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REPRODUCTION-DEVELOPMENT |
Department of Obstetrics and Gynecology and Center for Reproductive Sciences (R.C.Z., E.X., M.F.), College of Physicians and Surgeons, Columbia University, New York, New York 10032; and ImClone Inc. (P.B.), New York, New York 10014
Address all correspondence and requests for reprints to: Dr. Michel Ferin, Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, New York 10032. E-mail: . mf8{at}columbia.edu
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Abstract |
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Introduction |
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In a previous study, we have demonstrated that the final maturation stages of the dominant follicle during the late follicular phase in the rhesus monkey are delayed by the administration of anti-VEGF blocking antibodies (8). These results clearly support a role for angiogenesis, and for VEGF in particular, in the later stages of cyclic follicular maturation in the primate. These functional data are supported by observations that growth and maturation of the dominant follicle are accompanied by increased vasculature, forming a mesh of two concentric vascular networks in the theca interna and externa (9), and that greater amounts of VEGF m-RNA are detected in granulosa cells of the dominant follicle than of less advanced follicles (10). The objective of the present study is to investigate the role of angiogenesis in early cyclic follicle development in the same species and to determine whether this process and the selection of a dominant follicle can be delayed by interfering with VEGF action. Because VEGF receptor 2 (VEGF-R2) seems to mediate most of VEGF-dependent angiogenic activity (9, 11, 12), we have studied the effects of an antibody against VEGF-R2 administered in the early follicular phase on cyclic events. A similar antibody had previously been shown to be an effective inhibitor of angiogenesis during corpus luteum formation in the rodent (13). To specifically demonstrate an effect on the recruited follicular cohort and on follicle selection, daily measurements of inhibin B [a marker for the small antral follicles cohort (14)] and of estradiol (E2) [a marker for follicle selection and growth (3)] were obtained. The data suggest that angiogenesis is also a requisite for small antral follicle development during the early follicular phase in the nonhuman primate.
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Materials and Methods |
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Experimental protocol
The experiment was designed to investigate the role of angiogenesis in the selection and development of a dominant follicle in the primate. Two normal control ovulatory cycles were first documented in six monkeys. In the following cycle, the animals received repeated iv injections of anti-VEGF-R2 antibodies (2.5 mg/kg•injection). The antibody was directed against the extracellular domain of the human VEGF-R2 receptor (p1C11, ImClone Inc.) (15). Antibody injections were initiated on d 2–4 of the menstrual cycle and continued at 3-d intervals for a total of five injections. To monitor possible delayed effects of antibody treatment, the posttreatment cycle was also investigated. To evaluate nonspecific effects of the treatment antibody, all monkeys then received iv injections of nonspecific humanized mouse Ig (IgG; ImClone Inc.) using an identical starting point in the cycle, dosage, and regimen as that used for the treatment antibody. Daily blood samples for the measurement of E2, progesterone, FSH, and LH were obtained throughout the entire period to monitor cyclicity in the control, treatment, treatment, and control antibody menstrual cycles. Inhibin A and B were measured in control cycle 2, in the treatment cycle and in the control antibody cycle.
Because anti-VEGF-R2 antibody treatment consistently induced an increase in FSH and LH secretion, we have also evaluated whether this treatment has a direct stimulatory effect on the pituitary. LH and FSH responses to two iv injections (2.5 mg/kg•injection) of anti-VEGF-R2 antibody or control antibody, administered 3 d apart, were monitored in two estrogen-replaced ovariectomized monkeys. For estrogen replacement, one capsule (SILASTIC brand tubing: inside diameter, 3.3 mm; outside diameter, 4.6 mm; length, 30 mm; Dow Corning Corp., Midland, MI) containing E2–17ß (Steraloids, Wilton, NH) was implanted sc under ketamine (5–7 mg/kg) tranquilization, 1 wk before the experiment. Capsules were incubated in distilled water at least 24 h before implantation. Blood samples were obtained daily for gonadotropin measurement, until 3 d after the second antiserum injection.
