Here we describe the development and use of two ELISA assays that quantitatively measure sLHCGR and sLHCGR in complex with LH (LH-sLHCGR) in human serum from women seeking fertility treatment. In addition to serum, these ELISA assays could quantitatively measure sLHCGR and LH-sLHCGR in human plasma and follicular fluids.
Serum AMH is increasingly used in fertility treatment as an indicator of ovarian reserve. Very low (0-4 pmol/L) and very high (≥45 pmol/L) AMH concentrations are associated with reduced oocyte yields and PCO (risk of OHSS) following COS, respectively . Our data demonstrate the expected correlation between AMH levels and oocyte yield in women who exhibited OHSS and had all embryos frozen. However, high AMH (> 39 pmol/L) was a positive predictor for only 52% (12/23) of these patients; the remaining 48% (11/23) had AMH values ranging from 7.6-35 pmol/L. Regarding pregnancy, like many other reports [23–26], we found no correlation between AMH and the embryo implantation potential. We have described three categories of patients seeking fertility treatment with respect to the basal (pre-treatment) serum concentrations of sLHCGR/LH-sLHCGR: undetectable to very low (low), intermediate (optimal) and very high. Patients who were high (oocytes ≥15) and low responders (oocytes ≤7), had fresh transfers and became pregnant had soluble receptor levels that were significantly lower than those who did not become pregnant. Comparing pregnancy rates in low and high responders we found that the levels of LH complexed with sLHCGR were low in those who became pregnant (low and high responders) and higher in those who did not become pregnant. Although embryos were not transferred, the OHSS group (oocytes ≥15) also had low sLHCGR/LH-sLHCGR and FSH:LH ratio. A comparison of pregnancy rates among those producing an intermediate number of oocytes (8-14) revealed the opposite; those who became pregnant had higher levels of LH complexed to sLHCGR than did those who did not become pregnant. The results for complexed LH-sLHCGR amongst intermediate responders were not considered statistically significant and would require a larger study to verify a significant difference. Nevertheless, we believe that this observation merits discussion because, if verified, it could indicate that an optimum saturation level of sLHCGR that correlates with oocyte yields, is important for pregnancy. Moreover, if verified, this result might suggest an important biological function for sLHCGR in modulating LH availability, e.g. by acting as an LH reservoir as observed for IL-4 and growth hormone and tumor necrosis factor receptors [27–30]
Given that the production of soluble polypeptide hormone and cytokine receptors is regulated by alternative splicing, protease activation, secretion of membrane vesicles , ligand-mediated receptor activation  and stress , the higher concentrations of the sLHCGR/LH-sLHCGR that we have observed could be attributed to infection, endometriosis, ovarian and adrenal pathology, obesity, insulin resistance and other metabolic diseases. Notably, a recent report suggests hCG stimulates LHCGR expression in lymphocyte during controlled ovarian stimulation and is linked to improved implantation  On the other hand, undetectable to low circulating sLHCGR/LH-sLHCGR might reflect reduced hormonal activation and down regulation of sLHCGR synthesis as observed in Down's syndrome placenta [33, 34]. This may suggest that like other cytokine and hormone receptors [27–30], the production of sLHCGR is regulated.
