P-236 IL-6 levels in follicular-fluid and day5/6 spent culture drops indicate its role as a biomarker of oocyte maturation and an embryokine influencing embryo developmental competence
Authors: Chimote B N , Chimote N 
Published: June 2023

Abstract

Study question
To elucidate the role of IL-6 in folliculogenesis and embryo development during human in-vitro culture

Summary answer
High IL-6 levels in follicular-fluid positively correlate with oocyte maturity status whereas higher IL-6 secretome in day5/day6 spent culture media inversely correlates with blastocyst/ICM grades

What is known already
Although some studies have attempted to explore the correlation of FF IL-6 with fertilization and embryo development rates; the results have been inconclusive. Similarly, studies comparing FF IL-6 levels with endometrial receptivity and pregnancy outcomes have been ambiguous and contradictory. Recent research has reported improvement in bovine blastocyst ICM grades in culture media supplemented with IL-6. No study has yet evaluated the role of IL-6 as a biomarker of oocyte maturity nor investigated IL-6 as an embryokine that could regulate embryo developmental competence in humans. We have employed the FF metabolomic and Secretomic approach for IL-6 in human IVF cycles.

Study design, size, duration
Prospective study in women (25-35 years) undergoing IVF cycles (n = 256) for unexplained/tubal-factor infertility from 2018-2020. Only those women whose FF and day5/6 spent culture media sample could be obtained, were included. Women with endometriosis, PID, Genital Kochs were excluded. IL-6 levels were measured by diagnostic kit in spent micro-drops and FF (pooled per patient per cycle). Cycles were divided into Low and High IL-6 groups (FF: ≤ 47.5 and &gt;47.5pg/ml; Spent Medium: &lt;0.5 and ≥0.5pg/ml).

Participants/materials, setting, methods
Antagonist cycles involving COH with r-FSH and ICSI for all. Noted oocyte Nuclear maturity (GV/MI/MII). Oocyte Cytoplasmic maturity graded as Top and non-Top, based on presence of granulation, refractile bodies, sER, visco-elasticity. Post ICSI, oocytes cultured individually in 50µl single-step media micro-drops till day3 and shifted to fresh 50µl micro-drops for extended culture till day5/6. Blastocyst gradation was done using Gardner’s classification for expansion, ICM and trophectodermal cells. Statistical analysis done using graph-pad prism VI

Main results and the role of chance
High FF/IL-6 group (n = 103) had significantly higher proportion of MII (92.08 vs. 76.33%; p &lt; 0.001) and top-quality (88.10 vs. 70.0%; p &lt; 0.001) oocytes and fertilization rates (89.45 vs. 77.30%; p = 0.009) compared to Low FF/IL-6 group (n = 153) although total number of days of stimulation/gonadotropin dose, mean oocytes retrieved did not differ significantly between the two groups. Cleavage (84.64 vs. 75.92%; p = 0.061) and blastocyst formation (53.86 vs. 42.37%; p = 0.053) rate, although higher, did not differ significantly between the two FF/IL-6 groups. FF/IL-6 level correlated strongly with overall oocyte maturity status (Pearson r = 0.61, 95% CI 0.52-0.8). Threshold FF/IL-6 level for obtaining top-quality MII oocytes was &gt;39.2pg/ml (ROCAUC = 86%, Sensitivity = 78%, Specificity = 83%).
In D5/6 spent culture drops, likelihood of top-quality blastocyst formation (expansion grade ≥3 and ICM/TE grades AA/AB/BA) increased by 41% (odds ratio: 0.44, 95%CI: 0.4-0.5; χ2 p &lt; 0.0001) if IL-6 concentration was &lt;0.5 pg/ml. Lower culture-drop IL-6 levels correlated strongly with top ICM grades (Pearson r = −0.66, 95% CI 0.6-0.7). Highest IL-6 levels were found in spent culture-drops containing grade-C blastocysts whether on day5 or day6. IL-6 levels were comparatively higher in day6 than in day5 micro-drops, correlating with higher percentage of top-quality blastocysts on day5 than on day6. Threshold micro-drop IL-6 level for top-grade blastocyst &lt;0.42 pg/ml.

Limitations, reasons for caution
We estimated IL-6 levels in pooled FF (per patient, per cycle). Although cumbersome & time-consuming, it would be worthwhile to evaluate individual FF (per follicle, per patient, per cycle). Also, measuring IL-6 in spent media involving group embryo culture, may facilitate study of the probable paracrine effects of this ‘embryokine’.

Wider implications of the findings
IL-6 is a pleiotropic cytokine and its role/s in folliculogenesis, embryo development and endometrial receptivity is not yet clearly etched out. This study attempts to elucidate the functions of IL-6 in a systematic manner by deriving threshold values for obtaining top quality oocytes and blastocysts in human in-vitro culture conditions.

Trial registration number
NOT APPLICABLE