Performance evaluation of microbead and ELISA assays for follicular G-CSF: a non-invasive biomarker of oocyte developmental competence for embryo implantation.

G-CSF in individual follicular fluids correlates with the potential of the corresponding embryo to result in a live birth after transfer in IVF. To evaluate the requirements for routine follicular fluid G-CSF quantification, we compared follicular fluid G-CSF measurements made with two multiplexed microbead assays purchased from Bio-Rad Laboratories and R&D Systems, and a commercial G-CSF ELISA (R&D Systems). Individual follicular fluids (n=139) associated with transferred embryos were analysed to determine cytokine profile and the fate of each transferred embryo was recorded. The effect of multiplexing as well as comparison of the respective performances of the microbead assay with a flow cytometry assay was explored. Multivariable logistic regression analysis was performed and receiver operating characteristic (ROC) analysis was used to determine the performance and sensitivity/specificity of each method for individual follicular fluids. Covariate factors known to influence IVF outcome such as age, serum oestradiol and embryo score were systematically integrated in each analysis. The quantification of follicular fluid G-CSF using microbead assay methodologies, but not ELISA, yielded results showing the utility of follicular fluid G-CSF as a biomarker predictive of a successful delivery (Au(roc): 0.77 [0.68-0.84] (p=0.003) and 0.75 [0.66-0.82] (p=0.004) for Bio-Rad and R&D Systems microbead assays respectively), whereas follicular fluid G-CSF values quantified by ELISA were not predictive (Au(roc):0.61 [0.52-0.70] p=0.84). Microbead assay and flow cytometry appeared similarly efficient for quantifying follicular fluid G-CSF and multiplex versus single-plex assays did not influence the reliability of quantification

Patient characteristics for women recruited to Liège University. Values are Mean (S.E.M.).

<p>Patient characteristics for women recruited to Liège University. Values are Mean (S.E.M.).</p

Higher levels of sST2 are expressed in larger follicles.

<p>sST2 levels in individual follicles, classified as small (n = 16), medium (n = 35) or large (n = 80), were determined by ELISA. Means ± S.E.M.</p

sST2, but not IL-33, is increased in human FFs compared to plasma levels.

<p>FFs (at least 5 per patient) and plasma samples were collected from 21 women undergoing ART. Levels of sST2 and IL-33 were determined by ELISA. The mean values of FF levels were compared to the plasma levels, and the fold change in sST2 (A) and IL-33 (B) displayed.</p

sST2 is secreted by granulosa cells.

<p>Aspirated FFs contained a mixture of granulosa cells (CD45 negative) and leukocytes (CD45 positive) (A). Leukocytes were removed by filtration through a 70 µm filter which retained granulosa cells, resulting in >98% purity granulosa cells (B). Full length IL-33 and ST2 in the purified granulosa preparations can be detected by immunoblotting from three patients (1, 2, 3), PL = placenta lysate positive control (C). Purified granulosa secrete sST2 in culture (D), granulosa cell preparations from three patients (red diamonds, green triangles, blue circles) were cultured for up to 72 hours and supernatants assayed by ELISA for sST2. Growth media contained no detectable sST2.</p

sST2 levels are lower in follicles which produce average quality embryos compared to good quality embryos but levels do not predict IVF success, unlike G-CSF.

<p>FFs with known embryo outcome were analysed for levels of sST2 and G-CSF. Embryos were graded good (n = 52), average (n = 28) or poor (n = 38) and cytokine levels compared (A – sST2 and B – G-CSF). Levels of sST2 (C) and G-CSF (D) were compared to live birth outcome, (+; n = 25)) or not (−; n = 88), for all embryo grades, further designated by embryo quality (E and F respectively), good grade/+ (n = 15); good grade/− (n = 37); average grade/+ (n = 6); average grade/− (n = 22); poor grade/+ (n = 4); poor grade/− (n = 34). Bars = mean ± S.E.M (A–D) or scatter plot with bars = mean (E–F).</p

Patient characteristics for women recruited to the Oxford Fertility Unit. Values are Mean (S.E.M).

<p>Patient characteristics for women recruited to the Oxford Fertility Unit. Values are Mean (S.E.M).</p