Assessment of kisspeptin and trappin level in Iraqi women with early pregnancy loss: a cross sectional study

Introduction

Effective serological miscarriage indices can predict pregnancy outcomes, provide rescue scheduling, and alleviate patient and family stress. The determination of fetal viability is established through the sequential assessment of beta-human chorionic gonadotropin (hCG) concentrations in the bloodstream.¹ Blood beta-hCG levels increased in 20% of miscarriages. However, miscarriage diagnosis requires other signs.² Approximately 10–20% of clinical pregnancies end in miscarriage, with the most significant rate occurring within the first 12 weeks.^3,4 Identifying early pregnancy survival relies on the assessment of serum hCG and transvaginal ultrasound (TVUS). It should be noted that a consistently increasing hCG level is not always indicative of the possibility of pregnancy in women experiencing first-trimester blood loss.^3,5

Miscarriage is the most common complication of early pregnancy, affecting 20% of identified pregnancies and having potentially detrimental effects on expectant women’s health.^3,6,7 As some miscarriages are irreversible (except for threatened miscarriages), prevention is likely the only way to resolve this issue, and it may be possible to establish biochemical indicators with high diagnostic accuracy to predict or diagnose miscarriages.⁸ For example, evidence suggests that plasma or serum kisspeptin (kisspeptin-10 or kisspeptin-54) levels can distinguish spontaneous miscarriage from intrauterine pregnancy at diagnosis with equal or greater accuracy than plasma or serum progesterone levels.⁶

Biomarkers are routinely employed in current healthcare to improve diagnosis, disease or condition prognosis, and treatment outcome evaluation.⁹ Owing to its putative regulatory role in placentation and trophoblast function, kisspeptin is an emerging biomarker for distinguishing viable pregnancies.^3,7,10 Considerable interest has been devoted to evaluating its potential as a novel aspect of pregnancy.¹¹ A large-scale investigation to determine the typical serum or plasma kisspeptin levels throughout gestation at various gestational ages will aid in diagnosing or predicting these diseases or neonatal health. The kisspeptin assay can also be compared to other pregnancy outcome indicators, such as hCG or their combinations.¹²

Recent research indicates kisspeptin may influence placentation, ovulation, and puberty.¹³ Significant amounts of kisspeptin and its receptor are generated in the human placenta during gestation.^14–16 According to previous research, kisspeptin can potentially function as a biomarker for ectopic pregnancies.^7,15 Despite this, a recent editorial argued that additional evidence is necessary to corroborate its utility in miscarriage diagnosis.¹⁵ Kisspeptins, a family of neuropeptides, are crucial for female fertility and puberty. Plasma kisspeptin levels increase during pregnancy due to placental production. Plasma kisspeptin levels can be used to detect pre-eclampsia, fetal development, gestational trophoblastic neoplasia (GTN), and miscarriage.¹² Kisspeptin may stimulate oxytocin secretion during full-term pregnancy, facilitating labor.¹⁷ Pregnancy increases kisspeptin levels supplied by the placenta. The role of kisspeptin in fertility, puberty abnormalities, hypogonadotropic hypogonadism, and resistance-related diseases (insulin), such as type 2 diabetes, obesity, and polycystic ovary syndrome, has been intensively studied. Around the world, pregnancy-related diabetes mellitus, pulmonary embolism (PE), premature delivery, neonatal growth retardation, or spontaneous abortion impacts roughly 20% of pregnant women.¹⁸ Serum and plasma are used in clinical assays; however, the serum is more sensitive for biomarker detection.¹⁹ Serum kisspeptin levels have been reported to be minimal in non-pregnant individuals but are significantly elevated in viable pregnancies. In addition, the assay for serum kisspeptin was stable, in contrast to previous studies that revealed that serum kisspeptin degrades rapidly.²⁰

Kisspeptin levels also depend on sample collection, processing, and storage. If kisspeptin levels are to be used as a pregnancy indicator, a standard sample processing method would need to be developed. Since all kisspeptins strongly activate the KISS1R,²¹ multiple detections of these peptides are preferable for a single detection. Although a highly sensitive and specific method was used to identify kisspeptin grades (Kisspeptin-(10-14-54)), kisspeptin-13 levels were omitted.¹⁷ The kisspeptin ratio in the plasma or serum has not yet been investigated. Although there was a significant increase in either serum or plasma concentrations of kisspeptin at the start of the third trimester of pregnancy,^7,22,23 the onset of plasma kisspeptin levels remains unclear. Knowing how kisspeptin (serum or plasma) levels compare with hCG levels regarding sensitivity and specificity for detecting early pregnancy would be interesting.

