pro cedure: from 6˚C up to 95 ˚C at rate of
0.1˚C/sec, then cooled down to 40˚C, at the same time,
continuously monitored fluorescence signal changing, in
the progress of double chain degenerating gradually,
drew melting curve of products, in order to understand
the specificity of sample amplification, made sure mea-
surement results were precise and reliable. After the reac-
tion was finished, the computer according to amplifica-
tion of the quantitative standard template automatically
drew standard curve. According to the respective stan-
dard curve, the software automatically calculated accurate
content of the sample to be tested in the gene or house-
keeping gene.
3. Statistical Methods
Used SPSS12.0 statistical software to analyze data, Sp ea r-
man rank relation coefficient to analyze the relationship
of the mRNA expression level of p53, MDM2, VEGF
and HIF-la in vinous samples. ±SD was used as measure-
ment data, applied variance homogeneity test and single
factor variance analysis to compare measurement data
group mean. The two groups of measurement data were
compared by student T-test. The two each other in many
groups were compared with SNK-q test. Counted data
using × 2 test. P < 0.05 was considered obviously differ-
ent.
4. Results
The relationship of the mRNA expression level of p53,
MDM2, VEGF and HIF-la in vinous samples of sponta-
neous abortion mouse models were shown in Table 1.
The study found the relationship of the mRNA expres-
sion level of p53, MDM2, VEGF and HIF-la in vinous
samples of spontaneous abortion mouse models: in the
villous samples of spontaneous abortion mouse models,
the expression of p53 was positively correlated with the
expression of MDM2, H IF-la (r = 0.35; r = 0.63), and th e
relationship was significant (P = 0.01; P < 0.001); but
negatively correlated to the expression of VEGF (r =
−0.30), and the relationship was significant (P = 0.03).
The expression of MDM2 was positively correlated with
the expression of HIF-la (r = 0.28), and the relationship
was significant (P = 0.04); and negatively corr elated with
the expression of VEGF (r = −0.08), but the relationship
was not significant (P = 0.57). The expression of HIF-la
was negatively correlated with the expression of VEGF
(r = −0.37), and the relationship was significant (P =
0.007). Therefore, in the vinous samples of spontaneous
abortion mouse models the increased expression of p53,
MDM2 and HIF-la could make expression of VEGF de-
crease.
The relationship of the mRNA exp ression lev el o f p53,
MDM2, VEGF and HIF-1 a in vinous samples of normal
pregnancy models were shown in Table 2: in the vinous
samples of normal pregnancy models, the expression of
p53 was positively correlated with the expression of
MDM2, VEGF and HIF-la (r = 0. 31; r = 0. 48; r = 0. 67),
and the relationship was significan t (P = 0.03; P = 0.003;
P < 0.001).
The expression of MDM2 was positively correlated
with the expression of VEGF (r = 0. 23), but the rela-
tionship was not significant (P = 0.11); and negatively
correlated with the expression of HIF-la (r = −0.03), but
the relationship was not significant (P = 0.84). The ex-
Table 1. The relationship of the mRNA expression level of
p53, MDM2, VEGF AND HIF-la in vinous samples of spon-
taneous abortion mouse models.
TABLE HEAD P53 MDM2 VEGF HIF-1A
r P r P r P r P
P53 1.00 - 0.35 0.01 −0.30 0.03 0.63 <0.001
MDM2 - - 1.00 - −0.08 0.57 0.28 0.04
VEGF
- - - - 1.00 - −0.37
0.07

C. L. ZHOU ET AL.
373
Table 2. The relationship of the mRNA expression level of
p53, MDM2, VEGF and HIF-1a in vinous samples of nor-
mal pregnancy models.
TABLE HEAD
P53 MDM2 VEGF HIF-1A
r P r P r P r P
P53 1.00 - 0.31 0.03 0.48 0.003 0.67 <0.001
MDM2 - - 1.00 - 0.23 0.11 −0.03 0.84
VEGF
- - - - 1.00 - 0.35
0.01
pression of HIF-la was positively correlated with the
expression of VEGF (r = 0. 35), and the relationship was
significant (P = 0.01). Therefore, in vinous samples of
normal pregnancy models, the increased expression of
p53, MDM2 and HIF-la could make expression of VEGF
increase, too.
5. Discussion
Apoptosis is a process of regulating body growth, main-
taining homeostasis, actively removing damaged and non-
required cells controlled by genes. The imbalanced ex-
pression of related genes about cell apoptosis probably
leading to various pathological pregnancy is one of the
important reasons, placenta is the important position di-
rectly connecting the maternal and infant in vivo, it’s
normal development is crucial to maintain normal preg-
nancy, in order to better adapt to the needs of the fetus,
placenta reconstructs tissue by villou s apoptosis and pro-
liferation. There exists trophoblastic cells apoptosis to
some degree in normal pregnancy, in the process of emb-
ryo development, moderate apoptosis facilitates villous
blood lumen formation and the development of villus in
early pregnancy [1-3]. The progress of embryo develop-
ment and placenta germination is in a relatively hypoxic
environment. In the early pregnancy (first 12 weeks of
pregnancy), trophoblastic cells adapting to hypoxia en-
vironment is the key to successful pregnancy.
