An important objective of this study was to examine whether serum LHCGR could increase the screening efficiency of PAPP-A plus free--βhCG which are currently used as first trimester biochemical markers together with NT for prenatal risk assessment of fetal aneuploidy. In order to compare the relative screening efficiencies in this study, first the DR and FPR at fixed cut-off values for PAPP-A (≤0.5 MoM), βhCG (≥1.7 MoM), hCG-sLHCGR (≤2.0 & ≥ 20.0 MoM) were calculated. Out of all combinations, the DR for Down’s syndrome with PAPP-A plus hCG-sLHCGR was highest (57.5%) with FPR of 2.3%. These results were comparable (DR, 58.1% and FPR, 4.5%) to our published data on 43 Down’s samples from two sources . The additive effect of hCG-sLHCGR on PAPP-A and βhCG measurement was 35%, compared to 21% in published data, . The additive effect is defined as T21 pregnancies identified by the PAPP-A + hCG-sLHCGR which could not be detected by conventional PAPP-A and βhCG measurements. Additionally, hCG-sLHCGR, in combination with PAPP-A, βhCG and NT (≥.2.0 MoM), detected 95% (38/40) DS pregnancies. Together, the data presented here demonstrate that serum sLHCGR/hCG-sLHCGR alone or in combination with existing markers can increase the DR and reduce the FPR in Down’s screening.A limitation of this study is that the cut-off value for sLHCGR:hCG-LHCGR ratio of ≥ 2.0 was established on analysis of samples derived from a single center (Figure 1). Therefore, it must be emphasized that larger population-based future studies, involving both low and high-risk groups, are required to establish the cut-off values for serum sLHCG/hCG-sLHCGR to detect DS.
Currently, the routine non-invasive prenatal testing (NIPT) involves the biochemical screening of maternal serum biomarkers (PAPP-A and free - βhCG) at 9–14 wks of gestation as well the measurement of fetal NT. The algorithms based on these results, and other parameters including maternal age, body mass index (BMI), parity (twin or singleton) etc., are used to assess the risk for fetal aneuploidy. The biochemical and NT testing together could detect 79-90% of trisomy 21 at a FPR of 5% [1, 2]. The screen-positive pregnancies, following initial risk assessment, are referred to more invasive genetic and molecular testing by CVS/amniocentesis for definitive diagnosis of aneuploidy or other chromosomal abnormalities.
Current guidelines on NIPT proposed by the International Society for Prenatal Diagnosis and others [11–13] support the idea of cfDNA testing on ‘high-risk’ pregnancies only. The high-risk was defined on the basis of maternal age (≥35 yrs), screen-positives by biochemical and ultrasound testing, history of aneuploidy and parental balanced Robertsonian translocation associated with trisomy 13 and 21 . In the absence of sufficient validation of the cfDNA testing for fetal aneuploidy, it should be considered as ‘advanced screening test’ and is not fully diagnostic. Additionally, the current cfDNA testing for aneuploidy is insufficient to account for half the chromosomal abnormalities detected by molecular analysis of samples derived from CVS or amniocentesis .
While non-invasive fetal DNA sequencing provides the highest sensitivity for T21 screening, a major disadvantage is the cost of introducing fetal DNA sequencing as a universal screening method for low as well as high risk populations. As discussed recently [15, 16], a cost-effective prenatal screening strategy would initially employ the traditional first trimester screening and subsequently analyze only the high-risk pregnancies using the relatively expensive DNA-sequencing method. Therefore, increased sensitivity with simultaneous reduction in false positive rates in conventional prenatal screening could be the most economical avenue for successfully implementing the fetal DNA sequencing scheme for the diagnosis of trisomic 21 pregnancies in the general population.
Compared to the fetal DNA sequencing scheme, which is highly specific and sensitive [4, 5, 15, 16], sLHCGR biomarkers are more broad-ranging. The hormone hCG is the earliest embryonic signal which directly modulates placental growth, angiogenesis and fetal development. Our current and previous analyses of >1000 pregnancies suggest that hCG functions are partly regulated by circulating sLHCGR. The serum sLHCGR, at intermediate concentrations appears to be necessary for maintaining normal pregnancy. However, soluble LHCGR at very low and extremely high concentrations are strongly associated with adverse pregnancy outcome. The dramatic reduction of full-length LHCGR expression in Down’s syndrome chorionic villi [6, 7] points toward a physiological role for sLHCGR in hCG signalling at early pregnancy. While the precise nature of this role is not known, the impact of high serum LHCGR in pregnancy could be two-fold: reduced hCG bioactivity and aberrant systemic vasculo-endothelial and immune activation.
Given the fundamental role of hormone hCG and its receptor LHCGR throughout pregnancy, it is perhaps to be expected that measurement of sLHCGR forms may also detect other pregnancy pathologies, in addition to being a useful first trimester biochemical adjunct for the detection of DS. Indeed, sLHCGR forms have shown preliminary success as predictive diagnostics for a wide range of pregnancy pathologies including miscarriage, preeclampsia and pre-term delivery. The sLHCGR system may therefore complement, rather than directly compete with, existing and emerging technologies by providing early indication of those at most risk. It should be noted however that the diagnostic capacity of serum sLHCGR forms as biomarkers for other pregnancy pathologies, has yet to be extensively explored and requires full clinical validation.