Further research is needed to address these concerns.¹⁷ Trapping-2 (trappin), also known as pre-elafin, an endogenous inhibitor of neutrophil serine proteases, plays an essential role in maintaining the production of proteins, mainly through inflammatory reactions, and in conjunction with secretory leukocyte proteinase inhibitor, a protein with a similar structural design.²⁴ Recent data indicate that trappin-2 exhibits antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa.²⁴ Simultaneously, previous research has found that secretory leukocyte proteinase inhibitors exhibit antimicrobial and antifungal activities.²⁵ Trappin-2/Elafin (T-2/E) has been shown to have antimicrobial properties against fungi as well as Gram-negative and Gram-positive bacteria.²⁵ T-2/E is unusual because it may act as a secreted and cell-associated protein. Trappin-2 is a T-2/E precursor with a TSBD (transglutaminase substrate-binding domain) that allows T-2/E to be removed from the mature molecule.²⁵ The TSBD participates in covalent binding to extracellular matrix proteins such as collagen, IV laminins, elastin, fibrinogen, and fibronectin.^26,27 The soluble fragmented form may function at a distance, whereas the insoluble form may provide local protection from endogenous protease activity. T-2/E has been associated with psoriasis, other inflammatory skin illnesses, and chronic obstructive pulmonary disease (COPD).²⁸ The objective of the present study was to assess the capacity of kisspeptin, trappin, and hCG concentrations in Iraqi females who experienced a premature loss of pregnancy to distinguish between possible pregnancies during the initial trimester and early pregnancy loss (EPL) in a group of women with symptomatic pregnancy.²⁴

Methods

Sample size

The sample size was determined and computed using the computer application G*Power 3.1.9.7 (RRID: SCR 013726). The smallest total sample size was 96 patients, with an effect size of 0.35 and 95% power at a two-tailed alpha of 0.05 and a 95% confidence interval (f). Only 90 were selected for the research, while the rest fulfilled the exclusion criteria (Figure 1).

Figure 1. Study participants' flowchart.

Results

Table 2 displays the demographic details of the study population. The female participants in both cohorts belonged to the age bracket of 20 to 38 years.³⁴ The mean age for the patient women (PW) was significantly older than that of the control women (CW). The mean age of CW was 28.33 years (95% CI: 26.42–30.25), while the mean age of PW was 28.82 years (95% CI: 27.09–30.54). A significant statistical difference was observed in the maternal age among the groups. The study reports the mean gestational age for crown-rump length (CRL) and biparietal diameter (BPD) measurements. The mean CRL gestational age was 8.70 weeks with a 95% confidence interval (CI) ranging from 7.41 to 9.99 weeks. The mean BPD gestational age was 9.47 weeks, with a 95% CI ranging from 8.83 to 10.10 weeks. A significant statistical difference in gestational age was observed among the groups. Table 1 compares the median serum concentrations of kisspeptin, trappin, and hCG between the PW and CW groups. The results indicate that the median serum kisspeptin levels were significantly lower in the PW group (2.07867 ng/mL, 95% CI: 1.99625–2.16109) compared to the CW group (mean: 10.11957 ng/mL, 95% CI: 9.21594–11.02319), as depicted in Figure 2A. The results indicate that the levels of trappin were comparatively lower in the PW group (median 1.07548 ng/mL, 95% CI (.97873–1.17224)) in comparison to the CW group (mean 3.12220 ng/mL, 95% CI (2.90860–3.33580)) as illustrated in Figure 2B.

Figure 2. Stem and leaf plots for (A) kisspeptin, (B) trappin, and (C) hCG: human chorionic gonadotrophin.

hCG levels were significantly higher in the CW group (median, 93.89860 mIU/mL; 95% CI (87.82227–99.97493) than in the PW group (median, 15.67363 mIU/mL; 95% CI (13.19573–18.15154) (Figure 2C). Nevertheless, notable variations were detected in beta-hCG as compared to kisspeptin and trappin.² Nevertheless, the dissimilarities pertaining to trappin were not as conspicuous as those observed for hCG. This is evidenced by the linear regression analysis conducted on kisspeptin, trappin, hCG, and covariates. The analysis revealed that the model results solely consisted of the intercept, indicating that no effects were chosen. A forward stepwise linear regression analysis was conducted to ascertain the significance of kisspeptin and trappin in conjunction with hCG. The findings of the study revealed that kisspeptin exhibited the highest significance in relation to hCG, while trappin demonstrated the lowest significance, as illustrated in Figure 3 through the application of linear regression analysis.

Figure 3. Linear regression analysis to predict the relationship between kisspeptin and trappin concerning human chorionic gonadotrophin.

The accuracy of hCG, kisspeptin, and trappin in distinguishing EPL pregnancy was shown to be excellent through the examination of receiver operating characteristic (ROC) curves (Figure 4). According to the ROC curve analysis, kisspeptin exhibited superior diagnostic performance in relation to EPL compared to trappin.²

Figure 4. Receiver operating characteristic (ROC) curve analysis of the performance of serum (kisspeptin and trappin) and hCG: human chorionic gonadotrophin.

