P-695 Anti-muellerian Hormone (AMH) and Plasminogen Activator Inhibitor (PAI-1) are robust follicular-fluid (FF) biomarkers predictive of follicle maturity and oocyte quality in women undergoing IVF cycles
Authors: Chimote N.M , Chimote B 
Published: June 2022

Abstract

Study question
Is AMH the in-vivo trigger that correlates with PAI-1 in the follicular-fluid micro-milieu to influence maturity and quality of oocytes derived in vitro during IVF?

Summary answer
Lower FF-AMH levels correlate with top quality oocytes whereas Lower FF-PAI-1 levels correlate with follicle-maturity as reflected in mature MII oocytes retrieved in IVF cycles

What is known already
Ovulation is a time-coordinated tightly regulated proteolytic event. PAI-1, primarily produced by granulosa cells, is a major form of PAI in FF of human preovulatory follicles, allowing localized degradation and regulating ovulation.While ovulation process is rendered redundant in IVF cycles; nevertheless FF PAI-1 concentration may influence maturity of oocyte within the follicle. AMH is exclusively produced by granulosa cells of pre-antral & early-antral ovarian follicles in the adult female and secreted into FF. Although AMH is widely accepted as an ovarian-reserve marker, very few studies have explored direct putative correlation of FF-AMH with oocyte quality in stimulated IVF cycles.

Study design, size, duration
Prospective study of n = 1150 (Power of study &gt;85%) women (mean age 30.22±4.25 years, BMI 23.97±4.53, W/H ratio 0.88±0.06) undergoing antagonist stimulation protocol with r-FSH (September 2015-December 2018). On day of ovum pick-up, original aspirate per follicle was pooled per patient. AMH and PAI-1 were evaluated in pooled FF by ELISA method using diagnostic kits. These protein-biomolecules were expressed as ratio of total protein content. SerumE2, LH, P on d/hCG trigger were also estimated by RadioImmunoAssay.

Participants/materials, setting, methods
Elderly women (age&gt;35 years), women with polycystic-ovaries, endometriosis were excluded. Embryo-transfer (ET) was done either on Day3 or day5. Luteal-phase was supported with micronized progesterone.
All cycles were divided into Low FF-AMH (≤1.72 ng/mg protein), High FF-AMH (&gt;1.72ng/mg protein) and Low FFPAI-1 (≤592.1 ng/mg protein), High FFAI-1 (&gt;592.1 ng/mg protein) groups. Oocyte quality was graded as poor, average and top based on morphological assessment of COC-complex, zona-pellucida (thickness, regularity) and cytoplasm (granularity, vacuolation, refractile bodies).

Main results and the role of chance
Mean Age, Baseline D3-Serum AMH, E2 d/hCG and number of eggs retrieved did not differ significantly between low vs. High FF AMH groups as well as Low vs. High FF-PAI-1 groups.
Low FF AMH group displayed significantly higher percent of top-quality oocytes (70.38±11.5 vs. 52.16±8.3;p&lt;0.0001) and higher fertilization rates (88.68±10.7 vs. 72.5±9.2%; p = 0.004) compared to High FF AMH group. Incidences of total fertilization failure were significantly higher in high FF AMH (3.25% vs. 1.1%, p = 0.0002) compared to Low-FFAMH group.
FF-AMH correlated with oocyte-quality (Pearsonr=0.48); lower-levels increased the odds of top-quality oocytes. Thresholds of FF-AMH for top quality oocytes was &lt;1.8 ng/mg protein;ROCAUC:85%)
Low FF PAI-1 group had significantly higher Percentage of MII oocytes (93.55 vs. 74.8%; p &lt; 0.0001), Rates of Fertilization (86.35 vs. 71.04, p = 0.008) and cleavage (79.19 vs. 69.85;p=0.03) compared to High FF-PAI-1 group.
FF-PAI-1 correlated with oocyte-maturity (Pearsonr=0.51), lower-levels increased the odds of obtaining MII-oocytes. Cutoff of FF PAI-1 for mature MII oocytes was &lt;504.4 ng/mg protein;ROCAUC:76%).
FF-AMH showed strong positive correlation with FF-PAI-1 (Pearsonr=0.66)

Limitations, reasons for caution
Several other molecules in the follicular-fluid milieu may be responsible for maturity status and quality of oocytes. Although these are apparently the most plausible of markers; role of other factors cannot be ruled out. Also, it may be better to study mono-follicular aspirates rather than pooled FF for each patient.

Wider implications of the findings
FF-levels of AMH and PAI-1 as non-invasive endogenous biomarkers of oocyte maturity and quality in-vitro may facilitate better understanding of the physiological pathway governing in-vivo ovulation process. This study reiterates ‘Follicle-maturation’ is the rate-determining step that sets off a cascade mechanism triggering oocyte-maturation and quality further influencing fertilization, embryo development.

Trial registration number
In patients with polycystic ovarian syndrome (PCOS), are serum androgen levels (total and free testosterone) associated with the risk of endometrial hyperplasia or cancer?