Pregnancy involves the implantation of products of conception, primarily aiming for fetal growth and development within the uterus. This process leads to significant physiological changes in the mother to support the fetus and placenta. Adverse outcomes like intrauterine fetal death (IUFD), hypertensive disorders (HTD)  often linked to abnormal placentation and inflammation ^(1,2). Despite their prevalence, these risks are frequently overlooked in women's health, highlighting the need for better awareness and preventive strategies. IUFD has a global rate of 18.9 per 1000 live births, while HTD affects around 10% of pregnancies and is influenced by maternal age and obesity ^(3,4). Oxytocin (OT) is a significant peptide hormone involved in stimulating myometrial contractions during pregnancy . Its metabolism involves two primary peptidases: placental leucine aminopeptidase (P-LAP) and post-proline endopeptidase , P-LAP is zinc-dependent enzyme responsible for degrading OT activity , thus maintaining pregnancy. It was initially thought to be placenta-specific, but its distribution is now recognized throughout various tissues, implicating it in various pathophysiological processes ⁽⁵⁾.  P-LAP functions by breaking disulfide bonds in substrates like OT and vasopressin, selectively hydrolyzing those with a unique N-terminal ring structure while not affecting other similar hormones ⁽⁶⁾. Structurally, P-LAP shares considerable identity with insulin-regulated membrane aminopeptidase (IRAP) and consists of three domains: a cytoplasmic domain, a transmembrane domain, and a large extracellular domain, which includes a zinc-binding site essential for its enzymatic function ⁽⁷⁾.  During pregnancy, P-LAP serves a protective role by regulating uterine contractions; its activity increases with gestational age, presumably to counterbalance rising OT levels in the feto-placental-maternal unit. Elevated P-LAP activity in maternal serum is correlated with decreased risk of premature delivery. Notably, P-LAP activity is modulated under pathological conditions such as chorioamnionitis and pre-eclampsia, reflecting its adaptive involvement in counteracting vasopressin-mediated hypertension effect and inflammatory disruptions. Overall, P-LAP acts as a critical homeostatic regulator within the feto-placental-maternal unit, with potential implications for predictive biomarkers in pregnancy-related complications, ensuring both maternal and fetal health ⁽⁸⁾.   The role of progesterone in maintaining pregnancy is well established, once the fetal levels of glucocorticoids rise, enzyme induction begins, affecting progesterone metabolism ⁽⁹⁾.   In the induction of labor, there is a significant increase in oxytocin secretion and receptor density in the uterine muscle as term approaches. Elevated oxytocin levels and heightened uterine sensitivity to this hormone facilitate strong contractions required for fetal expulsion. The exact mechanisms behind uterine sensitivity to oxytocin are not thoroughly understood; however, it is noted that like oxytocin, angiotensin sensitivity in pregnancy increases due to changes in degrading enzymes ⁽⁵⁾. Monitoring P-LAP activity, an oxytocin degrading enzyme, reveals that it progressively rises during late pregnancy, peaking shortly before labor. Several studies indicate that P-LAP does not increase significantly as labor approaches, suggesting that it may no longer be able to balance the rising fetal oxytocin levels. This equilibrium persists until fetal maturation disrupts the hormonal balance, limiting P-LAP release just prior to labor onset ^(5,9). The current study aimed to evaluate the association level of maternal serum placental leucine aminopeptidase (P-LAP) between healthy pregnancies and adverse pregnancy outcomes (intrauterine fetal death, hypertensive disorders of pregnancy) to determine maternal serum placental leucine aminopeptidase (P-LAP) can serve as a predictive biomarker for adverse pregnancy outcomes.

Patients and Methods

 This is a case- control study was Conducted in the Obstetrics department  of Al- khansaa and Al Batool Maternity teaching hospitals between 1st Jan  to 30th June 2025, Performed on 150 pregnant women divided into 3 groups as follows:

Group 1 (Intrauterine Fatal Death IUFD (n-25)): Pregnant women  with intrauterine fetal death ≥ 28 week of gestation.

