Contributed equally
The aim of this study was to investigate the correlation between the expression of pregnancy-associated plasma protein A (PAPP-A) in basal decidual cells and recurrent spontaneous abortion (RSA). A total of 39 patients with a history of RSA were enrolled into the RSA group. A further 30 females who had experienced normal pregnancy were enrolled into the control group. The mRNA expression of PAPP-A in basal decidual cells was analyzed using real-time PCR. The distribution and expression of PAPP-A protein levels in basal decidual cells were analyzed by immunohistochemistry. The correlation between PAPP-A protein levels and RSA was analyzed. The levels of PAPP-A mRNA in the RSA group were significantly decreased, compared with the control group (P<0.05). Consistent with the mRNA levels, the protein levels of PAPP-A were also significantly lower in the RSA group compared with the control group (P<0.05). Multivariate logistic analysis indicated that the suppression of PAPP-A was one of the risk factors for RSA. Furthermore, Hosmer-Lemeshow analysis suggested that the expression levels of PAPP-A is an important factor for predicting RSA. In conclusion, the expression levels of the PAPP-A protein were significantly reduced in basal decidual cells of the RSA group compared with the control group. Therefore, PAPP-A is likely to play an important role in RSA.
Recurrent spontaneous abortion (RSA) is defined as three or more spontaneous abortions of a fetus before 20 weeks of gestation (
A total of 39 RSA patients from June 2010 to June 2012, termed the RSA group, were enrolled in this study. The criteria for enrolment were: i) Normal cytogenetic phenotype without any heritable disease or spontaneous abortion in the family history; ii) Negative physical examination of vaginal infection; iii) Negative for anticardiolipin antibodies, antinuclear antibodies, antisperm antibodies and antiendometrium antibodies; iv) No autogenous immune disease or endocrine disease; v) No vascular disease or infection disease history; vi) No reproductive disorder or sperm impairment of the fetus’ father; vii) No addiction to cigarettes or alcohol. The age range of the RSA group was 27–41 years, with an average of 33.1±5.4 years. The pregnancy time period was 5–7 weeks, with an average of 6.1±0.8 weeks. In addition to the RSA group, 30 patients who were experiencing normal pregnancy, but who were subjected to induced abortion, were enrolled as a control group. The age range of the control group was 23–40 years, with an average of 32.1±5.2 years. The pregnancy period time was 5–8 weeks, with an average of 6.5±0.9 weeks. No significant difference concerning age and pregnancy time period between the two groups was observed (P>0.05). This study was conducted in accordance with the declaration of Helsinki and with the approval from the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University. Written informed consent was obtained from all the participants.
Basal decidual tissue was obtained using vacuum suction and stored in liquid nitrogen. This tissue was then removed from liquid nitrogen and resolved in 1 ml TRIzol (Invitrogen, Carlsbad, USA). After resolving, 200
Basal decidual tissue from the RSA and control groups were paraffin-embedded. Deparaffinization treatment of a 5
The stained sections were analyzed using Motic Med 6.0 digital medicine image software. For each section, 10 views were randomly chosen under a microscope (magnification, ×400). The PAPP-A-positive cells were counted in all 10 views and the percentage of positive cells in the entire population was calculated. Four grades were defined to analyze the results: Negative(−): % of positive cells, <5%; weak positive(+): % of positive cells, 5–25%; medium positive (++): % of positive cells with a range of, 5–25%; positive(+++): % of positive cells was 25–50%; strong positive (++++): % of positive cells, >50%. Quantitative evaluation was achieved using Motic Med 6.0 digital medicine image software.
Data were shown as mean ± SD. For statistical analyses, the Student’s t-test and the Chi-Square test were used as appropriate by using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Multivariate non-conditional logistic regression analysis was used to evaluate the correlation between PAPP-A and RSA. A difference at P<0.05 was considered statistically significant.
The total RNA in decidual tissue was measured using ultraviolet spectrophotometry. The optical density (OD)260/280 values were in the range of 1.9–2.0. qPCR results demonstrated that the mRNA levels of PAPP-A in the RSA group were significantly decreased compared with the control group (t=5.204, P<0.05) (
Immunostaining showed that PAPP-A protein was expressed in the cytoplasm of basal decidual cells (
The correlation analysis between the PAPP-A protein levels and RSA was evaluated using multivariate logistic analysis. The results showed that the protein expression level of PAPP-A was highly related to RSA, indicating that PAPP-A is one of the major risk factors of RSA. The Hosmer-Lemeshow analysis showed that the protein expression level of PAPP-A is likely a good prognosis reference for RSA (χ2=3.158, P<0.05).
The incidence of RSA is approximately 1% in all females experiencing pregnancy. Therefore, it has been a hot topic in clinical and scientific research in recent years (
PAPP-A, which is secreted by placental syntrophoblastic cells and basal decidual cells, is one of the plasma glycoproteins involved in pregnancy. PAPP-A has previously been shown to regulate embryonic development (
The results have demonstrated that the level of PAPP-A mRNA in basal decidual tissue was significantly decreased in the RSA group patients compared with the control group. To obtain more detailed information on the difference in PAPP-A protein expression level, immunostaining with PAPP-A antibodies was performed. In agreement with the qPCR result, the PAPP-A protein expression levels were also decreased in the RSA group compared with the control group. Furthermore, results of the correlation and Hosmer-Lemeshow analyses suggested that the protein levels of PAPP-A could be used as an important reference in clinical RSA prognosis. The decrease in PAPP-A levels may also be a critical risk factor in RSA.
In conclusion, PAPP-A levels were significantly decreased in RSA patients compared with patients experiencing normal pregnancy. This abnormal expression may be one of the risk factors leading to RSA.
PAPP-A mRNA level of decidual in the RSA and control groups. *P<0.05.
Tissue distribution and quantitative results of the PAPP-A protein between the RSA and control group. *P<0.05.
PAPP-A protein expression level in the RSA and control groups.
Group | − | + | ++ | +++ |
---|---|---|---|---|
RSA | 2 | 19 | 8 | 1 |
Control | 0 | 7 | 11 | 21 |
PAPP-A, pregnancy-associated plasma protein A; RSA, recurrent spontaneous abortion.