This retrospective case-control study was conducted at Nguyen Dinh Chieu Hospital in Ben Tre Province (Mekong Delta, Vietnam) from June 2020 to December 2020. A total of 61 pregnant women diagnosed with threatened miscarriage and 61 healthy pregnant women were included.

Pregnant women who were selected for the control group included those who met all of the following criteria:

• Pregnant women without threatened miscarriage aged under 45 were 11–14 gestational weeks and received regular follow-ups according to their prenatal appointment schedules.

The disease group included individuals who were diagnosed with threatened miscarriage during the 11–14 gestational week period based on clinical features and imaging (ultrasound). For this retrospective case-control study, the control group was matched to the disease group concerning age, body mass index (BMI), maternal body weight, maternal body height, unhealthy habits, medical history, and maternal blood pressure.

The sample size was based on the study by Kaijomaa et al. [15], who reported 9 stillbirths (0.9%) among 961 patients with low PAPP-A, but none in 961 women from the control group. The following formula was used to calculate the sample size:

$$n=\frac{\left[p_1\left(1-p_1\right)+p_2\left(1-p_2\right)\right]\times {\left(z_{1-\frac{\alpha }{2}}+z_{1-\beta }\right)}^2}{{\left(p_2-p_1\right)}^2}$$

In this formula, n is the total number of miscarriage women; $z_{1-\frac{\alpha }{2}}=2.576(\alpha =0.01)$; $z_{1-\beta }$ = 1.282 (1-$\beta$ = 0.90); p₁= 0.49 is the percentage of non-miscarriage women with low PAPP-A; p₂ = 1 is the percentage of miscarriage women with low PAPP-A. Thus, the total number of miscarriages in women was 15. Additionally, Manning and Kuhn [16] reported a minimum miscarriage rate among pregnant women of 13.5%. Consequently, the sample size for the disease group was calculated by: (15/13.5%)/2 = 56. In this study, 61 patients were included in the disease group.

Besides time in set sampling, pregnant women in the control group are often matched for each case concerning important confounding factors regarding age, weight, height, BMI, smoking status, maternal blood pressure, and heart rate. The sample size ratio in the disease group and the control group is 1:1 in a pair-matched case-control study, with both groups selected from pregnant women who visited the obstetrics clinic of Nguyen Dinh Chieu Hospital. Statistical comparisons, including the Chi-square test, Fisher’s exact test, Student’s t-test, and Mann-Whitney U test, were used to assess the differences between the disease and control groups.

According to the American College of Obstetricians and Gynecologists, threatened miscarriage is defined as a nonviable intrauterine pregnancy that may involve either an empty gestational sac or a gestational sac with an embryo or fetus, but without fetal cardiac activity, occurring within the first 12 weeks and 6 days of gestation and the terms “miscarriage” and “early pregnancy loss” are used interchangeably [17]. Additionally, Bell et al. [18] defined threatened miscarriage as mainly a clinical term used when a pregnant woman presents within the first 20 weeks of gestation with spotting, mild abdominal pain and contractions, and a closed cervical os.

The inclusion criteria are formulated based on the standards of the Ministry of Health (Vietnam) [19] and the Radiopaedia article [18].

Pregnant women who were ineligible for the current study included those with at least one of the following:

• Women aged 45 years and above.

Pregnant women were screened with clinical tests such as ultrasound scans, NIPT, and testing for rubella antibodies. Participants underwent all clinical examinations voluntarily, and the results were available before study initiation. Prior to signing the consent forms, the participants were informed about the study details, including the fact that their PAPP-A levels were not used for their pregnancy monitoring.

Information was collected from consenting patients through direct interviews or by reviewing medical records. The data collected for the disease and control groups included general patient information such as age, weight, height, and BMI according to the World Health Organization categorization for Asian adults [20]. Also recorded were risk factors for miscarriage (unhealthy habits and historical factors), blood pressure, gestational age, CRL value, nuchal translucency (NT-value), and fetal heart rate.

PAPP-A concentrations were measured in both the disease and control groups. Venous blood samples were collected from participants, and the plasma samples were stored at –20 °C before quantitative PAPP-A analysis. The samples were thawed only once prior to testing.

PAPP-A levels were measured using an enzyme-linked immunosorbent assay (ELISA) on the Cobas PAPP-A platform. The ELISA kit included standards (PAPP-A, Lot Number: 37356201, Roche Diagnostics, Mannheim, Germany) and a test machine (Cobas 6000, Hitachi-Roche, Tokyo, Japan). The results were reported in mIU/mL, with a measurement range of 0.025–10 mIU/mL and a sensitivity of 0.025 mIU/mL. The final PAPP-A level was measured and converted to the MoM value by dividing the absolute concentration by the median concentration at a specific gestational age and correcting for maternal age.