Group 2 (Hypertensive Disorder of Pregnancy (HTD) (n=25)): Pregnant woman  presented with any hypertensive disorder of Pregnancy including ( preeclampsia ,eclampsia or chronic hypertension with superimposed preeclampsia) ≥ 28 week of gestation.

Group 3 (Control Group (n=100)):Include healthy pregnant women with full term pregnancy without any complication during Pregnancy.

To evaluate maternal Serum placental leucine amino peptidase (P-LAP) levels as predictors of pregnancy outcome.

Inclusion Criteria : Singleton pregnancy (viable and non-viable according to study outcome definition), gestational age between 28 to 41week, confirmed by reliable last menstrual period and/or by first-trimester ultrasonography, any maternal age, any parity, and absence of major congenital fetal anomalies.

Exclusion Criteria : Multiple gestation, uncertain gestational age, pre-existing chronic maternal  diseases unless specifically included in the study design, assisted reproductive technology (ART) pregnancies, placental structural abnormalities , known chromosomal or major congenital fetal anomalies, Rh incompatibility, maternal malignancy, recent administration of medications known to influence placental function (e.g., corticosteroid, anticoagulants, low dose aspirin), sever maternal anemia (Hb less than 8 g /dl).

Ethical approval:

Official approval was obtained by the Scientific Council of Obstetrics and Gynecology specialization/ Iraqi board for medical specializations and  Nineveh  Health directorate.

Verbal permission and written consent were obtained from each participant  prior to data collecting and explain the aim of study and the date were exclusively used for the Purpose of this study.

Data Collection:

All pregnant women included in this study was obtained detailed history , general and obstetrical examination was done and routine and  specific investigation include Trans abdominal ultrasonography, Cardiotocography and laboratory investigations including: hemoglobin (Hb), platelet count (PLT), renal function tests (RFT), liver function tests (LFT).

By collecting  5 ml of whole blood to be send to the private laboratory.

After delivery record information include mode of delivery  (vaginal or Cesarean) and Cesarean whether  elective or emergency and neonatal outcome include newborn sex, APGAR scores in 1 and 5 minute, birth weight, newborn admission to neonatal care unit or not.

Sample collection and assay:

venous blood sample of 5 ml were collected at the time of labors Prior to delivery in all Patients. Initially skin in cleaned with 70 % alcohol, by using disposable plastic syringe ,about 5 ml of blood were withdrawn in sterile gel tube (tube with anticoagulant) following incubated at room temperature for  10-20  minutes, tubes are Centrifuged for 20 min at (2000-3000 rpm) and Serum separated and stored at -60°C or below until the analysis was performed P-LAP concentrations were measured using enzyme linked immunosorbent assay (ELISA) kit. The kit instruction human Leucyl-Cystinyl Aminopeptidase (LNPEP) ELISA kit (Sunlong Biotech Co., Ltd) according to the kit leaflet (3) and the principle applied in this kit is Sandwish enzyme immunoassay.

Statistical analysis:

Data were entered into Microsoft Excel 2010 and analyzed using IBM SPSS version 26. Normality was assessed with the Shapiro–Wilk test, and parametric tests were used when assumptions were met. Means and standard deviations were calculated for numerical data. The nominal data were described by number and percentages. The One-way ANOVA test was used for numerical to find the mean differences among the multiple groups (more than 2 groups) under the study, with post hoc test to find the honestly significant difference among the significant results of one way ANOVA. Student t test used for difference between two means. For the nominal data, Chi square was performed and the Freeman-Halton Exact test was used instead of Chi square test if any cell has an expected value less than 5 for the tables more than 2 by 2.  ROC analysis (Receiver Operating Characteristic) was used as an indicator for S.leucine for discrimination of pregnancy outcomes. The p value ≤0.05 was considered significant.

RESULTS

The current study included 150 pregnant women. Participants were classified into three groups: intrauterine fetal death, hypertensive disorders of pregnancy, and a healthy control group. The comparison of mean maternal age among participants groups was demonstrated in table (1) which revealed that the highest mean age was among group 1 and 2 while the lowest mean age was reported for those among the control group (24.13±5.200 years); the difference in participants groups found to be statistically significant (p=0.000), the post hoc test elicited that the real difference was just between the control group and each of the cases groups. Regarding the BMI, showed a statistically significant difference (p=0.017); Participants in control Group (29.53±2.437) had a significantly lower BMI compared to the studied groups.