What could be the patho-physiological significance of sLHCGR in the context of COS and implantation of the embryo? There are two possible mutually non-exclusive ways the sLHCGR could affect the LH/hCG functions: sLHCGR is intrinsically a specific serum binding protein [28–30] which stabilizes or prevents degradation of the LH/hCG until the hormone is delivered to the traditional membrane-bound receptor. In extreme conditions (low or undetectable sLHCGR), leading to OHSS and high response might reflect an unregulated burst of stimulation resulting from hormonal induction. This is consistent with fast and slow disappearance (double exponential curves) of LH and hCG reported a few decades ago . Accordingly, very short serum half-life of variably glycosylated LH (20-80 min, [35, 36]) compared to FSH (several hrs, ) and hCG (1-3 days, ) suggests that combined and sustained LH-FSH induction of ovarian functions would require receptor-mediated stabilization, specifically of LH enhancing its resistance to clearance. Therefore, in addition to OHSS, our data demonstrate the increased release of oocytes (≥ 15) on COS is associated with low sLHCGR/LH-sLHCGR concentrations (Figure 5 and Table 1). The possibility that an optimal level of sLHCGR complexed with LH exists that promotes pregnancy in those producing 8-14 oocytes might support a physiological reservoir role for sLHCGR in regulating LH availability. In an alternative scenario [27, 39, 40] the mobile sLHCGR may compete with membrane-associated counterparts for binding to circulating LH/hCG. Therefore, increased levels of serum sLHCGR modulate the LH/hCG activity by inhibiting their interactions with cell surface receptors. In general, high serum sLHCGR concentrations are linked to reduced production of oocytes as well as poor implantation (Figures 5, 6 and Table 1). This could be again linked to the inhibitory effect of circulating sLHCGR on LH/hCG functions.
Pre-treatment serum sLHCGR/LH-sLHCGR and LH levels could provide an indication of functional LH levels that would allow the adjustment of hormone dose prior to ovarian induction. This could be an important step towards avoidance of OHSS, particularly for patients whose AMH levels do not correlate with high oocyte yield and potential OHSS. It should be emphasized that we have measured baseline, pre-treatment sLHCGR concentrations and that during fertility treatment and prior to embryo transfer, these concentrations could alter. Indeed, this might explain both positive and failed implantation when the pre-treatment serum LH receptor concentrations in some women were high and low, respectively (Figure 6). Therefore, the true effect of circulating sLHCGR in modulating implantation and early pregnancy would require its estimation before and immediately after embryo transfer. Future experiments are needed to test this hypothesis.
Critical clinical parameters that predict reduced response to COS are age, BMI, early cycle FSH, LH, inhibin B, antral follicle counts (AFC) and AMH concentrations [5, 41]. Of these, serum AMH and AFC are recognized as superior predictors of ovarian reserve and OHSS . Our study indicates that in the absence of AMH data, measurement of pre-treatment circulating sLHCGR/LH-sLHCGR together with FSH:LH ratios, age and AFC might provide sufficient information on aberrant ovarian response to establish individualized hormone combinations, daily doses and duration of treatment prior to COS.
The improvement of pregnancy outcome could be another potential benefit of measuring pre-treatment serum sLHCGR. Following a decline in LH support, hCG is the foremost leutotropic paracrine signal produced by the embryo, well before the implantation begins . This hCG signaling critically influences the blastocyst development, uterine receptivity through stromal fibroblast proliferation, secretion of IGF binding protein-1, NK cell activation and apoptosis (Fas-FasL), transient immune tolerance through activation of regulatory T-cells and dendritic cells (MHC class II, IL-10 and IDO expression), and endometrial angiogenesis through secretion of VEGF [42, 43]. Furthermore, an increased LH surge is associated with a high rate of miscarriage . Therefore, measurement of sLHCGR/LH-sLHCGR following embryo transfer together with targeted hCG therapy could improve pregnancy outcome by extending the window of implantation and simultaneously reducing the frequency of miscarriage.
The bioactive serum/plasma LH concentrations, conventionally measured by Leydig cell assay  have been shown to be significantly lower than the immunoreactive hormone in various clinical conditions affecting ovarian functions and fertility [46, 47]. Whether the ELISA assays described here for measuring the soluble LH receptor and the hormone-receptor complex would be comparable to the Leydig cell assay with respect to LH bioactivity requires a separate investigation. The link between hypogonadism and LHCGR functions was first demonstrated by partial purification of Mr 30 K-60 K serum proteins that were found to competitively inhibit the hormone binding to the LH receptor and affect testosterone production in uremic boys .