Circulating LH/hCG receptor (LHCGR) may identify pre-treatment IVF patients at risk of OHSS and poor implantation

Abstract

Background: Successful pregnancy via in vitro fertilization (IVF) depends on the recovery of an adequate number of healthy oocytes and on blastocyst implantation following uterine transfer. Two hormones, LH and hCG, utilize a common LH/hCG receptor (LHCGR), variations in which have profound implications in human reproduction. Soluble LHCGR (sLHCGR) is released from experimental cell lines and placental explants and it can be detected in the follicular fluid and serum.

Methods: To evaluate the impact of circulating soluble LHCGR (sLHCGR) in fertility treatment, we measured sLHCGR and LH-sLHCGR complex in serum from women seeking IVF using specifically developed quantitative enzyme-linked immunosorbent assays (ELISA). Following an IVF cycle of treatment, patients were grouped according to oocyte yield into low (lower than or equal to 7 oocytes), intermediate (8-14 oocytes) and high (greater than or equal to 15 oocytes) responders and pregnancy outcome noted.

Results: Pre-treatment sLHCGR identified many women at risk of ovarian hyperstimulation. Low levels of sLHCGR were associated with pregnancy in both high and low responders but sLHCGR did not significantly affect the treatment outcome of intermediate responders. Low responders who failed to become pregnant had high levels of circulating sLHCGR bound to LH (LH-sLHCGR).

Conclusions: Pre-treatment measurement of sLHCGR could be used to tailor individual fertility treatment programs and improve outcomes by avoiding ovarian hyperstimulation and poor embryo implantation.

Figure 1

The sensitivity of sLHCGR and LH-sLHCGR ELISAs. The purity of the affinity purified recombinant LHCGR protein extracellular domain (a, indicated by an arrow) and LH-sLHCGR (d, indicated by arrows) proteins were examined in SDS-polyacrylamide gels-stained with coomassie blue. The small arrows above the major band in d) are either glycosylated or dimers because these bands react with anti-FLAG and LHR29 monoclonal antibodies. The sensitivity of the ELISAs was verified by linear responses to the dilution effect of sLHCGR (b) and LH-sLHCGR protein calibrators (e). The sensitivities of these assays were further examined by assaying serially diluted serum samples from three patients with known concentration of sLHCGR (c) and LH-LHCGR (f). Each data point in b and e represents mean value obtained from six independent dilution of the standards.

Figure 2

High serum FSH and low sLHCGR are inversely related to the pre-treatment serum AMH concentrations. The basal AMH concentrations at ≤ 4 pmol/L and ≥ 45 pmol/L were compared with corresponding FSH (a & c) and sLHCGR (b & d) concentrations. The correlation coefficient (r) and the level of significance (P value) are shown in each case.

Figure 3

A large proportion of women (> 30%) seeking fertility treatment have > 20 pmol/mL, high pre-treatment sLHCGR. The frequency and distribution of the concentrations of a) LH and b) sLHCGR measured in 274 women seeking fertility treatment are shown. Additionally, the LH concentrations ≥ 9.1 IU/L are inversely related to the sLHCGR levels.

Figure 4

Younger women among low responders to COS have higher serum sLHCGR. Patients yielding a) ≤ 7, b) ≤ 5 and c) ≤ 4 oocytes with respect to their age were plotted. The correlation between sLHCGR and age among high responders (≥ 15 oocytes) with OHSS is shown (d). The correlation coefficient (r) and the level of significance (P value) are shown for each group.

Figure 5

Quantitative inhibition of the release of oocytes by high pre-treatment serum sLHCGR following COS. The ratio, sLHCGR:Oocyte, was determined by calculating the sLHCGR molecules (pmol/mL) per oocyte in women who had ≥ 15 oocytes (a) and those who yielded b) < 15 and ≤ 5 oocytes. The percentage of sLHCGR bound to LH (sLHCGR/LH-sLHCGR%) with respect to the yield of oocytes and treatment outcome (c & d). The level of significance (P values) determined by comparing different groups are shown. NP, Not pregnant; P, Pregnant.

Figure 6

High pre-treatment serum sLHCGR and LH-sLHCGR reduce embryo implantation. The serum sLHCGR (a & b) and LH-sLHCGR (c & d) concentrations were plotted for patients yielding ≥ 15 oocytes (a & c) and < 15 oocytes (b & d). Each data set was plotted in duplicate (open circle and triangle), where extremely high values (open circle) in each data set were excluded (open triangle) in order to compare the data representing > 90% of the patients. The cut-off values for a) and b) were 50 and 100 pmol/mL, respectively and for c) and d) were 10 and 20 pmol/mL, respectively. The bar indicates the mean value for each condition below the cut-off values. The level of significance (P values) determined by comparing different groups are shown. NP, Not pregnant; P, Pregnant.