Recurrent pregnancy loss (RPL) is a very difficult and emotional condition that affects approximately 1-2% of women of reproductive age.  Defined by two or more consecutive pregnancies failing prior to fetal viability, RPL can be caused by a combination of genetic abnormalities, anatomical defects, and hormonal imbalances, among other immunological dysfunctions. Despite medical advances, a portion of RPL cases remains unexplained, known as unexplained recurrent pregnancy loss (URPL). This diagnostic uncertainty often leaves patients and clinicians searching for reliable markers and treatments [1].

In recent decades, scientific inquiry into immunological processes during pregnancy has provided new approaches to understanding RPL. The regulation of the maternal immune system, which must tolerate the semi-allogeneic fetus, is crucial. Dysregulation of this balance has been linked to several pregnancy complications, including RPL, prompting researchers to focus on immune markers as potential predictors of pregnancy outcome [2]. Pentraxin-3 (PTX3), an acute-phase protein in the long pentraxin family, is a promising marker. As a key mediator of the innate immune system, PTX3 regulates inflammation and immune responses. Unlike C-reactive protein (CRP), produced in the liver, PTX3 is synthesized locally at inflammation sites, allowing it to reflect tissue-specific immune activity and making it a valuable biomarker for inflammatory conditions, including pregnancy-related complications [3]. PTX3 plays a role in placental development, including angiogenesis and tissue remodeling. Elevated PTX3 levels have been linked to pregnancy complications such as preeclampsia, intrauterine growth restriction (IUGR), and preterm birth. Excessive inflammation, common to these conditions, disrupts fetal development. As a result, PTX3 is emerging as a potential biomarker for predicting adverse pregnancy outcomes [4]. In the context of URPL, elevated PTX3 levels may signal an exaggerated immune response at the maternal-fetal interface, impairing implantation and placentation, and leading to pregnancy loss. Although the mechanisms by which PTX3 contributes to URPL are not fully understood, it is hypothesized that higher PTX3 levels during early pregnancy reflect an immune dysregulation, where pro-inflammatory signals outweigh anti-inflammatory ones. This imbalance could hinder trophoblast invasion and vascular remodeling, critical for sustaining a healthy pregnancy [5]. While evidence supporting inflammation’s role in RPL is growing, there is no consensus on how to incorporate immune markers like PTX3 into clinical practice. Most diagnostic approaches focus on anatomical or genetic causes of RPL, leaving a gap in understanding immune dysregulation in recurrent miscarriage. Further research is needed to confirm PTX3 as a reliable biomarker for URPL and to explore the immune mechanisms involved [6]. This study aims to fill this gap by investigating maternal serum PTX3 levels in women with primary URPL during the first trimester and comparing them with healthy pregnant women. By exploring PTX3’s relationship with recurrent miscarriage, the study seeks to provide insights into immune dysregulation in URPL and evaluate PTX3 as a predictive marker. Understanding the inflammatory processes in RPL could enhance diagnostics and lead to targeted immunotherapies for women affected by recurrent pregnancy loss [7].  The aim of this study was to assess first-trimester maternal serum PTX3 levels in women with primary unexplained recurrent pregnancy loss and compare them with healthy pregnant controls, and to evaluate the potential diagnostic value of PTX3 in predicting URPL.

Patients and Methods

This prospective case–control study was conducted at Tikrit Teaching Hospital, Iraq, over a one-year period from January 2024 to December 2024. The study aimed to evaluate the association between first-trimester maternal serum Pentraxin-3 (PTX3) levels and primary unexplained recurrent pregnancy loss (URPL).

Ethical approval for the study was obtained from the Ethics and Scientific Research Committee of Tikrit Teaching Hospital, and all procedures were conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants prior to enrollment.

Study Population

A total of 80 pregnant women were enrolled in the study and divided into two groups:

• Patient Group (URPL group): Forty women diagnosed with primary unexplained recurrent pregnancy loss.

• Control Group: Forty healthy pregnant women with a history of at least one previous uncomplicated pregnancy and no history of recurrent pregnancy loss.

Both groups were recruited during the first trimester of pregnancy during routine antenatal visits.