In the progress of pregnancy, with the level of oxygen
changing, angiogenesis of placenta trophoblastic vascular
network will change, too [4]. Trophoblastic cells come
from embryonic trophectoderm, they play an important
role in process of embryo implantation, its proliferation,
functional diso rders are closely cor related with o ccurrence
and development of multiple pregnancy diseases. Suc-
cessful pregnancy depends on right fetal placenta vascu-
lar development in trophic layer, only in this way can the
oxygen and nutrients translate from the gap of trophob-
lastic cells to the fetus [5]. In early embryo development,
angiogenesis is the most basic condition of embryo im-
plantation and development of uterine endometriumt, an-
giogenesis requires multiple factors inter action, inc luding
a variety of hormones, growth factors, soluble extracel-
lular matrix molecules [6]. VEGF is one of the most im-
portant angiogenesis regulation factors [7], the normal
expression of VEGF is an important factor to maintain
pregnancy. The decreased expression of VEGF may be a
danger signal about abortion, its implantation is a com-
plex, ambiguous process, VEGF has a crucial role in the
process of blastocyst implantation and placenta growth
and development [8] VEGF is a highly conserved glyco-
protein, composed of two identical subunits (Mr23,000)
cross-linking to gether with d isu lfide, the molecu lar weight
is between 34,000 - 45,000, Isoelectric point 8.5. VEGF
has four different variants, the gene is located on the
sixth chromosome long arm, including the alternative
splicing 8 exons and 7 introns, the biological activity of
the four VEGF is similar, in human cells, the amino acid
residues are 121, 165, 189 and 206. The variants about
121, 165 of amino acid residues are easy to arrive the
target cell, pregnancy is inseparable from the rich blood
supply from implantation to placenta formation, then to
delivery, and is inseparable from the angiogenesis, per-
meability changing. When the fertilized egg implantation,
blastocyst is not closed with maternal blood, human
blastocyst development is in a relatively hypoxic state, so
hypoxia can make VEGF activity increased [9], VEGF
can promote angiogenesis, thereby improving blood flow,
increasing supply of oxygen in hypoxia tissue [10]. Shi-
raish [11] uses immunological technique to discover
VEGF expression of syncytiotrophoblast and extravillous
trophoblast cells during the pregnancy process, and VEGF
also expresses in villous stromal and decidual cells. The
expression of VEGF is not balanced in early pregnancy
in syncytiotrophoblast cells, at near vascular buds the
concentration is higher, morphological analysis shows
masculine expression area of VEGF is villous blood ves-
sel formation region, explaining placenta trophoblasts ge-
nerating VEGF to promote the development of placenta
vascular network. Use immunohistochemical method to
find the VEGF expression decreases in trophoblastic cell
in patients with missed abortion. Vuorela [12] uses im-
munohistochemical method to find the VEGF expression
decreases in trophoblastic cells in patients with missed
abortion.
Fertilized ovum from implantation to delivery, oxygen
concentration of placenta is changing. Human placenta
development is divided into two stages, early stages of
pregnancy (first 3 months of pregnancy), is placenta for-
mation stage, the oxygen concentration of placenta site is
low. When embryo inserts endometria, oxygen concen-
tration is only 10 - 15 mmHg, embryo relies on the secre-
tion of uterine gland to provide nutrients, increased flow
of uterine placenta blood is closely related to the changes
of trophoblast penetrating endometrium and uterine spir-
al artery. First 12 weeks of pregnancy, the villous space
bending and uterine spira l artery diameter being relative-
ly narrow, limit blood flowing into the villous space, the
site of placenta implantation is a low oxygen concentra-

C. L. ZHOU ET AL.
374
tion. After 12 weeks of pregnan cy, trophoblastic cell im-
merse into uterine spiral artery, remodel the vascular lay -
er, make narrow artery expanded, placenta blood flow
increased, corresponding placenta site oxygen concentra-
tions also increased [13]. Therefore, early trophoblast de-
velopment is in hypoxia environment, hypoxia has effect
on trophoblast immersion.
At present people have reached an agreement, placenta
trophoblast angiogenesis is strictly regulated by the oxy-
gen concentration. Hypoxia may lead to hypoxic cell
stressed, performing for the role of hypoxia in cells has
effect on oxygen sensors, initiates intracellular signal
transduction pathways, activate as a transcription HIF-1 a,
increased HIF-I a expression [14].