Table (1): Comparison of maternal age and anthropometric measurements among the studied groups.

The Obstetrical parameters were compared among the studied groups. Regarding in primigravidity found among the 20.0%, 28.0%, and 35.0% of group 1,2, and control respectively while the multigravida ≥5 found among 36.0%, 48.0%, and 15.0% in that order; this difference was statistically significant (p=0.000). Parity >1 found in 60.0% of group 1, 56.0% of group 2, and 42.0% of control in compared to 20.0%, 36.0%, and 37.0% in nullipara; this difference was statistically significant (p=0.003). The variation of abortion among the studied  groups was statistically significant (p=0.024) as shown in table (2).

Table (2): Comparison of Obstetrical parameters among the studied groups.

Spontaneous delivery in all control group while induction of delivery was more frequent among the group 1 and group 2; this difference was statistically significant (p=0.000). Concerning the mode of delivery, vaginal deliveries were common among the all studied groups in compared to emergency and elective CS; a difference which was statistically not significant, as shown in table (3).

Table (3): Comparison of delivery parameters among the studied groups.

Table (4) demonstrated the comparison of  postpartum hemorrhage and mothers admission among the studied groups. This table revealed  significant statistical variation among the studied groups concerning the history of postpartum hemorrhage. Most of the mothers had no admission to ICU among all the studied groups with a statistical significant difference (p=0.008).

Table (4): Comparison of postpartum hemorrhage and mothers admission among the studied groups.

Regarding the neonatal outcomes, the admission to NCU was reported among the 32.0% of group 2 with a statistically significant difference (p=0.000). All the neonates were alive for all the studied groups apart from group1 and this variation showed a statistically significant difference (p=0.000). The Apgar score at 1 and 5 min showed significant highest mean among the control in compared to the studied groups, as shown in table (5).

Table (5): Comparison of neonatal outcomes among the studied groups.

The Comparison of Serum P-LAP among the studied groups showed a statistically significant difference. The mean level of Serum P-LAP among the group 1 (292.48±92.450) was significantly lower than those among the remaining cases group 2 (632.29±121.633 pg/ml) and the controls which showed the highest mean level (1112.00±145.085 pg/ml), as shown in table (6).

Table (6): Comparison of Serum P-LAP among the studied groups.

Receiver operating characteristic (ROC) analysis showed an area under the curve (AUC) of 0.999 (SE = 0.001), indicating excellent discrimination (p < 0.001).” as shown in table (7) and figure (1). 

Table (7): ROC test and area under the curve for the serum P-LAP.

Figure (1): Area under the curve for the serum P-LAP.

Comparison of Serum P-LAP among the studied groups in relation to gestational age was demonstrated in table (8). This table elicited that the mean Serum P-LAP of patients with 28-32 weeks gestational age among the HTD group (458.03±42.692) was significantly higher (p=0.000) than that among the IUFD group (222.64±51.426). Among the 33-36 weeks gestational age, the difference in mean Serum P-LAP was statistically significant (p=0.000); the mean among the IUFD (316.99±30.393) was lower than those among the HTD (645.52±63.300). Regarding those with gestational age ≥36; the difference in the mean Serum P-LAP was statistically significant (p=0.000); the higher mean level was among the control group while the lowest level was among the IUFD group.

Table (8): Comparison of Serum P-LAP among the studied groups in relation to gestational age.

Direct significant correlations of Serum P-LAP with GA were showed for  IUFD and HTD versus moderate indirect correlation with control group. Regarding the Apgar scores, only HTD group showed strong direction correlations, as shown in table (9). 

Table (9): Correlations of Serum P-LAP among the studied groups in relation to studied variables.