Inclusion Criteria

Patient Group (Primary Unexplained Recurrent Pregnancy Loss)

Women were included in the URPL group if they met the following criteria:

• History of three or more consecutive first-trimester miscarriages without a previous live birth, consistent with primary unexplained recurrent pregnancy loss

• Confirmation of early pregnancy failure by transvaginal ultrasonography

• Normal glucose metabolism, confirmed by an oral glucose tolerance test (OGTT), excluding gestational diabetes

• Negative antiphospholipid antibody profile, including lupus anticoagulant and anticardiolipin IgG/IgM, to rule out antiphospholipid syndrome (APS)

• Normal thyroid function tests, indicating euthyroid status

• Normal parental karyotype analysis, excluding chromosomal abnormalities or balanced translocations that may contribute to recurrent miscarriage

Control Group (Healthy Pregnant Women)

Participants in the control group met the following criteria:

• Pregnant women with at least one previous successful pregnancy and live birth

• No history of recurrent pregnancy loss or adverse obstetric outcomes

• Good general health without known medical or obstetric conditions that could affect pregnancy outcomes

Exclusion Criteria

Participants were excluded from the study if any of the following conditions were present:

• Active smoking, due to its association with adverse pregnancy outcomes

• Multifetal pregnancy, because of the increased risk of obstetric complications

• Chronic hypertension or a history of hypertensive disorders of pregnancy, including preeclampsia

• History of intrauterine growth restriction (IUGR)

• History of preterm birth, including cases associated with preterm premature rupture of membranes (PPROM)

• History of intra-amniotic infection or other inflammatory obstetric conditions that might influence systemic inflammatory biomarkers

Procedure

Patients were recruited at the first-trimester visit to an outpatient antenatal clinic. The gestational age was estimated according to Naegele's rule and confirmed by obstetric ultrasound. Maternal blood samples for the study were collected post inclusion and exclusion criteria fulfilled as 5 ml of venous blood by complete aseptic technique before any surgical / medical intervention.

Samples Collection and Analysis

Whole blood samples were collected in EDTA tubes, centrifuged at 1500 × g and for 15 min. The isolated plasma was aliquoted into fresh tubes and stored at -20°C for further analysis. Levels of PTX3 were measured with an enzyme-linked immunosorbent assay kit using reagents provided by Quantikine R&D International, Inc. (R & D Systems Inc., Minneapolis, MN). The intra-assay and inter-assay coefficients of variation for PTX3 were .1% and 3.9%, respectively, with a detection limit level of sensitivity at 0.025 ng/ml.

Statistical Analysis

SPSS software was used for statistical analysis. Data are described as mean (standard deviation). Normality of data was checked through Shapiro-Wilk and Kolmogorov-Smirnov tests. For non-parametric data, comparison between two groups was carried out using the Mann-Whitney U test. Statistical Analysis Correlation analysis between variables was done using the Spearman Rank correlation coefficient. Statistical significance was defined as p<0.05 Are presented as charts and graphics - using Excel 2010 software

Ethical Considerations

Study purpose and procedures were explained to all the participants before a written informed consent was taken. The study was carried out in compliance with the Declaration of Helsinki. This will add valuable information to the field of reproductive medicine on a potential role for PTX3 in primary unexplained recurrent pregnancy loss, obtained by following methodologically structured study.

The study showed that maternal demographic and clinical characteristics were comparable between the control and patient groups, as no statistically significant differences were observed in maternal age (29.31 ± 3.94 vs 27.93 ± 4.98 years, p = 0.095), duration of marriage (4.44 ± 1.21 vs 4.18 ± 1.02 years, p = 0.146), height (p = 0.883), weight (p = 0.265), or BMI (p = 0.185). In addition, socioeconomic status (p = 0.517), residence (p = 0.371), smoking status (p = 0.748), and gestational age at sampling (p = 0.083) did not differ significantly between groups.

However, PTX3 levels were markedly higher in the URPL patient group (12.00 ± 4.07 ng/mL) compared with the control group (1.69 ± 0.91 ng/mL), showing a highly significant difference (p<0.001). This result suggests that elevated PTX3 levels are strongly associated with unexplained recurrent pregnancy loss (Table 1).

Table 1: Comparison of Maternal Characteristics and PTX3 Levels

The comparison of PTX3 levels between study groups revealed a statistically highly significant elevation ofPTX3 among patients with URPL compared to healthy controls (12.00 ± 4.07 vs 1.69 ± 0.91 ng/mL, p<0.001). This finding confirms the potential role of PTX3 as a biomarker associated with inflammatory mechanisms contributing to recurrent pregnancy loss (Table 2).