Comparative analysis of the control and disease groups was performed according to the flowchart shown in Fig. 1.

Fig. 1.

Flowchart of the research steps in this study. PAPP-A, pregnancy-associated plasma protein A.

Data was collected in “.xlsx” format using Microsoft Office Excel software version 16.96.1 (Microsoft Corporation, Redmond, WA, USA) and analysed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). Qualitative variables were expressed as a frequency and percentage. The Kolmogorov-Smirnov test was used to determine whether a variable had a normal distribution, which was indicated by a p-value $>$ 0.05. The mean and standard deviation ($\overline{X}$ $\pm$ SD) were used to describe variables with a normal distribution, while the median, minimum-maximum, first-quartile, and third-quartile values were used to describe variables with a non-normal distribution. Statistical analyses were performed to identify significant associations between variables, including the Chi-square test, Fisher’s exact test, Student’s t-test, Mann-Whitney U test, and Z-test analysis. The Chi-square test was used to compare the observed counts in each category with the expected counts, except if the expected count was 5, in which case Fisher’s exact test was applied. The Z-value was used to assess the degree of deviation between an observed value and the mean of the distribution. The odds ratio (OR) with 95% confidence interval (CI) was calculated in univariate and multivariate logistic regression analysis. A multiple linear regression model with correlation coefficient (r) was applied for the quantitative factors. In this study, a p-value of $\le$0.05 was considered statistically significant. Additionally, receiver operating characteristic (ROC) curve analysis was employed to determine the area under the curve (AUC). The coordinates of the curve were used to describe the relationship between sensitivity and specificity at different cut-off points. The Youden index (J) was used to determine the PAPP-A level with the highest sensitivity and specificity. The Youden index is the maximum value of the sum of sensitivity and specificity minus 1: J = max (Se + Sp – 1), where Se is the sensitivity, and Sp is the specificity.

Table 1 shows the characteristics and clinical history of women in the two groups. Most of the pregnant women in this study had a normal or overweight BMI. The disease group contained more underweight women than the control group (p = 0.024), as well as significantly longer gestational age and greater CRL and NT values (p = 0.003, 0.005, and 0.002, respectively). Other characteristics, including maternal age, weight, height, BMI, wine and/or beer consumption, clinical history, blood pressure, and fetal heart rate, were not significantly different between the disease and control groups (p $>$ 0.05). The median PAPP-A concentration in the disease group was lower than in the control group (0.63 MoM and 1.09 MoM, respectively; p 0.001). Multivariable logistic regression shows that PAPP-A levels and NT value are independently negative and positive factors of threatened miscarriage with OR = 0.207 and 3.678 (p 0.05), respectively.

Table 2 shows that the PAPP-A level has moderate predictive value for threatened miscarriage in two groups of pregnant women, with many relevant indexes such as the Youden index, cut-off point, sensitivity, specificity, AUC, and others.

When evaluating the disease and control groups, the optimal cut-off point for MoM of 0.8255 yielded a sensitivity of 73.8% and a specificity of 80.3% (Table 2), with the ROC curve having an AUC of 0.789 (Fig. 2). This indicates the PAPP-A test has moderate value for differentiating between women with and without threatened miscarriage.

Fig. 2.

ROC curves and AUC for PAPP-A in the disease and control groups. ROC, receiver operating characteristic; AUC, area under the curve; PAPP-A, pregnancy-associated plasma protein A.

The majority of pregnant women in this study were younger than 35 years. Magnus et al. [21] reported that the risk of miscarriage in women aged 45 years and over was more than half. Therefore, only pregnant women aged 45 years were included in the present work in order to avoid bias due to the increased risk of miscarriage associated with older age. Age 45 years also helped to increase the sample size of the study, because the wider the age range selected, the more options there are for samples.

Viral infection, particularly human papillomavirus (HPV), was one of the exclusion criteria. Therefore, participants underwent interviews, gynecological examinations, and HPV testing using polymerase chain reaction analysis of cervical fluid to ensure accurate data collection [22].

The gestational age at the time of study participation was slightly higher in the disease group compared to the control group. The disease and control groups were not significantly different in terms of age, BMI, maternal body features, unhealthy habits, clinical history, and maternal blood pressure. This suggests the two groups were appropriately selected and therefore suitable for analysis of PAPP-A concentrations, which was the primary focus of this study. The results are likely to be more robust without interference from confounding variables. However, the disease and control groups showed significant differences in gestational age, CRL, and NT values. Since these differences may affect the PAPP-A value, this biomarker was measured in pregnant women at 11–14 gestational weeks and converted into the MoM to avoid distortion.

