P-571 The newly developed highly sensitive reagent GL13 indicates extensively increased cellular senescence in the follicular fluid of poor responder patients: A prospective observational study
Authors: Simopoulou M , Maziotis E , Veroutis D , Grigoriadis S , Giannelou P , Chronopoulou M , Sfakianoudis K , Pantos K , Evangelou K , Gorgoulis V 
Published: June 2022

Abstract

Study question
Do women of diminished ovarian reserve and poor ovarian response (POR) present with increased lipofuscin follicular fluid (FF) levels, indicating increased ovarian cellular senescence?

Summary answer
Poor responders present with a six-fold increased lipofuscin FF levels indicating extensive senescence in POR ovaries. Lipofuscin may be a new sensitive biomarker for POR.

What is known already
Lipofuscin is a nondegradable substrate of metabolism accumulating in cells due to impaired mitochondrial/lysosome/proteasome function, upon stress or damage. It is well-documented that lipofuscin constitutes a highly sensitive biomarker of cellular aging and senescence. Recently, a novel reagent (GL13) coupled by a hybrid histochemical–immunohistochemical method were developed for detecting and measuring soluble lipofuscin levels in biological fluids, ascertaining high sensitivity and specificity. Data indicate that lipofuscin levels are associated with oocyte competence and age-related infertility. However, hitherto no data has been published indicating the value of lipofuscin FF levels as a biomarker towards accurately predicting ovarian reserve and response.

Study design, size, duration
This prospective observational study was collaboratively conducted between November 2020 and September 2021 at the Athens University Medical School and at Genesis Athens Clinic. A total of 32 patients undergoing IVF treatment were enrolled. The study group comprised of 16 POR patients defined according to the Bologna criteria. The control group consisted of 16 normal responder women undergoing IVF due to tubal factor or/and mild male factor infertility. Patients with other infertility aetiologies were excluded.

Participants/materials, setting, methods
Participants in both groups received the standard short GnRH-antagonist protocol. The FF samples were collected as part of the oocyte retrieval process. Lipid and protein parts of lipofuscin were isolated from FF samples and stained with GL13. The complex of lipofuscin-GL13 was labeled with an anti-biotin HRP conjugated antibody and detected employing a chemiluminescence reaction. Luminescence was measured and signal intensity was corresponding to lipofuscin concentration. Statistical analysis was performed employing R Programming Language.

Main results and the role of chance
Poor responders presented with a statistically significant six-fold higher lipofuscin FF levels in comparison to the normal responder group (869.21 ± 501.87 vs 146.6 ± 107.64 RLU; P-value &lt;0.0001). Lipofuscin levels were negatively correlated with AFC (Spearman’s Rho: -0.68; P-value &lt;0.0001), AMH levels (Rho: -0.61; P-value =0.0002), estradiol levels on triggering day (Rho: -0.68; P-value &lt;0.0001), number of oocytes retrieved (Rho: -0.54; P-value =0.001), number of mature metaphase II (MII) oocytes obtained (Rho: -0.58; P-value =0.0008), number of normally fertilized (2PN) zygotes (Rho: -0.51; P-value =0.003), number of cleavage stage embryos (Rho: -0.48; P-value =0.005) and number of blastocyst stage embryos (Rho: -0.41; P-value =0.02). These correlations remained statistically significant when adjusting for ovarian stimulation response status. No association was established between Lipofuscin levels and embryo quality neither on Day 3 nor on Day 5. Lipofuscin levels, with a cut-off value at 294, were able to predict ovarian stimulation response status with an area under the curve (AUC) at 0.96. The sensitivity was 0.875, the specificity was 0.938 and the accuracy was 0.906. The positive predictive value was 88.24% and the negative predictive value was 93.33%.

Limitations, reasons for caution
Limitations of our study refer to the limited size of the studied population, as well as to the lack of data referring to pregnancy outcomes. Moreover, molecular data with regards to the possible mechanisms leading to the observed increased senescence are required. Future studies are needed to verify these findings.

Wider implications of the findings
Data presented herein indicates, for the first time in literature, that lipofuscin FF levels measured via the GL13 method may be a promising and sensitive tool for predicting POR and stimulation outcome. Lipofuscin could further serve as a valuable novel biomarker indicating ovarian senescence, ovarian reserve status and oocyte competence.

Trial registration number
Not Applicable