Table 2: PTX3 Level Comparison Between Groups

The distribution of the number of previous miscarriages among the URPL patients demonstrated that 27.5% had four miscarriages and another 27.5% had six or more miscarriages, while 25% experienced three miscarriages and 20% experienced five miscarriages. This distribution highlights the severity of reproductive history among the studied patients and provides a basis for evaluating the relationship between miscarriage frequency and PTX3 levels (Table 3).

Table 3: History of Previous Miscarriages in Patients Group

The diagnostic performance analysis of PTX3 showed that different cutoff values yielded varying sensitivity and specificity. At a cutoff value of 4.5 ng/mL, the sensitivity was 92.5% and specificity 85.0%. Increasing the cutoff to 6.0 ng/mL resulted in 87.5% sensitivity and 90.0% specificity, providing the most balanced diagnostic accuracy. At 8.0 ng/mL, specificity increased to 95.0%, although sensitivity decreased to 72.5%. These results indicate that 6.0 ng/mL represents the optimal cutoff value for predicting URPL (Table 4).

Table 4: Diagnostic Accuracy of PTX3

Stratification of PTX3 levels according to the number of previous miscarriages revealed a progressive increase in mean PTX3 concentration with increasing miscarriage frequency. Women with three miscarriages had a mean PTX3 level of 9.7 ± 2.8 ng/mL, while those with four miscarriages showed 11.9 ± 3.5 ng/mL. The mean value increased further to 13.4 ± 3.1 ng/mL among women with five miscarriages and reached 14.7 ± 2.6 ng/mL in those with six or more miscarriages. This stepwise increase supports the hypothesis that higher PTX3 levels are associated with greater severity of recurrent pregnancy loss (Table 5).

Table 5: PTX3 Level Distribution by Number of Miscarriages

The comparison of PTX3 levels across different maternal age groups showed consistently higher PTX3 concentrations in URPL patients compared with controls in all age categories. For example, in women aged 20–25 years, the mean PTX3 level was 13.0 ng/mL in patients compared to 1.5 ng/mL in controls, while in the 26–30 year group, levels were 11.8 ng/mL vs 1.7 ng/mL, respectively. Similar patterns were observed in the 31–35 year group (12.4 vs 1.9 ng/mL) and among women older than 35 years (10.6 vs 1.6 ng/mL). These findings suggest that the elevated PTX3 levels observed in URPL patients are independent of maternal age (Table 6).

Table 6: PTX3 Levels by Maternal Age Groups

During patient recruitment, several cases were excluded based on predefined criteria to minimize confounding variables that could influence pregnancy outcomes. The most common exclusion factor was a history of IUGR or preeclampsia (6 cases), followed by hypertension (5 cases) and smoking (4 cases). Additional exclusions included multifetal pregnancy (3 cases) and preterm premature rupture of membranes (PPROM) (2 cases). These criteria ensured a more homogeneous study population focusing specifically on unexplained recurrent pregnancy loss (Table 7).