DISCUSSION

This study evaluated maternal serum placental leucine aminopeptidase (P-LAP) as a biomarker for predicting adverse pregnancy outcomes linked to placental dysfunction. Significant differences in maternal age were found among the groups in compared to control with the HTD group having the highest mean age. This aligns with previous research conducted by Glick et al. ⁽¹⁰⁾,  Tagami et al. ⁽¹¹⁾,  and Ito et al., ⁽¹²⁾ indicating that incidence of hypertensive disorders increases with advanced maternal age. The IUFD group also had a higher mean age than controls, reaffirming the established link between maternal age and perinatal complications, including increased risk of intrauterine fetal demise (IUFD). The second statistically significant finding in present study is the difference in Body Mass Index (BMI) among the groups. The mean BMI was highest in the HTD group is  supported by  Zhang et al. ⁽¹³⁾ found that high BMI in early pregnancy was a risk factor for HTD.

The finding of the current study suggested that  highly significant differences in gravidity (p=0.000) and parity (p=0.003). The adverse outcome groups (IUFD and HTD) are dominated by women of high gravidity and parity, while the control group has a much larger proportion of women with fewer or no prior pregnancies, this study  supported by Muniro et al. ⁽¹⁴⁾  and Dasa et al. ⁽¹⁵⁾  confirmed that grand multiparity increased the risk of adverse maternal outcomes. It is important to note that nulliparity is not without risk. Lin et al. ⁽¹⁶⁾   reported that nulliparous women are at a higher risk for different sets of complications  .  The present study found a statistically significant difference in the history of prior abortion (p=0.024). The rate of previous abortion was substantially higher in the  HTD and  IUFD groups compared to the control group. This finding  supported by Hautamäki et al. ⁽¹⁷⁾   study found that women with a history of recurrent pregnancy loss (RPL) have higher risks for hypertensive pregnancy disorders in their subsequent pregnancies.

The finding in this study show highly statistically significant difference in the onset of labor. Spontaneous labor is the hallmark of an uncomplicated, physiological pregnancy that proceeds to term without the need for intervention. Conversely, IOL was the most common mode of onset in the IUFD group and HTD group, this agree with Tsakiridis et al, ⁽¹⁸⁾ recommends that induction of labor should be offered to the patient, as expectant management can increase the risk of maternal complications such as infection or coagulopathy. Also a systematic review by Shami et al. ⁽¹⁹⁾ reviewed various methods for IOL after fetal demise, confirming that induction is the standard management approach.

Hypertensive disorders, particularly preeclampsia, are a leading indication for IOL. The decision to induce is often made to prevent the progression to more severe disease (e.g., eclampsia, HELLP syndrome) or in response to evidence of fetal compromise (e.g., growth restriction, abnormal Doppler studies). This agree with  a study by Swift et al. ⁽²⁰⁾ noted that from 1997 to 2018, hypertensive disorders were an increasingly common indication for IOL, accounting for 7.0% to 11.1% of all inductions. 

The mode of delivery was not statistically significant, the high rate of emergency CS in the HTD group is well-documented. Preeclampsia is a major risk factor for failed induction, non-reassuring fetal status, and placental abruption, all of which can lead to emergency CS. This agree with  Pasokpuckdee et al. ⁽²¹⁾ found that preeclampsia significantly increased the rate of overall cesarean section.  

The post-partum hemorrhage (PPH) was statistically significant, the HTD group demonstrated the highest proportion rate followed by IUFD groups. The PPH rate in the HTD group is (12%) also in line with expectations. While severe preeclampsia is a known risk factor for PPH (often due to coagulopathies like HELLP syndrome or thrombocytopenia), many cases of gestational hypertension are milder ⁽²²⁾. This agree with Cagino et al.,⁽²³⁾ study  noted that only preeclampsia with severe features was associated with an increased risk of clinically meaningful hemorrhage. Lowest PPH Rate in IUFD (8%) this rate is remarkably consistent with study by Pekkola et al. ⁽²⁴⁾ specifically examining maternal complications in stillbirth found a PPH rate of 10%. The slightly lower rate of 8% in this study is well within the expected range. PPH in stillbirth can occur due to retained products of conception or, a consumptive coagulopathy, but it is not as common as in cases of macrosomia or severe preeclampsia.