Discussion

The objective of this investigation was to evaluate the diagnostic significance of serum kisspeptin, serum trappin, and serum beta-hCG during the initial weeks of conception in discerning the dissimilarities between women whose pregnancies will result in miscarriage and those whose pregnancies are anticipated to be viable. Nevertheless, it seems that neither exclusive nor consecutive maternal kisspeptin and trappin assessments exhibit a greater prognostic value for abortion than beta-hCG.²⁸ Early pregnancy loss, commonly known as miscarriage, is a distressing occurrence that usually takes place prior to the completion of 12 weeks of the gestational period.²⁹ The identification of pregnancies that are at a heightened risk of abortion necessitates uncomplicated and reliable testing methods. Several hormone tests, including beta-hCG and progesterone, have been developed to aid in predicting pregnancy outcomes.³⁰ Apart from beta-hCG, no other marker has been validated as an accurate predictor of early pregnancy loss. Kisspeptin has been identified as a promising biomarker for discerning viable pregnancy due to its potential regulatory function in trophoblast activity and placentation.

However, the effectiveness of its usage as a serum indicator during the initial stages of pregnancy lacks clarity and necessitates further validation through a prospective cohort study prior to its implementation in clinical settings.²⁸ Kisspeptin is a peptide hormone that is genetically encoded by the KISS1 gene. The polypeptide precursor of Kisspeptin-54, consisting of 145 amino acids, undergoes cleavage and hydrolysis to yield various smaller human variants that exhibit physiological activity. Kisspeptin-10 and other forms of kisspeptin exhibit identical C-terminal amino acid sequences consisting of 10 amino acids.¹⁴ In order to ascertain the reliability of kisspeptin as a potential indicator of miscarriage, a comprehensive analysis of all functional variants was conducted on the specimens. Two prior studies^3,7 have demonstrated that plasma kisspeptin is a reliable biomarker for miscarriage in pregnant women with a gestational period of at least 6 weeks, and in some cases, it has been found to be more effective than hCG. The present investigation conducted a comparative analysis of kisspeptin, beta-hCG, and trappin as predictors of pregnancy in patients experiencing early first-trimester EPL, as reported previously.²⁸ In accordance with prior studies, it was observed that females who had clinical pregnancies exhibited elevated levels of serum kisspeptin in comparison to those who underwent biochemical pregnancy loss or were unable to conceive. In contradistinction to the aforementioned research, our study revealed that kisspeptin exhibited superior diagnostic efficacy as compared to beta-hCG in the prognostication of miscarriage.

The observed dissimilarities between our results with those of prior research may be attributed to variances in detection timing and ethnic diversity. The markers were assessed during the pregnancy testing phase, which occurred notably before the onset of the sixth week of gestation, during which the beta-hCG and kisspeptin expression patterns were ascending. The clinical outcomes may be influenced by the diverse bioactive forms of kisspeptin and their corresponding clinical manifestations. For example, Sullivan-Pyke et al.³ conducted a study investigating the serum kisspeptin-54 level in women presenting to the emergency department with symptoms of bleeding, discomfort, or both. The study revealed a noteworthy correlation between kisspeptin and beta-hCG in women who experienced unplanned abortions, while no such association was observed in pregnant women.

Additionally, the administration of supplementary progesterone to our in vitro fertilization (IVF) group subsequent to embryo transfer may have impeded the production of kisspeptin. It is plausible to consider that IVF activation protocols may have an impact on the levels of serum kisspeptin.²⁸ After pregnancy, there is extensive production of kisspeptin by placental syncytiotrophoblasts. The available evidence indicates that kisspeptin plays a critical role in regulating pregnancy-associated occurrences, including the process of embryo implantation. The successful establishment of placentation and maintenance of a healthy pregnancy is contingent upon the essential process of extravillous trophoblasts penetrating the maternal uterine wall. There exist noteworthy similarities between invasive cytotrophoblasts and invasive cancer cells.³¹

Contrary to the unregulated invasion of tumors, the interaction between trophoblasts and uterine cells is subject to strict temporal and spatial regulation. According to previous studies, it has been observed that kisspeptin has the ability to regulate reactions by inhibiting the activity of matrix metalloproteinases.^10,15,32 Furthermore, it has been observed that kisspeptin facilitates the adherence of extravillous trophoblasts to type I collagen through the activation of ERK1/2 and protein kinase C signaling pathways. This finding provides evidence in favor of the hypothesis that kisspeptin plays a role in regulating normal placentation by promoting adhesion of extravillous trophoblasts to the substrate, thereby limiting their migration and invasion.²⁸ It is suggested that kisspeptin has the potential to initiate immune tolerance in pregnant women. The results of the linear regression analysis indicated a significant correlation between the levels of kisspeptin and hCG. The findings suggest that hCG and kisspeptin may be separate biological pathways linked to the initiation of early pregnancy. Thus, it is plausible that kisspeptin could offer distinct information that may augment diagnostic accuracy beyond the utilization of hCG in isolation.³