HIF-1 is a major hypoxia-inducible transcription factor,
with one a subunit and one B subunit, HIF-1a is an ad-
justable function unit, regulated by the oxygen concen-
tration, in recent years, studies have shown that HIF-1a
under certain condition has effects on apoptosis. Under
hypoxia state, HI F-1 makes hig her express ion of P53 a nd
inducts p21 synthesis. Suppress activity of cell cycle de-
pendent kinase, makes cells stay in the G1 period, pro-
motes cell apoptosis, HIF-1 as the organism regulation
factor under hypoxic conditions, mediates hypoxia-in-
duced apoptosis, hypoxia can increase the wild-type em-
bryonic stem cell (ES, HIF-1a) apoptosis, but can not
promote embryonic stem cells (ES, HIF-1β) of gene de-
letion apoptosis, they incubated tumor derived from em-
bryonic stem cell, also showed that HIF-1a genotype
tumor apoptosis less than HIF-1β genotype hypoxic in-
ducing apoptosis is mediated by HIF-1. Moritz also found
early cell apoptosis is related to high expression of HIF-
1 when pancreas transplantation is for type I diabetes.
VEGF is the major transcription factor of HIF-1a.
Under anoxic state, the high expression of HIF-la can
induce higher expression of VEGF, but the too high ex-
pression of HIF-la could inhibit angiogenesis [14]. HIF-
la induces apoptosis in hypoxia lying in p53 protein
through combining the oxygen degradation domain, ODD
and HIF-1 a, compound formation, enhance the stability
of p53. The p53 protein is a short-lived protein, the intra-
cellular concentration is strictly regulated by the negative
accommodation factor mouse double minute gene 2
(MDM2) gene [15-17]. MDM2 is an important intracel-
lular regulating p53 con centration and its activity, as p53
important activity molecules to involved in cell growth
inhibition, apoptosis, regulation of cell cycle process.
The expression of wild type P53 protein can stimulate
high expression of MDM2 protein. Based on the p53 and
MDM2 negative feedback loop, study found increased
MDM2 protein can inhibit p53 protein function, thus in-
creasing the negative feedback loop function. Factors
that can affect the p53, MDM2 or P53-MDM2 interac-
tion have an important effect on for cell fate (apoptosis,
cell cycle arrest, etc.) [18]. The changing of MDM2 gene
promoter sequence change can alter the expression of
MDM2 protein. The expression of MDM2 protein im-
proves, make p53 reduce stability, therefore change the
apoptotic reaction of p53 mediating on DNA damage.
[17,19].
If MDM2 identifies target p53, meanwhile combines
with HIF-1a subunit, it can mediate HIF-1 a degradation,
thereby affecting the expression of VEGF. Our study
found, the expression of p53, MDM2 and HIF-1 are
higher in villous samples of spontaneous abortion mouse
models than normal pregnancy models, however the ex-
pression of VEGF is lower in villous samples of sponta-
neous abortion mouse models than normal pregnancy
models. We hypothesized that in the progress of normal
early villous samples development, Chorionic villus in is
the hypoxic microenvironment, hypoxia makes the ex-
pression of HIF-la gene higher, leading to the lower ex-
pression of VEGF, thus prompting villus angiogenesis,
ensuring the success of early pregnancy. However in the
progress of spontaneous abortion, Chorionic villus cells
suffer a more severe hypoxia and DNA damage, make
higher expression of p53, make higher expression of
MDM2 by the positive feedback regulation, and hypoxia
can cause expression of HIF-la increased further, but
MDM2 distinguishes target p53, meanwhile combines
HIF-1 a subunit, instead mediates HIF-1 a degradation,
making the expression of VEGF lower and angiogenesis
decreased, at the same time making cell cycle arrested,
apoptosis increased, eventually leading to the occurrence
of spontaneous abortion. Therefore, based on the study of
chorionic, we hypothesized that the p53, MDM2 gene
plays a role in regulating during early pregnancy villous
samples development, finally influencing the pregnancy.
REFERENCES
[1] M. Jerzak and P. Bischof, “Apoptosis in the First Trimes-
ter Human Placenta: The Role in Maintaining Privilege at
the Matemal-Foetal Interface and the Trophoblast Remo-
deling,” The European Journal of Obstetrics & Gynecol-
ogy and Reproductive Biology, Vol. 100, 2002, pp. 138-
142. http://dx.doi.org/10.1016/S0301-2115(01)00431-6
[2] D. Michael and M. Oren, “The p53-Mdm2 Module and
the Ubiquitin System,” Cancer Biology, Vol. 13, 2003, pp.