Table 7: Summary of Exclusion Reasons

The present study demonstrated that first-trimester maternal serum pentraxin-3 (PTX3) levels were significantly elevated in women with primary unexplained recurrent pregnancy loss (URPL) compared with healthy pregnant controls. Furthermore, PTX3 concentrations increased progressively with the number of previous miscarriages, suggesting that inflammatory activation may correlate with the severity of reproductive loss. These findings support the hypothesis that dysregulated innate immune responses contribute to the pathophysiology of unexplained recurrent pregnancy loss and that PTX3 may represent a promising biomarker for early risk identification [5–8]. The association between inflammation and recurrent pregnancy loss has been extensively documented in recent reproductive immunology research. Several studies have shown that immune imbalance—characterized by altered cytokine expression, impaired regulatory T-cell activity, and increased pro-inflammatory mediators—plays a crucial role in pregnancy failure [6–9]. PTX3 is a long pentraxin family protein that functions as an acute-phase inflammatory mediator and is produced locally at sites of inflammation by endothelial cells, macrophages, and trophoblasts. Unlike classical inflammatory markers synthesized in the liver, PTX3 reflects tissue-specific inflammatory responses, making it particularly relevant in evaluating inflammatory processes occurring at the maternal-fetal interface [7,8]. Our findings are consistent with earlier investigations that identified PTX3 as a potential biomarker in pregnancy-related disorders. Yamashita et al. reported that elevated maternal PTX3 levels were associated with adverse pregnancy outcomes in women with recurrent pregnancy loss, suggesting that abnormal inflammatory signaling may contribute to early pregnancy failure [9]. Similarly, Liu et al. demonstrated that PTX3 is associated with early placental dysfunction in recurrent pregnancy loss, reinforcing the hypothesis that inflammatory pathways and impaired trophoblast invasion may underlie pregnancy loss in affected women [10]. These findings collectively indicate that PTX3 may reflect pathological immune activation occurring during the early stages of pregnancy. The biological plausibility of this relationship is supported by the critical role of inflammatory regulation during implantation and placental development. Successful implantation requires controlled inflammatory signaling, trophoblast invasion, angiogenesis, and immune tolerance. Disruption of these processes may lead to abnormal placentation and early pregnancy failure. PTX3 has been implicated in extracellular matrix remodeling, complement activation, and angiogenesis, suggesting that excessive PTX3 expression may disturb normal placental development and contribute to pregnancy loss [7,11,12,13]. Consequently, elevated PTX3 concentrations observed in URPL patients may indicate excessive inflammatory activity at the maternal-fetal interface. The inflammatory basis of pregnancy loss is further supported by studies investigating other biomarkers associated with miscarriage. For example, Khudhur and Al-Karim reported significantly elevated serum interleukin-15 levels in women with ectopic pregnancy and missed abortion compared with normal pregnancies, highlighting the contribution of inflammatory cytokines in early pregnancy failure [13]. Likewise, Enadi and Abdul-Karim demonstrated that altered serum levels of CA-125 and progesterone were associated with threatened abortion and could serve as potential predictors of early pregnancy loss [14]. These findings emphasize that both inflammatory mediators and hormonal factors interact in determining pregnancy outcomes.

Our study also showed that PTX3 levels increased as the number of previous miscarriages increased. This stepwise increase suggests that PTX3 may reflect disease burden and severity. Recent literature on recurrent pregnancy loss biomarkers has emphasized the importance of identifying markers that not only differentiate affected women from controls but also help predict disease progression and recurrence risk [15-22]. The positive correlation between PTX3 concentration and miscarriage frequency observed in our study therefore supports its potential role as a prognostic biomarker. In addition to recurrent miscarriage, elevated PTX3 levels have been reported in other obstetric complications characterized by placental inflammation and vascular dysfunction. Increased PTX3 concentrations have been observed in preeclampsia, intrauterine growth restriction, and preterm labor, conditions that share similar pathophysiological mechanisms involving endothelial dysfunction and exaggerated inflammatory responses [23-26]. These observations suggest that PTX3 may represent a common inflammatory pathway linking multiple pregnancy complications. The diagnostic performance observed in our study further supports the clinical relevance of PTX3. The optimal cutoff value demonstrated good sensitivity and specificity for identifying URPL cases, suggesting that PTX3 could potentially be incorporated into early pregnancy screening strategies. Recent systematic reviews have highlighted the need for reliable biomarkers capable of improving the diagnostic evaluation of unexplained recurrent pregnancy loss [6,15]. PTX3 may therefore serve as a complementary marker alongside clinical history and other laboratory parameters in assessing women at risk. Despite these promising findings, several limitations must be considered. First, the sample size was relatively small, which may limit the statistical power of the study. Second, the research was conducted at a single center, and therefore the results may not be fully generalizable to other populations. Third, variations in assay methods and laboratory techniques may influence PTX3 measurement and interpretation. Similar limitations have been noted in previous studies investigating biomarkers in recurrent pregnancy loss [27-30]. Future research should focus on large multicenter studies to validate the predictive value of PTX3 and determine standardized cutoff levels. Longitudinal investigations evaluating PTX3 changes throughout pregnancy may also provide valuable insights into the temporal relationship between inflammation and pregnancy outcomes. Additionally, mechanistic studies exploring PTX3-mediated immune pathways could help identify potential therapeutic targets for improving pregnancy outcomes in women with unexplained recurrent pregnancy loss.

Overall, the findings of the present study contribute to the growing body of evidence supporting the role of inflammatory biomarkers in reproductive medicine. Elevated first-trimester PTX3 levels appear to be associated with unexplained recurrent pregnancy loss and may reflect abnormal inflammatory activation during early pregnancy. With further validation, PTX3 may become a useful biomarker for early detection and risk stratification in women with recurrent pregnancy loss.

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