A study conducted by Cavoretto et al. [23] showed that the PAPP-A level for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was lower in comparison with controls. This confounder and moderator in the PAPP-A level may come from alterations in the placentation of IVF/ICSI pregnancies.

Yaron et al. [24] also found that women with a low PAPP-A level had significantly higher risks of fetal growth restriction and spontaneous miscarriage. It should be noted, however, that the definition of “low” PAPP-A differs between studies, thereby impacting the statistical and clinical significance of the results. Low PAPP-A has been defined as a range or threshold value, including percentages or arbitrary cut-offs ranging from 0.25 MoM to 0.50 MoM.

Using a test with the highest diagnostic accuracy, Swiercz et al. [25] reported a great accuracy for predicting preterm births occurring before 37 weeks of gestation. In comparison, our study found a moderate AUC for the threatened miscarriage group. The calculated cut-off value for PAPP-A in the study by Swiercz et al. [25] did not have satisfactory accuracy for predicting preterm birth occurring before 35 weeks. The best diagnostic accuracy achieved for predicting the preterm premature rupture of membranes (pPROM) was relatively high. Their findings demonstrated that low PAPP-A levels in the first trimester were associated with an increased risk of preterm birth and pPROM before 37 weeks [25]. Additionally, these authors showed that PAPP-A levels during the first trimester were associated with neonatal outcomes such as pH 7.2 and Neonatal Intensive Care Unit hospitalization. Neonates who scored 8 on the Apgar scale at birth or were born with an umbilical blood pH 7.2 showed lower normalized PAPP-A concentrations [26].

Jindal et al. [27] reported a significant association between low PAPP-A levels and various adverse pregnancy outcomes, such as intrauterine growth restriction, intrauterine death, pregnancy-induced hypertension, and other pregnancy-related complications.

Hanita et al. [28] found that low PAPP-A in women with threatened miscarriage was associated with pregnancy failure. The median PAPP-A level was significantly lower in patients with miscarriage than in those with successful pregnancies. In the present study, the median PAPP-A level in the disease group was significantly lower than in the control group. Hanita et al. [28] reported the best sensitivity and specificity at a lower cut-off value, while our study achieved the highest sensitivity and specificity at a higher cut-off value.

Movahedi et al. [29] found that the frequency of miscarriage during recent pregnancies in mothers with a low PAPP-A was significantly higher than in mothers with normal or elevated PAPP-A. Moreover, pregnant women with low or normal PAPP-A had a lower BMI than those with higher PAPP-A values.

Sun et al. [30] reported that pregnant women with a history of miscarriage had a higher risk of experiencing premature delivery, gestational diabetes mellitus (GDM), placenta abnormality, placenta previa, placenta accreta, and placenta adhesion compared to women with singleton pregnancies and without a history of miscarriage. Yu et al. [31] found that women with a history of induced abortion were more likely to experience preterm births before 37 weeks and 34 weeks [31]. A systematic review and meta-analysis of observational studies conducted by Woolner et al. [32] revealed that women who had experienced a miscarriage were more likely to report a family history of miscarriage.

Borna et al. [33] reported that PAPP-A values are also correlated with GDM, with GDM cases having a considerably lower PAPP-A level compared to the control group. Women with a low PAPP-A level had a significantly shorter gestational age at delivery, lower birth weight, and higher intrauterine growth restriction. ROC analysis revealed the PAPP-A level was weakly predictive for GDM [33]. Chen et al. [34] found that the PAPP-A level had a value for predicting hypertensive disorders of pregnancy.

Therefore, the current research helps clarify the observed differences in the threshold values. A low maternal serum PAPP-A concentration in the first trimester can predict not only chromosomal abnormalities but also adverse pregnancy outcomes.

Despite producing novel insights, this study had only limited ability to comprehensively assess the predictive value of PAPP-A for the early diagnosis of threatened miscarriage. Our analysis was restricted to a single biomarker, PAPP-A, and did not consider the potential utility of a panel of markers or other clinical factors. Moreover, the results of this study about the diagnostic accuracy of PAPP-A may not be generalizable to all clinical settings, as the performance of the test could be influenced by factors such as gestational age and underlying maternal conditions. Further research involving larger, more representative patient cohorts and a broader range of diagnostic approaches is required to more thoroughly evaluate the role of PAPP-A for early identification of threatened miscarriage.