Performance of plasma kisspeptin as a biomarker for miscarriage improves with gestational age during the first trimester

Abstract

Objective: To compare the performance of kisspeptin and beta human chorionic gonadotropin (βhCG), both alone and in combination, as biomarkers for miscarriage throughout the first trimester.

Design: Prospective, nested case-control study.

Setting: Tertiary Centre, Queen Charlotte Hospital, London, United Kingdom.

Patient(s): Adult women who had miscarriages (n = 95, 173 samples) and women with healthy pregnancies (n = 265, 557 samples).

Intervention(s): The participants underwent serial ultrasound scans and blood sampling for measurement of plasma kisspeptin and βhCG levels during the first trimester.

Main outcome measure(s): The ability of plasma kisspeptin and βhCG levels to distinguish pregnancies complicated by miscarriage from healthy pregnancies unaffected by miscarriage.

Result(s): Gestation-adjusted levels of circulating kisspeptin and βhCG were lower in samples from women with miscarriages than in women with healthy pregnancies by 79% and 70%, respectively. The area under the receiver-operating characteristic curve for identifying miscarriage during the first trimester was 0.874 (95% confidence interval [CI] 0.844-0.904) for kisspeptin, 0.859 (95% CI 0.820-0.899) for βhCG, and 0.916 (95% CI 0.886-0.946) for the sum of the two markers. The performance of kisspeptin in identifying miscarriage improved with increasing length of gestation, whereas that of βhCG worsened. A decision matrix incorporating kisspeptin, βhCG, and gestational age had 83% to 87% accuracy for the prediction of miscarriage.

Conclusion(s): Plasma kisspeptin is a promising biomarker for miscarriage and provides additional value to βhCG alone, especially during later gestational weeks of the first trimester.

Keywords: Kisspeptin; miscarriage; pregnancy.

Figure 1

Kisspeptin and βhCG levels throughout the first trimester in healthy controls and women with miscarriages. (A) Scatterplot of plasma kisspeptin levels in healthy controls (gray) (n = 265 women providing 545 samples) and women with miscarriages (red) (n = 95 women providing 149 samples) over gestational age (weeks) calculated by LMP, during the first trimester. The data were analyzed by simple linear regression (r² = 0.31 for controls and 0.14 for women with miscarriages). ∗∗∗∗P<.0001 by analysis of covariance. (B) Median (IQR) plasma kisspeptin levels in healthy controls (gray) (n = 265 women providing 545 samples) and women with miscarriages (red) (n = 95 women providing 149 samples) over gestational age (weeks) calculated by LMP, during the first trimester. ∗∗P<.01, ∗∗∗∗P<.0001. (C) Scatterplot of plasma βhCG levels in healthy controls (gray) (n = 265 women providing 557 samples) and women with miscarriages (red) (n = 95 women providing 173 samples) over gestational age (in weeks) calculated by LMP, during the first trimester. (D) Median (IQR) plasma βhCG levels in healthy controls (gray) (n = 265 women providing 557 samples) and women with miscarriages (red) (n = 95 women providing 173 samples) over gestational age (weeks) calculated by LMP, during the first trimester. ∗P<.5, ∗∗P<.01, ∗∗∗∗P<.0001. βhCG = beta human chorionic gonadotropin; IQR = interquartile range; LMP = last menstrual period.

Figure 2

MoM values for plasma kisspeptin and βhCG in healthy controls and women with miscarriages and their diagnostic performance. (A) Scatterplot of median (IQR) MoM of gestation-specific kisspeptin values in healthy controls (n = 265 women providing 545 samples) (gray) and women with miscarriages (n = 95 women providing 149 samples) (red). The groups were compared by the Mann-Whitney U test. ∗∗∗∗P<.0001. (B) Scatterplot of median (IQR) MoM of gestation-specific βhCG values in healthy controls (n = 265 women providing 557 samples) (gray) and women with miscarriages (n = 95 providing 173 samples) (red). The groups were compared by the Mann-Whitney U test. ∗∗∗∗P<.0001. (C) Scatterplot of median (IQR) MoM of gestation-specific kisspeptin and βhCG values in healthy controls (n = 265 women providing 538 samples) (gray) and women with miscarriages (n = 95 women providing 130 samples) (red). The groups were compared by the Mann-Whitney U test. ∗∗∗∗P<.0001. (D–F) ROC curves for MoM of kisspeptin (D), βhCG (E), and kisspeptin and βhCG (F). AuROC = area under the receiver-operating characteristic curve; βhCG = beta human chorionic gonadotropin; IQR = interquartile range; MoM = multiple of median. ROC = receiver-operating characteristic curve.

Figure 3

Proportion of women with miscarriages with different MoM cutoffs and time to diagnosis of miscarriage. (A–C) Mean of the proportion of women with miscarriages with different cutoffs of multiples of gestation-specific median for kisspeptin (A), βhCG (B), and the sum of kisspeptin and βhCG) (C). (D–F) Mean (95% CI) of the multiples of gestation-specific median for kisspeptin (D), βhCG (E), and the sum of kisspeptin and βhCG (F) by the time to confirmation of miscarriage (in weeks). The miscarriage groups were compared by the Kruskal-Wallis test. ∗∗P<.01, ∗∗∗P<.001. βhCG = beta human chorionic gonadotropin; CI = confidence interval; MoM = multiple of median.