49-58. http://dx.doi.org/10.1016/S1044-579X(02)00099-8
[3] G. Beckman, R. Birgander, A. Sjalander, et al., “Is p53
Polymorphism Maintained by Natural Selection?” Human
Heredity, Vol. 44, 1994, pp. 266-270.
http://dx.doi.org/10.1159/000154228
[4] L. V. J. Schaffer, C. Breymann, M. Gassmann and H. H.
Marti, “Preserved Placental Oxygenation and Develop-
ment during Severe Systemic Hypoxia,” The American
Journal of Physiology Regulatory, Integrative and Com-
parative Physiology, Vol. 290, No. 3, 2006, pp. 844-851.
http://dx.doi.org/10.1152/ajpregu.00237.2005

C. L. ZHOU ET AL.
375
[5] A. C. K. Ajakuma r, “Expression, Ontogeny, and Regula-
tion of Hypoxia-Inducible Transcription Factors in the
Human Placenta,” Biology of Reproduction, Vol. 63, No.
2, 2000, pp. 559-569.
http://dx.doi.org/10.1095/biolreprod63.2.559
[6] D. E. F. J. Ingber, “Mechanochemical Switching between
Growth and Differentiation during Fibroblast Growth
Factor-Stimulated Angiogenesis in Vitro: Role of E xtr a-
cellular Matrix,” The Journal of Cell Biology, Vol. 109,
No. 1, 1989, pp. 317-330.
[7] M. D. L. D. Mueller, E. Garrett and R. N. T ay lor, “Neu-
trophils Infiltrating the Endometrium Express Vascular
Endothelial Growth Factor: Potential Role in Endometrial
Angiogenesis,” Fertility and Sterility, Vol. 74, No. 1,
2000, pp. 107-112.
[8] S. SK, “Angiogenesis and Implantation,” Human Repro-
duction, Vol. 15, No. S6, 2000, pp. 59-66.
[9] L. O. A. Hefler, P. Husslein, C. Kainz and C. Tempfer,
“Vascular Endothelial Growth Factor Serum Levels in
Pregnancy and Preeclampsia,” Acta Obstetricia et Gyne-
cologica Scandinavica, 2000, pp. 77-78.
[10] H. H. R. W. Mart i, “Angiogenesis in Ischemic Disease,”
Vol. 82, 1999, pp. 44-52.
[11] S. N. K. aishi, N. Kinukawa, H. Nakano and K. Sueishi,
“Immunohistochemical Localization of Vascular Endo-
thelial Growth Factor in the Human Placenta,” Placenta,
Vol. 17, No. 2-3, 1996, pp. 111-121.
[12] P. C. O. Vuorela, M. Tulppala and E. Halmesmaki,
“VEGF, Its Receptors and the Tie Receptors in Recurrent
Miscarriage,” Molecular Human Reproduction, Vol. 6,
No. 3, 2000, pp. 276-282.
[13] G. J. H. J. Burton and A. L. Wets on, “Maternal Arterial
Connections to the Placectal Intervil Lous Space during
the First Trimester of Human Pregnancy,” American Jour-
nal of Obstetrics & Gynecology, Vol. 181, 1999, p. 71.
[14] H. D. Z. J. Skinner, J. Fang, F. Agani and B. H. Jiang,
“Vascular Endothelial Growth Factor Transcriptional Ac-
tivation Is Mediated by Hypoxia-Inducible Factor Lalpha,
HDM2, and p74S6K1 in Response to Phosphatidylinosi-
tol 3-Kinase/AKT Signaling,” The Journal of Biological
Chemistry, Vol. 279, No. 44, 2004, pp. 45643-51.
[15] M. Thomas, A. Kalita, S. Labrecque, et al., “Two Poly-
morphic Variants of Wild-Type p53 Differ Biochemically
and Biologically,” Molecular and Cellular Biology, 1999,
pp. 1092-1100.
[16] P. Dumont, J. I. Leu, A. Cr. Della Pietra, et al., “The Co-
don 72 Polymorphic Variants of p53 Have Ma rkedly Dif-
ferent Apoptotic Potential,” Nature Genetics, Vol. 33,
2003, pp. 357-365.
[17] G. L. Bond, W. Hu, E. E. Bond, et al., “A Single Nucleo-
tide Polymorphism in the MDM2 Promoter Attenustes the
p53 Tumor Suppressor Pathway and Accelerates Tumor
Formation in Humans,” Ce ll, Vol. 119, 2004, pp. 591-
602.
[18] D. Michael and M. Oren, “The p53-Mdm2 Module and
the Ubiquitin System,” Cancer Biology, Vol. 13, 2003, pp.
49-58.
[19] Y. Hong, X. Zhang, et al., “The Role of p53 and MDM2
Polymorphisms in the Risk of Esophagel Squamaous Cell
Carcinoma,” Cancer Research, Vol. 65, 2005, pp. 9582-
9587.