FOLLICULAR FLUID HYALURONIC ACID POLYDISPERSITY IS ASSOCIATED WITH AGE AND IN VITRO FERTILIZATION CYCLE OUTCOME: A PILOT STUDY.

Human follicular fluid is rich with Hyaluronic acid (HA). HA is synthesized as a high-molecular weight (HMW) linear disaccharide polymer. It is fragmented into low-molecular weight (LMW) HA by the activity of hyaluronidases and reactive oxygen species. HA size determines its biological function: HMW HA (>1000kDa) displays anti-inflammatory and immunosuppressive properties, and LMW HA (generally <500kDa) can induce the release of pro-inflammatory cytokines. Our group has previously demonstrated that LMW HA fragments compromise estradiol production and gamete quality of mouse follicles, in vitro. The objective of this study was to evaluate the association of human follicular fluid HA size distribution (polydispersity) with reproductive aging and IVF cycle outcome using recently reported novel method of label-free detection and molecular weight discrimination with solid-state nanopore sensor. Follicular fluid (FF) samples from 30 participants from Northwestern University Reproductive Tissue Library were used to measure HA polydispersity in 3 age groups (<34yo, 36-38yo, >39yo). Participants in these 3 age groups (n=10, each) had similar AMH levels, BMI, days of ovarian stimulation and peak estradiol levels. FF samples were previously collected from 18-20 mm follicles under IRB approved protocol. Equal volumes of FF samples were pooled, and 2 replicates were tested per age group. Following proteinase digestion, separation and purification steps, HA molecular weight distribution was analyzed using solid-state nanopore devices consisting of a single pore (6–8 nm diameter). Electrophoretic translocations of HA through the pore were identified as transient reductions in the ionic current for each molecular translocation and converted to MW using a calibration standard. >39yo age group showed a trend toward smaller HA fragments compared to 36-38yo and <34yo (weight average 2228±213kDa, 5532±914kDa, 4945±779kDa, respectively, p=0.2 and number average 345±90kDa, 805±161kDa, 631±34kDa, respectively, p=0.2). Polydispersity index also showed a trend towards more homogenous molecules in the >39yo cohort compared to others (6.5±0.02, 6.9±0.2, 7.8±0.3, respectively, p=0.07). In addition, comparison of HA polydispersity of pooled FF samples within each age cohort based on IVF cycle outcome (clinical pregnancy) showed significant differences in <34yo (219±1880kDa vs. 199±1418kDa, p= 0.045) and 36-38yo group (227±1592kDa vs. 253±2318kDa, p=0.007), however, no significant difference was observed within >39yo cohort (193±725kDa vs. 194±879kDa, p=0.89) Human follicular fluid HA molecular weight distribution shows a trend towards smaller fragments in older patients. HA polydispersity is associated with the outcome of the IVF cycle in patients <34yo and 36-38yo.