The maternal ICU admission occurred exclusively in the HTD group. This difference is statistically significant and clinically profound. In obstetrics, the most common indication for ICU admission is severe hypertensive disorders of pregnancy and their sequelae. This agree with  Rossi et al. ⁽²⁵⁾ developed a predictive model for maternal ICU admission and identified gestational hypertension and preeclampsia as major risk factors.

The comparison of neonatal birth weight reveals a highly significant difference. The IUFD and HTD groups were characterized by significantly lower birth weights in contrast to the control, this agree with  Kim et al. ⁽²⁶⁾ found low birth weight infants born to mothers with pregnancy-induced hypertension.

The rates of admission to the Neonatal Intensive Care Unit (NICU) were also highly significantly different. NICU admission was showed in the HTD, while no neonates in the control group required NICU care.

Neonates born to mothers with hypertensive disorders are at high risk for NICU admission due to a combination of factors, including prematurity (often from iatrogenic early delivery), low birth weight, and complications related to placental insufficiency, such as respiratory distress syndrome. This agree with  Kim et al. ⁽²⁶⁾ found that preterm infants of mothers with pregnancy-induced hypertension had an increased risk of neonatal morbidities.

 The Apgar scores, which assess the newborn's condition at 1 and 5 minutes after birth, were significantly lower in the HTD group compared to the control group. This agree with Simpson et al, ⁽²⁷⁾  reported that an Apgar score below 7 at 5 minutes is considered abnormal and indicates that the newborn is having difficulty adapting to extrauterine life. The lower scores in the HTD group are a direct reflection of the neonatal morbidities. Factors such as prematurity, respiratory distress and birth trauma can all lead to lower Apgar scores.

This study  suggested  the central finding of this research  the comparison of maternal serum Placental Leucine Aminopeptidase (P-LAP) levels across the three study groups. The results show a highly statistically significant difference, graded reduction in Serum P-LAP levels that corresponds directly to the severity of the pregnancy outcome. Control Group had the highest Serum P-LAP levels, representing the normal physiological state. HTD Group  showed significantly reduced in Serum P-LAP levels compared to controls. IUFD Group had the lowest Serum P-LAP levels from other groups. These results are remarkably consistent with  landmark study by Tian et al., ⁽²⁸⁾ which investigated the same three groups and found an identical pattern of Serum P-LAP reduction. Normal Pregnancy Serum P-LAP is produced by the placenta and its levels are known to rise progressively throughout a healthy pregnancy. The high levels in the control group are therefore expected ⁽²⁹⁾.

In this study the moderately reduced levels of Serum P-LAP in the HTD group are indicative of placental dysfunction. As shown in  Mizutani et al. ⁽³⁰⁾ as the severity of preeclampsia progresses, Serum P-LAP decreases due to placental damage.

Extremely Low Levels of Serum P-LAP in IUFD group signify severe placental failure. The placenta is the source of Serum P-LAP, and its near-absence in the maternal serum is a direct biochemical reflection of a non-functioning or deceased feto-placental unit. This agree with Tian et al. ⁽²⁸⁾ found that serum P-LAP was a highly sensitive (100%) and specific (78.9%) predictor of fetal death.