The current investigation revealed that the levels of trappin were comparatively less significant in relation to hCG when compared to kisspeptin levels. However, there was a notable difference in the surge of levels between the two groups. According to a study conducted by Abbott et al.,³¹ the levels of trappin were found to be three times higher in women who eventually experienced cervical shortening and premature birth compared to the control group. The present study reveals that the concentration of Trappin-2 in the CW group exhibited a twofold increase. The present research provides empirical evidence in support of the conjecture that Trappin-2 levels are significantly diminished in asymptomatic women with a high risk of developing a certain condition, as opposed to those with a lower risk. The detection of Trappin-2 has been utilized in a limited number of published studies as a sole predictor of EPL. The predictive accuracy of trappin for preterm birth in high-risk asymptomatic individuals is notably higher during the 14–16 weeks of the gestation period compared to later stages of pregnancy. The present investigation aimed to compare the prognostic precision of heightened Trappin-2 concentrations with other predictors that have been examined in the scholarly literature.³³

Trappin exhibits favorable comparability with other predictors in regards to accuracy, ease of sample acquisition, safety, and non-intrusiveness. This predictive model has the ability to anticipate the occurrence of spontaneous preterm birth (PTB) during the early stages of pregnancy, with a lead time of approximately 6–8 weeks in comparison to other predictive models. Trappin levels can be utilized to differentiate asymptomatic high-risk women during the early stages of the second trimester. This provides a sufficient duration for the implementation of preventive measures, interventions, or the transfer of the patient to a specialized medical facility. Furthermore, in the event of the availability of a commercial test product, healthcare professionals can utilize Trappin-2 to detect women with abnormal cervical cells (ANC) who are at a heightened risk. Future multicenter studies are planned to assess the predictive capacity of PTB in the context of universal screening.³³

Developing a prompt screening examination to identify patients with a heightened susceptibility to EPL (miscarriage) would be beneficial for various reasons. Miscarriage is an agonizing experience for the patient, and a prognostic test with a negative outcome could provide patients with better psychological preparation. In the interim, this measure will curtail the superfluous elongation of progesterone supplementation. The determination of early pregnancy viability is reliant on the variations in sequential serum beta-hCG levels and transvaginal ultrasound observations. The investigation of intra-individual differential variations in kisspeptin over time could potentially serve as a noteworthy biomarker for risk assessment. However, further research is required to validate this claim, particularly over a more extended time frame during early pregnancy than what was observed in our study. The integration of beta-hCG and kisspeptin assays may enhance the precision of diagnoses in clinical contexts. In order to develop a precise predictive tool, it is necessary to validate our results in larger study populations and across multiple time points during the early stages of pregnancy.

The study’s strengths are attributed to the timely collection of serum, which serves to authenticate the measurement’s efficacy in patients exhibiting symptomatic pregnancies during the initial stages of gestation. Furthermore, the examination of the kisspeptin assay in a potential cohort with precisely defined phenotypes enables the validation of outcomes, albeit with inherent selection bias. Two limitations were identified in the study, namely the cross-sectional nature and limited sample size. Although the sample size is limited, the utilization of a case-control design featuring patients with well-defined phenotypes is suitable for the characterization of biomarkers. This facilitates the identification of variations in serum kisspeptin levels among distinct cohorts.

Conclusion

The implementation of an initial screening examination to identify individuals who are susceptible to abortion may offer benefits in terms of supplementary psychological assistance and therapeutic intervention. The present investigation provided evidence that kisspeptin can be identified in the serum of early pregnancy and exhibits superior discriminatory ability compared to trappin in distinguishing between early pregnancy loss and human chorionic gonadotropin. The predictive accuracy of pregnancy outcomes is comparatively lower with sequential measurements of serum kisspeptin levels as compared to hCG. An inverse correlation was observed between elevated levels of kisspeptin and the incidence of miscarriage. Kisspeptin has been identified as a potential indicator of placental function. However, there is a lack of adequate data regarding the levels of circulating kisspeptin in women experiencing antenatal complications during the first trimester. Additional investigation is necessary to determine the potential utility of serum kisspeptin levels as a diagnostic and prognostic tool for assessing the well-being of expectant mothers and their offspring. Despite this, kisspeptin exhibits potential as a biomarker for assessing the viability of pregnancy and could prove to be a valuable tool in the development of a precise diagnostic test for early pregnancy prognosis.