Assays and statistical analysis
Blood samples were centrifuged, and sera were kept at -20 C until assay. To monitor the follicular process and luteal function, concentrations of E2 and of progesterone were measured daily by chemiluminescent immunoassays (Immulite; Diagnostic Products, Los Angeles, CA). Interassay coefficients of variation (CVs) were 11.9% and 11.1% for E2 and progesterone, respectively. To detect potential effects of antibody treatment on the early follicular growth process (recruitment and selection), inhibin B was measured during control cycle 2, the treatment cycle and the control antibody cycle, by a commercial 2-site enzyme-linked immunoassay (Oxford Bio-Innovation LTD, Oxford, UK) (16, 17). Daily measurements were started in the late luteal phase (2 d before menses) and continued throughout the subsequent follicular phase until ovulation. To characterize (together with E2) the mature follicle, inhibin A levels were also measured by a commercial 2-site enzyme-linked immunoassay (Diagnostic Systems Laboratories, Inc., Webster, TX) (18, 19). Daily inhibin A measurements were started on d 1 of the follicular phase, continued until ovulation, and performed in control cycle 2 and the treatment cycle. Intraassay and interassay CVs, respectively, were 10.8% and 11.6% for inhibin A and 11.6% and 12.5% for inhibin B. LH was measured with a recombinant homologous RIA, as described previously (20). Assay sensitivity (at 95% binding) was 0.06 ng/ml. Intra- and interassay CVs were 7.9 and 13.1%, respectively. FSH was measured with a recombinant cynomolgus monkey FSH RIA kit (provided by Dr. A. F. Parlow, Pituitary Hormones and Antisera Center, Harbor-University of California-Los Angeles Medical Center, Torrance, CA). Synthetic cynomolgus monkey FSH (Genzyme, Cambridge, MA; AFP-6853A) was used for reference and iodination, whereas rabbit antirecombinant cynomolgus monkey FSH antibody (AFP 782594) was used as the first antibody, at a dilution of 1:750,000. In the 10 most recent assays, mean total binding was 27.8%, whereas the slope of the dose-response curve was -2.37. Assay sensitivity (at 95% binding) was 0.045 ng/ml, and mean intra- and interassay CVs were 5.0% and 6.1%, respectively. Dilution curves with pools of sera from ovariectomized rhesus monkeys were parallel to standard curves.
Blood samples were tested for immunogenicity to the anti-VEGF-R2 antibody 1C11, 1 wk before initiation of the study and 1 wk and 6 months after the last dose of antibody was administered. The anti-1C11 assay was a nonspecies specific, double-antigen radiometric assay specific for 1C11. 1C11-coated beads were first incubated with monkey serum samples. Anti-1C11 antibody present in the sample binds bead-bound 1C11 to form an anti-1C11 antibody/1C11 complex. After washing off unbound anti-1C11, 125I-1C11 was then added. Assay results were expressed as nanograms per milliliter of 125I -IMC-1C11 bound and were calculated using the specific activity of the 125I -1 C11. For a positive anti-IMC-1C11 response, the posttreatment value must be 2-fold that of pretreatment baseline.
Cycle parameters, such as length of the follicular and luteal phase, were calculated and compared between control and treatment cycles. Integrated luteal progesterone values (as calculated by trapezoidal analysis of the areas under the daily luteal phase progesterone curves) were compared in control, treatment, and posttreatment cycles. Daily mean FSH, LH, and inhibin B levels and mean preovulatory E2 peaks were also calculated. Comparisons between control and experimental cycles were made by using multiple ANOVA, followed by the Tukey test. Mean inhibin A values in control 2 and treatment cycles were compared by Student’s t test. The level of significance was set at P < 0.05.
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Results |
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). However, follicular phase length in posttreatment and in IgG cycles was similar to that of control cycles. All control and treatment cycles were ovulatory. Mean preovulatory E2 and inhibin A peaks were similar in control and antibody treatment cycles. There were no differences in luteal phase length or in integrated luteal progesterone values among control, treatment, posttreatment, and IgG cycles.
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). Inhibin B levels were 181.0 ± 29.6 on the day before antibody administration, and 66.3 ± 18.4 and 44.5 ± 13.1 pg/ml on d 1 and 2 of treatment, respectively (picograms per milliliter; mean ± SE; P < 0.05 vs. d 1). No significant decrease was observed after IgG treatment. Anti-VEGF-R2 antibody treatment also delayed the normal follicular-phase E2 rise (Fig. 2). Whereas the first significant increase in mean E2 concentration occurred on d 8 and 6 in control cycles 1 and 2, this was delayed to at least d 13 in the treatment cycle. Posttreatment and IgG cycles were similar to control cycles. No preovulatory E2 peaks were reached while animals were under anti-VEGF-R2 antibody treatment.
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). LH concentrations (data not shown) also increased significantly over baseline within 24 h after anti-VEGF-R2 antibody treatment in parallel with FSH. Preovulatory FSH and LH peak levels in the treatment cycle, although delayed, were similar to those in control, posttreatment, and control antibody cycles.
