P-681 Balance of Estrogen:Androgen ratio (Estradiol E2: Dehydroepiandrosterone-sulphate DHEAS) and its symbiotic correlationship with Vascular-Endothelial Growth-Factor (VEGF) critically foments implantation and pregnancy outcomes in IVF cycles
Authors: Chimote B.N , Chimote N 
Published: June 2022

Abstract

Study question
Is the estrogen/androgen balance vital for implantation and does this E2:DHEAS ratio coordinate with neoangiogenesis/neovascularisation (VEGF) to facilitate establishment of viable pregnancy in IVF cycles?

Summary answer
The Estradiol E2:DHEAS ratio independently as well as in symbiotic correlationship with VEGF is strongly predictive of invasive implantation and pregnancy outcomes in IVF cycles

What is known already
Decidualization of endometrial stroma and vascularization at feto-maternal interface is necessary for implantation. Estrogen Estradiol E2 is among the factors widely known to regulate these processes. Androgen Dehydroepiandrosterone (DHEA) is the designated primary precursor for natural estrogens. DHEA-induced PCOS mice have demonstrated adverse impacts on Embryo implantation and decidualization. However, role of DHEA/S in human implantation has not yet been explored. Vascular-Endothelial growth-factor (VEGF) is known to influence vascularization, angiogenesis, endometrial receptivity. Higher VEGF expression in endometrium is associated with pregnancy. However endometrial-biopsy for VEGF estimation is an invasive method for implantation studies and cannot be applied to ongoing cycles.

Study design, size, duration
Prospective study of n = 235 (Power of study&gt;85%) non-PCOS, normal-responder infertile females (mean age 31.77±2.42 years, BMI 24.4±4.1, W/H ratio 0.82±0.07) undergoing fresh IVF treatment cycles using standard antagonist stimulation protocol at our fertility clinic during January 2019-December 2020. Elderly women (age&gt;35 years), women with polycystic-ovaries, endometriosis and frozen-thawed cycles were excluded.7 cycles were cancelled owing to no embryo transfer (ET). Day3-cleavage stage or day5-blastocyst stage embryos were transferred (n = 228). Luteal-phase was supported with Micronized progesterone.

Participants/materials, setting, methods
Serum levels of Estradiol E2 and DHEAS were measured by Radio-Immunoassay and VEGF estimation by ELISA on day ET, day7, day14 post ET. Cycles were divided into Pregnant (P:n=73) and Non-Pregnant (NP=n=155) groups. Main outcome measures: Clinical Pregnancy (CPR:n=70=30.70%), Live-Birth (LBR:28.94%) rates. Secondary outcome measures: Biochemical-pregnancy (BCP:n=3=1.31%), Early-Miscarriage (EM:n=4=1.75%) rates. Pregnant=Serum βhCG&gt;50 on d14ET. BCP=No exponential rise/tapering βhCG levels post d14ET, CP=positive cardiac activity at 6-8 weeks gestation, EM=non-viable intra-uterine pregnancy upto 12 weeks gestation.

Main results and the role of chance
dET: E2 did not differ significantly (780±50 vs. 750±30 pg/ml;p=0.61) whereas DHEAS = (212±11 vs. 170±6 ng/dL;p=0.0005), E2:DHEAS = (3.72±0.13 vs. 4.65±0.17;p=0.0011), VEGF = (658±20 vs. 495±16;p&lt;0.0001) differed significantly between P vs. NP groups; indicating importance of E2/DHEAS ratio.
D7ET: E2 = (790±57 vs. 222±19;p&lt;0.0001), DHEAS = (381±28 vs. 1262±70;p&lt;0.0001), E2:DHEAS = (2.37±0.25 vs. 0.30±0.029;p&lt;0.0001), VEGF = (636±23.61 vs. 480±10.13;p&lt;0.0001) displayed statistically robust differences between P vs. NP groups.
d14ET: E2 = (855±73.45 vs. 193.2±13.31;p&lt;0.0001), VEGF = (544.2±15.80 vs. 452.3±16.77;p=0.0011) displayed statistically robust differences between P vs. NP groups. Interestingly, although DHEAS = (685±41.05 vs. 806±55.37;p=0.18) did not differ significantly, E2:DHEAS = (1.643±0.26 vs. 0.45±0.048;p&lt;0.0001) showed highly significant difference; again highlighting importance of E2/DHEAS balance.
Ratio E2:DHEAS on d7ET correlated strongly with d7VEGF (Pearson r = 0.31,p&lt;0.0001). ROC curve for d7 E2:DHEAS had AUC 78%, p &lt; 0.0001

Hormone values and their rising trends (d/ET to d7/ET to d14/ET) varied with pregnancy outcome status
E2= LB:(d/ET:780±50 to d7/ET:790±57 to d14/ET:855±73.45) vs. BCP:(d/ET:723.3±157.6 to d7/ET:743.3±159 to d14/ET:770±153) vs. EM:(d/ET:906.3±104 to d7/ET:1150±113 to d14/ET:1758±127)
DHEAS= LB:(d/ET:212±11 to d7/ET:381±28 to d14/ET:685±41.05) vs. BCP:(d/ET:248.3±55 to d7/ET:266.7±71.2 to d14/ET:400±120.3) vs. EM:(d/ET:243.8±27 to d7/ET:303.8±11.43 to d14/ET:391.3±49)
E2:DHEAS= LB:(d/ET:3.72±0.13 to d7/ET:2.37±0.25 to d14/ET:1.643±0.26) vs. BCP:(d/ET:2.93±0.15 to d7/ET:2.97±0.4 to d14/ET:2.12±0.32) vs. EM:(d/ET:3.78±0.40 to d7/ET:3.77±0.26 to d14/ET:4.68±0.59)
Decreasing E2:DHEAS ratio (d/ET-d7/ET-d14/ET) is vital for successful implantation/Live-birth
VEGF= LB:(d/ET:658±20 to d7/ET:636±23.61 to d14/ET:544.2±15.80) vs. BCP:(d/ET:513.3±37.56 to d7/ET:628.3±40.45 to d14/ET:468.3±35.63) vs. EM:(d/ET:647.5±78.8 to d7/ET:887.5±60 to d14/ET:1138±94.4)

Limitations, reasons for caution
This study is limited by small sample size and is not a randomized controlled trial. We have taken only representative molecules into consideration. There is a plethora of factors involved during the implantation process. Multi-centric systematic studies are needed to corroborate these findings and account for the probable confounding factors.

Wider implications of the findings
This novel study explores and highlights significance of maintenance of a critical estrogen/androgen ratio (E2:DHEAS), rather than individual estrogen or androgen markers, in the implantation process. We have also underscored how this ratio coordinates symbiotically with VEGF as a critical non-invasive determinant of implantation and pregnancy-outcomes in ongoing IVF cycles.

Trial registration number
Not Applicable