The association between circulating SIGLEC6 and preeclampsia: observational studies of seven cohorts

Abstract

Background: Preeclampsia is a serious complication of pregnancy.

Methods: We did an observational study using seven tissue bank/cohorts to examine the association between circulating SIGLEC6 and preeclampsia. We included samples from participants with preterm disease (delivering <34 weeks gestation in Australia), examined whether levels altered with clinical disease severity (samples collected in South Africa) and whether there were alterations preceding disease onset using samples collected at 15- and 20-weeks' gestation in New Zealand, samples collected between 26 and 34 weeks in the UK and samples collected at 28 or 36 weeks gestation in Australia. Circulating SIGLEC6, sFlt-1, and PlGF were measured via ELISA or a electrochemiluminescence immunoassay platform.

Findings: SIGLEC6 was elevated 9.5-fold (23,397 pg/ml, IQR 16701-32,267) in preterm preeclampsia (<34 weeks gestation), compared to normotensive pregnancies (2441 pg/ml, IQR 871.9-6547; p = 6.3 × 10^-9). SIGLEC6 levels correlated with disease severity: compared to preeclampsia without severe features, SIGLEC6 was raised 1.5-2.5-fold with eclampsia, or preeclampsia with life-threatening complications. There was a stepwise increase in SIGLEC6 with increasing numbers of maternal complications, accentuated when expressed as a SIGLEC6/PlGF ratio (10.7-fold rise with ≥3 maternal complications, versus no complications). Circulating SIGLEC6 concentrations were significantly increased among those later diagnosed with preeclampsia in samples collected at 36 weeks (n = 1032; Australia), 26-34 weeks (n = 235; UK), 28 (n = 283; Australia), and 20 weeks' gestation (n = 1945; New Zealand).

Interpretation: SIGLEC6 is elevated with preeclampsia and levels correlate with disease severity.

Funding: National Health and Medical Research Council (#1065854) and the Norman Beischer Medical Research Foundation. Additional sources of funding for the biobank from South Africa was received from the Swedish Medical Society, Märta Lundqvist Foundation, Swedish Foundation for International Cooperation in Research and Higher Education, Jane and Dan Olssons Foundation, Mercy Perinatal (Australia), the Swedish Research Council (Vetenskaps-rådet), Sweden, and the Center for Clinical Research Dalarna, Sweden. The MAViS study (UK) was funded through National Institute Health Research (NIHR-CS-011-020). MUMS was funded by a St George and Sutherland Medical Research Foundation of Australia grant. Salary or scholarship support was received from: Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG) Taylor Hammond Scholarship to TM; National Health and Medical Research Council Fellowships to ST (#2017897) and DMK (#2008017); Australian Research Council Future Fellowships to TKL (FT230100125) and NJH (FT210100193), Senior Medical Research Fellowship from the Sylvia and Charles Viertel Charitable Foundation Fellowship and a National Heart Foundation Future Leader Fellowship (#105663) to FZM.

Keywords: Biomarker; Placenta; Preeclampsia; Pregnancy; SIGLEC6.

Fig. 1

Explanation of screening pipeline.In silico analysis of publicly available data repositories allowed us to screen for proteins expressed at high levels in the placenta or on the syncytiotrophoblast surface. To date, we have screened 567 such molecules via enzyme linked immunosorbent assay (ELISA). Of these proteins, 302 were excluded due to poor ELISA quality, due to proteins being undetectable in plasma, or because the ELISAs required high volumes of plasma. We have measured 265 of the proteins in a case/cohort of samples at 36 weeks' gestation—examining the outcome of whether the participants later developed preeclampsia or not. Each ELISA was run individually with its own standard curve and QCs (not multiplexed). Of the 265 proteins, 60 individual proteins were identified as significantly different when measured via ELISA (p < 0.05 on students t test or Mann–Whitney U test) and 205 were unchanged. Of the 60, we proceeded to validation to assess whether we could still find significant changes when the molecules were measured in a population cohort (∼1000 unselected samples collected at 36 weeks' gestation). Of the 60, 17 validated in a population cohort (one of those being SIGLEC6) and we have not tested the other 43. This screening pipeline is ongoing and this flow chart shows how many we have screened as of May 2024.