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). In pattern 1 (n = 3), inhibin B levels completely recovered by d 3–5 of treatment, to again reach preinjection concentrations. In this group, E2 reached its preovulatory peak 17–20 d after initiation of treatment. In pattern 2 (n = 3), the inhibin B response differed, as concentrations remained at a nadir for a longer period of time, and recovery was delayed. In this group, E2 attained its preovulatory peak 22–43 d after initiation of treatment. Peak follicular inhibin A levels were similarly delayed in both groups. In contrast, the pattern of the FSH response was similar in all six animals, in that FSH levels remained elevated until the rise in E2.
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illustrates longitudinal hormone changes in the successive two control, anti-VEGF-R2 antibody treatment and posttreatment cycles, and in the IgG cycle in an individual monkey. Treatment with the anti-VEGF-R2 antibody, but not with the control nonspecific IgG, delayed the process of follicular maturation by 27 d in the treatment cycle, compared with the two preceding control cycles, the posttreatment cycle, and the control antibody cycle.
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Anti-VEGF-R2 antibody1C11 antibody formation was detected in the serum of one animal 6 months after the last dose of antibody had been given; all other results were negative.
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Discussion |
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The rise in inhibin B typically observed during the normal luteal-follicular transition period and continuing in the early follicular phase probably reflects the participation of small antral follicles in the process of follicle recruitment (14, 21). Granulosa cells from such follicles are indeed very active in secreting inhibin B in vitro (14). Thus, inhibin B provides a useful functional marker for the activity of the recruited cohort in the early follicular phase. Administration of anti-VEGF-R2 antibody results in a steep decline in inhibin B within 24 h, suggesting that the antibody interferes with the ability of these small antral follicles to produce inhibin B. Because no such decrease follows control IgG administration, one must conclude that angiogenic activity at this early stage of the follicular phase is a requisite for the normal early development of the recruited cohort of follicles.
Anti-VEGF-R2 antibody treatment results in a rapid, 2.5-fold increase in FSH, most probably reflecting the sudden decline in inhibin B. In the normal cycle, the rise in inhibin B during the luteal-follicular transition period is dependent on FSH (21, 22), whereas elevated inhibin B during the early- to midfollicular phase return FSH levels back to baseline (21). The absence of a inhibin B response to increased FSH stimulation in anti-VEGF-R2 antibody-treated animals suggests an inability of the recruited follicle pool to quickly respond to the stimulus. This presumably reflects an interruption of the normal angiogenesis process in these small antral follicles. Subsequently, two distinct response patterns of inhibin B to these antagonistic signals (local inhibition of angiogenesis and pituitary stimulation) emerge. In one group of animals, there is a recovery of inhibin B within 2–3 d, probably reflecting the continuing stimulation by supraphysiological FSH levels and presumably the continuing viability of the antral follicles. In these animals, there is only a moderate delay in the follicular phase. It is of note that the recovery of inhibin B secretion was not able to normalize FSH levels, which remain elevated. This unusual constellation of elevated inhibin B and FSH levels may indicate that other ovarian substances besides inhibin B and E2 might play a role in the feedback regulation of pituitary FSH secretion. In a second group, inhibin B secretion does not recover fully and rapidly, notwithstanding high FSH levels, presumably reflecting a greater degree of dysfunction of the small antral follicles in response to the inhibition of angiogenesis by the antibody. In this group, the follicular phase is delayed for a longer period. After discontinuation of antibody treatment in group 1, there is a rapid rise in E2 to preovulatory levels, indicating that the selection process of a dominant follicle was able to occur rapidly and suggesting relatively minor damage to the recruited follicular cohort. The preovulatory E2 increase in the second group was delayed for a longer period. The absence of an inhibin B response to increased FSH stimulation in these animals suggests a greater damage to the recruited follicle pool, making it unable to respond to the stimulus and perhaps the need to recruit a new cohort. LH levels also rise concomitantly with FSH, although more modestly, after antibody treatment. The reason for this effect is not clear. A pilot experiment, testing for direct pituitary effects of the treatment in two estrogen-replaced monkeys, indicates that anti-VEGF-R2 treatment does not alter LH or FSH release in those animals. Thus, even though the pituitary gland produces VEGF (23), this angiogenic factor does not seem to locally regulate pituitary FSH and LH secretion. This seems to indicate that other ovarian factors might play a role in regulating LH secretion.