Figure 4

ROC curves for plasma levels of kisspeptin, βhCG, and kisspeptin and βhCG for gestational weeks left panel) and eight or more weeks (right panel). For the left panel, the AUCs are 78.4% (kisspeptin), 79.5% (βhCG), and 79.7% (both factors). For the right panel, the AUCs are 90.9% (kisspeptin), 79.6% (βhCG), and 92.3% (both factors). Gestational ages were determined by LMP. AUC = area under the curve; βhCG = beta human chorionic gonadotropin; KP = kisspeptin; LMP = last menstrual period; ROC = receiver-operating characteristic.

Supplementary Figure 1

Comparison of gestational age and plasma kisspeptin levels by Crown-Rump Length and Last Menstrual Period. A. Scatterplot of gestational age as estimated by Crown-Rump Length (CRL) (in days) over gestational age as estimated by Last Menstrual Period (LMP) (in days) in healthy controls (in grey) (n=101 women) and women with miscarriage (in red) (n=81 women). Data was analysed by simple linear regression (r²=0.82 in controls and r²=0.54 in miscarriage); ∗∗∗∗P<0.0001. B, C. Spaghetti plots of plasma kisspeptin with gestational age (in weeks) as estimated by Crown-Rump Length (CRL) (B) and Last Menstrual Period (LMP) (C). Red bars are 95% confidence limits for a mixed-effects linear regression model fitted to the data; blue bars are 95% prediction limits for the same model. Intraclass Correlation Coefficients (ICC) are calculated from the random effects variance for the models. ICC for panel B is 19.9% and ICC for panel C is 30.3%, indicating that 20% - 30% of the variation in kisspeptin can be explained by subject difference.

Supplementary Figure 2

Classification tree presenting the risk of miscarriage on the basis of gestation-adjusted i.e. multiple of median (MoM) of kisspeptin and βhCG levels. The number of samples and the proportion that are from women with control pregnancies, or from women affected by miscarriage is presented in the box. From the ‘start’, the risk of miscarriage can be estimated by following the arrows according to the MoM of plasma kisspeptin, for which the categories are shown in blue. The risk of miscarriage is first categorised according to MoM kisspeptin levels; for example 68.7% of samples with a low MoM kisspeptin level of <0.12 were from women who suffered miscarriage, whereas only 1.5% were if MoM kisspeptin > 1.0. Women with MoM kisspeptin levels between 0.12 and 1.0 were further risk-stratified using MoM βhCG levels (red). The area under receiver operating characteristic curve (auROC) for correct categorization by this decision tree is 0.90 (95% CI: 0.87 – 0.93). MoM, Multiple of gestation-specific median values of women with healthy pregnancies; βhCG, beta chorionic gonadotropin (iU/L).

Supplementary Figure 3

Plasma kisspeptin by type of miscarriage and relationship with βhCG in women with miscarriage. A. Scatterplot median (IQR) of multiples of gestation specific medians of plasma kisspeptin in samples from women with complete (n=12), incomplete (n=9) and missed (n=57) miscarriage. Groups were compared by Kruskal Wallis test with post hoc Dunn’s multiple comparison test. ∗P<0.05, ∗∗P<0.001. B. Scatterplot of plasma kisspeptin versus plasma βhCG levels in women with miscarriage (n= 95 women providing 132 samples). Values analysed by simple linear regression, r²= 0.6

Supplementary Figure 4

Decision trees to predict the risk of miscarriage. Only the initial blood sample taken at first presentation was used to generate this decision tree. The risk of miscarriage (between 0 and 1) is presented within each node (rectangles with rounded corners coloured in either green (starting), blue (decision) or red (terminal). The proportion of all women who would reach that node is presented above each node. At the starting node (green), the risk of miscarriage is 0.28. The arrows can then be followed according to the level of unadjusted βhCG (IU/L), kisspeptin levels (pmol/L) and gestational age by last menstrual period (days). The final miscarriage risk is indicated once the terminal nodes (red) have been reached. For example, a pregnant woman that has a βhCG level <14,000 iU/L will have a 0.81 risk of miscarriage and this risk will be present in 17% of the total study population (Decision Tree A). The left panel (Decision Tree A) has an accuracy for correctly classifying women of 87.4% (95% CI: 83.4% - 90.7%), but this complex model could be susceptible to overfitting (i.e. may perform with less accuracy when used in other datasets). The right panel (Decision Tree B) is a parsimonious (simplified) model with an 83.1% accuracy (95% CI: 78.6% - 86.7%) that is more likely to maintain similar performance when applied to other datasets. βhCG, beta chorionic gonadotropin (iU/L); KP, Kisspeptin (pmol/L).