Intravenous neutralization of vascular endothelial growth factor reduces vascular function/permeability of the ovary and prevents development of OHSS-like symptoms in rhesus monkeys

Abstract

Background: Ovarian hyperstimulation syndrome (OHSS) is a disorder associated with elevated serum VEGFA following chorionic gonadotropin (hCG) exposure in controlled ovarian stimulation (COS) cycles in women. In this study, we tested the effect of intravenous VEGFA neutralization on OHSS-like symptoms and vascular function in rhesus macaques during COS cycles.

Methods: Monkeys (n = 8) were treated with 3 COS protocols and assigned randomly to groups as follows: 1) COS alone (Control, n = 5); 2) COS + VEGF mAb Avastin 19 ± 5 h before hCG (Avastin pre-hCG; n = 6); 3) COS + Avastin 3-4 days post-hCG (Avastin post-hCG; n = 4); 4) COS + Simulated Early Pregnancy (SEP n = 3); or 5) COS + SEP + Avastin (SEP + Avastin n = 3). Follicles were aspirated 36 h post-hCG, fluid was collected from one follicle for analysis of steroid and vascular hormone content. Remaining follicles were aspirated, and luteinized granulosa cells (LGCs) cultured for 24 h. Ovarian/uterine vascular flow (VF) and blood volume (BV) were analyzed by contrast enhanced ultrasound (CEUS) before hCG bolus and 6-8 days post-hCG bolus/time of peak SEP response. Ovarian permeability to albumin was analyzed by Dynamic Contrast Enhanced-MRI (DCE-MRI) post-hCG.

Results: Abdominal fluid was present in 4/5 Control, 2/6 Avastin pre-hCG, and 3/4 Avastin post-hCG females. Neutralization of VEGFA before hCG reduced ovarian VF, BV, and permeability to albumin (P < 0.05), while only ovarian VF and permeability were reduced in Avastin-post hCG group (P < 0.05). There was no effect of Avastin on ovarian vascular function during COS + SEP. VEGF levels in follicular fluid were reduced 78-fold by Avastin pre-hCG, and LGCs exposed to Avastin in vivo also released 4-fold less VEGF into culture media (P < 0.05). Culture medium of LGCs exposed to VEGFA neutralization in vivo had lower levels of P4 and ANGPT1, and an increased ratio of ANGPT2/1 (P < 0.05). Uterine VF was reduced by SEP + Avastin in the basalis/junctional zone (P < 0.05).

Conclusions: Avastin treatment before hCG prevents the development of symptoms associated with ovarian hyperstimulation syndrome. In vitro data suggest neutralization of VEGFA alters expression of other vascular factors typically induced by hCG in the luteinizing follicle. Neutralization of VEGFA action alters the vascular function of the basalis zone of the uterus during simulated early pregnancy, indicating a potential effect on embryo implantation.

Keywords: Chorionic Gonadotropin; Controlled ovarian stimulation; Ovarian vascular function; Uterine vascular function; VEGFA.

Fig. 1

Rhesus macaques show symptoms of moderate OHSS following hCG administration in COS cycles. Magnetic resonance imaging (MRI) depicting pelvic anatomy before (a) and after (b) hCG administration in COS cycles in one representative macaque female (tissue density is depicted in grey scale). The reproductive tract (uterus and vaginal canal) is highlighted by light grey line. Presence of ascites (fluid is bright white) is depicted in this female after exposure to hCG (c) and indicated by blue arrow

Fig. 2

Effects of VEGFA neutralization during COS cycles; ovarian vascular flow/volume. Serum progesterone (P4; a), ovarian blood volume (BV; b), and ovarian vascular rate of flow (VF; c) in control COS cycles and COS cycles + anti-VEGFA (Avastin) treatment pre-hCG (Avastin Pre) and post hCG (Avastin Post). Values observed during natural menstrual cycles in these same females (NC) are depicted as a reference. For details of treatments see Materials and Methods. Upper case letters indicate differences (P < 0.05) between COS treatment groups at mid-luteal phase and lower case letters indicate differences (P < 0.05) within each treatment group between ovulation and mid-luteal phase

Fig. 3

Effects of VEGFA neutralization during COS cycles; ovarian permeability. DCE-MRI analyses at mid-luteal/luteal phase of albumin flux through ovarian tissue in COS control cycles and COS cycles with and without Avastin treatment pre-hCG (Avastin Pre) and post-hCG (Avastin Post). Data from the CL of natural menstrual cycle (NC) are included as reference. a Estimation of ovarian permeability to albumin (K^trans) and b estimate of gadolidium-conjugated albumin present in extravascular/extracellular space (v_e). Different uppercase letters indicate differences (P < 0.05) between COS Control, Avastin Pre and Avastin Post treatment groups

Fig. 4

Local effects on ovarian angiogenic factors by VEGFA neutralization in COS cycles. Intrafollicular VEGFA (a), and LGC production of VEGFA (b), progesterone (P4; c), ANGPT1 (d), ANGPT2 (e), and ratio of ANGPT2:1 (f) as detected in media after 24 h of culture in aspirates from control COS and anti-VEGFA (Avastin) treated females. See Methods for further details. Different uppercase letters indicate significant (P < 0.05) differences between Control and Avastin Pre-CG treatment groups

Fig. 5

Effects of VEGFA neutralization during COS + SEP cycles. Serum progesterone (P4; a), ovarian blood volume (BV; b), and ovarian vascular rate of flow (VF; c), uterus VF (d), junction (basalis) VF (e), and endometrial VF (f) in control COS + SEP cycles and COS + SEP cycles + anti-VEGFA (Avastin) treatment. For details of treatments see Methods. Upper case letters indicate differences (P < 0.05) between COS + SEP and COS + SEP + Avastin treatment groups