Data in this study support conclusions in two previous reports of a critical role of angiogenesis in cyclic ovarian function in the primate. In our laboratory, we had demonstrated that the short-term administration of blocking antibodies to VEGF during the late follicular phase in the rhesus monkey interferes with the final growth stages of the dominant follicle and delays ovulation (8). Other investigators have shown that administration of a VEGF trap at the time of ovulation in the marmoset monkey prevents normal luteal function (24). More specifically, these reports and the present one emphasize the primordial role of VEGF, an important angiogenic factor, and of the VEGF/VEGF-R2 pathway, at critical stages of the menstrual cycle. Observations in the rodent confirm the role of VEGF-R2 in endothelial cell proliferation in corpora lutea (25). In general, it is known that antiangiogenic treatment acts by interfering with the formation of new blood vessels (11). This is also true for follicular and luteal structures in the monkey: for instance, the administration of a VEGF trap during the luteal phase decreases endothelial cell area in corpora lutea as well as in the tertiary follicles present at that time of the cycle (2, 24). In gonadotropin-stimulated hypophysectomized mice treated with anti-VEGF-R2 antibodies, expansion of the follicular vasculature failed to occur (13). Similar studies have not been performed in the early follicular phase. However, because the development of follicles at the early antral stages is absolutely dependent on FSH (26), the persistence of low inhibin B secretion in the face of high FSH levels in the initial days of treatment strongly suggests that the anti-VEGF-R2 antibody rapidly impedes access of FSH to the ovary. If this had not been the case, we would have observed multiple codominant follicles in our animals, as reported by other investigators in normal animals after supraphysiologic FSH levels in the range seen in our experiment (21, 27). This interpretation is in agreement with observations made in an hypophysectomized mouse model, where gonadotropin-driven follicle growth to the preovulatory stage is readily blocked by anti-VEGF-R2 antibody (13). Because the VEGF/VEGF-R2 pathway is not only involved in the regulation of endothelial proliferation but also alters vascular permeability, it is also possible that some of the observed effects in our animals may be related through changes in vascular permeability (9).
Reasons for early recovery of folliculogenesis in some animals may reflect: 1) an antibody dose insufficient to completely block ovarian angiogenesis throughout the whole experiment; 2) ovarian counterregulatory processes activated by persistent FSH and LH stimulation [because gonadotropins have been shown to increase local production of VEGF (28), it is conceivable that supraphysiological gonadotropin levels may have partially overridden the blocking effect of the anti-VEGF-R2 receptor antibody]; and 3) compensatory activity by the VEGF-R1 pathway or by other ovarian angiogenic factors like angiopoietins or e.g. VEGF (7, 29). It has, for instance, been shown that inhibition of VEGF during the luteal phase results in an increased localized expression of angiopoietin-2 mRNA and its receptor, Tie-2 (24). Another reason, i.e. that recovery during treatment may reflect the production of antibodies against the anti-VEGF-R2 antibody, is unlikely because no such antibody was detected during this period. It is important to note that, even though recovery lagged past the end of treatment in half the animals, overall, all monkeys developed an ovulatory follicle. Function of the succeeding corpus luteum was normal, as judged by integrated luteal progesterone levels comparable with those in control and IgG cycles. As opposed to the response to a short-term stress challenge, in which damage frequently extends to the poststress cycle (30), all posttreatment cycles after anti-VEGF-R2 antibody were normal. Thus, antiangiogenic effects in the ovary seem to be entirely reversible, and no long-term adverse effects on ovarian function of this treatment were seen. This finding of complete reversibility is relevant to potential antiangiogenic therapy for different types of cancer, in regard to ovarian effects (31).
In summary, our data clearly indicate that growth of the recruited cohort of small antral follicles in the early follicular phase and the subsequent selection of a dominant follicle are angiogenesis-dependent processes and suggest a specific role for the VEGF/VEGF-R2 pathway in these processes in the nonhuman primate. These data complement a previous report indicating that the final growth stages of the dominant follicle during the late follicular phase are also VEGF-dependent (8). We therefore conclude that angiogenesis and the angiogenic factor VEGF are critical components in the events that lead to normal follicle development during the menstrual cycle.
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Acknowledgments |
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Footnotes |
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Abbreviations: CV, Coefficient of variation; E2, estradiol; VEGF, vascular endothelial growth factor; R2, receptor 2.
Received January 9, 2002.
Accepted for publication March 19, 2002.
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