In this study the corresponding  ROC curve represent the statistical culmination of the study's primary aim: to evaluate serum P-LAP as a biomarker. The result is striking: an Area Under the Curve (AUC) of 0.999, which is exceptionally high and indicates that serum P-LAP has near-perfect discriminatory ability. Given the dramatic drop in serum P-LAP levels in the IUFD group, this ROC analysis most likely evaluates the ability of serum P-LAP to diagnose Intrauterine Fetal Death (IUFD). In this context, an AUC of 0.999 means that if you were to pick one random patient with IUFD and one random patient from the other groups, there is a 99.9% chance that the IUFD patient would have a lower serum P-LAP level. This finding is strongly supported by  Tian et al.  al  ⁽²⁸⁾. Also Mizutani et al, ⁽³⁰⁾ serum P-LAP is produced by the placenta. Therefore, in cases of fetal demise, the cessation of placental function leads to a collapse in serum P-LAP levels, making it a direct biochemical indicator of placental and fetal death. At the cutoff of 812.7566 pg/ml, serum P-LAP demonstrates excellent performance across all key metrics Sensitivity 99.0%, the test correctly identifies 99% of patients with the adverse outcome (likely IUFD), meaning there are very few false negatives. It is highly reliable for not missing the condition. Specificity  97.3%, the test correctly identifies 97.3% of healthy patients, indicating a very low rate of false positives. This is crucial to avoid unnecessary anxiety and interventions. Positive Predictive Value (PPV)  98.0%, a positive result (serum P-LAP< 812.76 pg/ml) carries a 98% probability that the patient truly has the adverse condition. Negative Predictive Value (NPV)  96.3%, a negative result (serum P-LAP ≥ 812.76 pg/ml) provides a 96.3% probability that the patient is free of the condition, making it a powerful "rule-out" test. Accuracy: 98.15%  the overall correctness of the test is exceptionally high. This agree with the landmark study by Tian et al. ⁽²⁸⁾ also identified a cutoff for serum P-LAP in predicting fetal death, reporting a sensitivity of 100% but a much lower specificity of 78.9%.

In the current study, significant variations in Serum P-LAP levels between groups according to gestational age were discovered by the investigation. The mean Serum P-LAP levels in the HTD group at 28–32 weeks were substantially greater than those in the IUFD group. IUFD was significantly lower than HTD group during 33–36 weeks with significance at p=0.000. The control group had the greatest mean Serum P-LAP at gestational age ≥37 weeks, whereas IUFD had the lowest. There is insufficient evidence in the provided literature to directly compare Serum P-LAP at 28–32 weeks of gestation among IUFD, gestational hypertension, and healthy control groups. None of the supplied abstracts report Serum P-LAP concentrations or statistically tested differences for all three groups specifically within the 28–32 week GA window.

The gestational hypertension versus control metabolomics study in the corpus sampled at mid-gestation and therefore does not provide Serum P-LAP data in HTD cases.  In late-third-trimester (33–36 weeks) Serum P-LAP comparison for gestational hypertension or intrauterine fetal death, were not available. The IUFD-focused paper in the corpus reports clinical parameters (blood pressure, urine protein, renal markers). In pregnancies with and without IUFD but does not report amino acid or Serum P-LAP measurements ^(31,32).

In the current study, The IUFD and HTD groups showed significant associations between Serum P-LAP and GA, but the control group showed a moderate indirect correlation. In terms of Apgar ratings, only the HTD group showed significant directional relationships. Longitudinal studies conducted by Wang et al.,⁽³³⁾  and Shirota et al., ⁽³⁴⁾ done in healthy pregnancies report decreases in Serum P-LAP with advancing gestation, consistent with an inverse relation between Serum P-LAP and gestational age in controls. Several studies as Mokkala et al., ⁽³⁵⁾ and Li et al., ⁽³⁶⁾ reported higher Serum P-LAP early in pregnancy and changes across trimesters, but none in the supplied set state “no significant correlation” of Serum P-LAP with gestational age within a GDM subgroup in the exact terms requested. A study conducted by Razo-Azamar et al., ⁽³⁷⁾ among newborns of hypertensive mothers reports increased preterm birth and lower Apgar scores among cases but does not report maternal or neonatal Serum P-LAP measurements or correlations with gestational age or Apgar.

CONCLUSION

The study concluded that there was a notable distinctions included maternal age, BMI, Obstetrical history (gravidity, parity, abortion) and lab values (blood urea, serum creatinine, uric acid, RBS). The neonatal outcomes  varied  with case groups showing lower birth weights and Apgar scores, and higher NCU admission rates compared to controls. The P-LAP level among IUFD group was the lowest than HTD, while the control group reported the highest level with significant statistical difference. There is significant positive correlation between serum P-LAP level and gestational age among IUFD and HTD group, while there was significant negative correlation in control group. The cut-off point of serum P-LAP for discrimination of pregnancy with complicated groups from control healthy pregnancy was 812.7566 with high validity indexes.

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