Fig. 2

SIGLEC6 is increased in preterm preeclampsia. Circulating SIGLEC6 was (a) increased in 41 women who birthed at <34 weeks because of preterm preeclampsia compared to 26 women who birthed healthy babies at term. (b) Circulating SIGLEC6 levels did not significantly alter across gestation for the preeclampsia (n = 41) or control group (n = 26). (c) Placental SIGLEC6 mRNA expression from 62 women with preterm preeclampsia was increased compared to 16 gestational age matched, normotensive preterm controls. Placental SIGLEC6 protein from 82 women with preterm preeclampsia was (d, e) non-significantly increased compared to 20 preterm controls when measured via Western blot (p = 0.058), but (f) significantly increased when placental SIGLEC6 protein was measured via ELISA. All samples were matched for gestation at sampling as shown in Tables S1, S2A–S2C. Data expressed as median ± interquartile range and statistically analysed using a Mann–Whitney U test. ∗∗∗∗p < 0.0001. Panel e shows a representative Western blot. PE = preeclampsia, pg = picogramme, ml = millilitre, ug = micrograms.

Fig. 3

SIGLEC6 is increased with disease severity. Relative to participants with preeclampsia and no severe features (n = 111), circulating SIGLEC6 (expressed as multiples of the median (MoM)) was (a) elevated in those who developed eclampsia (n = 36, p = 1.7e10⁻³), severe organ involvement (n = 23, p = 4e10⁻⁴) or severe hypertension (n = 135, p = 1.8e10⁻⁶) but not changed in a small group who had pulmonary oedema (n = 14, p = 0.34). When we grouped all patients according to whether they delivered a fetal growth restricted (FGR) infant or not (b) we found circulating SIGLEC6 was elevated in those delivering with FGR (p = 2.7e10⁻³). There was a modest and significant association with both diastolic (c) and systolic (d) blood pressure (BP) and SIGLEC6 and higher levels were associated with smaller babies (e). Data expressed as median ± interquartile range and statistically analysed using a Kruskal–Wallis with Dunn's multiple comparisons post hoc test (a) or a Mann–Whitney U test (b). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ng = nanogrammes.

Fig. 4

SIGLEC6 at 36 weeks' predicts preeclampsia. Biomarkers were measured at 36 weeks in 41 women who developed preeclampsia compared to 951 who did not. Circulating SIGLEC6 was (a) elevated at 36 weeks' gestation in the women who developed preeclampsia compared to those who did not; an (b) AUC of 0.67. (c) sFlt-1 was also elevated in those who later developed preeclampsia in this cohort; an (d) AUC of 0.79. (e) A ratio of SIGLEC6/PlGF was elevated among those who later developed preeclampsia; (f) an AUC of 0.76. (g) Similarly, the sFlt-1/PlGF ratio was increased in those who later developed preeclampsia relative to those who did not; an (h) AUC of 0.79. Data expressed as median ± interquartile range and statistically analysed using Mann–Whitney U tests. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. AUC = Area under the curve, pg = picogrammes, ng = nanogrammes.

Fig. 5

SIGLEC6, sFlt-1, and PlGF across the second trimester. In samples collected in Melbourne, Australia, circulating SIGLEC6 was (a) elevated at 28 weeks' gestation in 93 women who developed preeclampsia compared to 190 who did not; an (b) AUC of 0.61. (c) sFlt-1 was also increased with preeclampsia in this cohort; an (d) AUC of 0.65. (e) SIGLEC6/PlGF ratio was increased in this cohort; an (f) AUC of 0.66. (g) sFlt-1/PlGF ratio was also increased in this cohort; an (h) AUC of 0.71. In a different cohort (samples from Auckland, New Zealand), circulating SIGLEC6 was (i) elevated at 20 weeks' gestation in 62 women who developed preeclampsia, and 20 women had preeclampsia and birthed small for gestational age infants, compared to 1863 who remained normotensive. The ROC for the preeclampsia/SGA group relative to controls is shown, with an AUC of 0.70. Also in samples collected in Auckland, New Zealand, SIGLEC6 was (j) non-significantly increased at 15 weeks' gestation in 84 women who developed preeclampsia, compared to 1923 who did not (p = 0.058). Data expressed as median ± interquartile range and statistically analysed using or Mann–Whitney U tests or a Kruskal–Wallis with Dunn's multiple comparisons post hoc test (i). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. AUC = Area under the curve, ng = nanogrammes, pg = picogrammes.