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Medical Research Council Human Reproductive Sciences Unit (H.M.F., H.W.), Centre for Reproductive Biology, Edinburgh EH16 4SB, United Kingdom; and Regeneron Pharmaceuticals (J.S.R., S.J.W.), Tarrytown, New York 10591
Address all correspondence and requests for reprints to: Hamish M. Fraser, Ph.D., D.Sc., MRC Human Reproductive Sciences Unit, The University of Edinburgh Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, United Kingdom. E-mail: h.fraser{at}hrsu.mrc.ac.uk.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In these experiments, the VEGF pathway has been inhibited using antibodies directed against VEGF itself (8, 9), antibodies to the VEGF receptor VEGFR-2/Flk (10, 11), a VEGF receptor tyrosine kinase inhibitor (12), or by decoy VEGF receptors (13, 14). We employed highly potent receptor-based VEGF antagonists. Initial studies in marmosets used a prototypical receptor-based antagonist, VEGF TrapA40, which comprised the immunoglobulin domains 1–3 of VEGFR-1 fused to the Fc portion of human IgG. Surprisingly, acute systemic administration during the early luteal phase inhibited not only luteal angiogenesis (15) but also follicular angiogenesis (16). In subsequent studies a successor molecule, VEGF TrapR1R2, was employed to evaluate the effects of inhibition of VEGF throughout the follicular phase. These studies confirmed that selective inhibition of VEGF markedly attenuated thecal angiogenesis and restricted follicular growth in the marmoset (17).
The small size of the marmoset restricts the number of blood samples that may be obtained to monitor the concomitant effects of VEGF inhibition on pituitary and ovarian hormones. In contrast, the stump-tailed macaque is an old world primate with a body weight of 12–15 kg and menstrual cycles similar to the human female, whose hormone profiles can be determined by well-established assays from blood samples collected at close intervals. We used this species previously to evaluate the effects of GnRH analogs (18) before initiating clinical investigations in women (e.g. Ref.19). The first objective of the current study was to assess the acute and longer-term effects of a single, iv injection of the VEGF TrapR1R2 at the midfollicular phase in the macaque. This phase was selected for detailed study because the follicle that will eventually ovulate is being selected at this time. Our earlier studies in the marmoset suggested that VEGF-mediated angiogenesis is of crucial importance in the selection and growth of the dominant follicle and that if VEGF action were effectively abrogated, the follicular cycle could not continue and a new phase of follicular recruitment would have to be initiated. A second objective was to determine the minimal dose of VEGF TrapR1R2 that would be required to interrupt follicular development and whether the duration of the subsequent suppression of ovarian function would also be dose related. A third objective was to assess the effects of acute VEGF inhibition during the late follicular phase when the thecal vasculature of the ovulatory follicle is already fully developed.
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Materials and Methods |
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Thirteen adult female stump-tailed macaques aged 7–22 yr and weighing 10–16 kg used in the study were captive bred in the United Kingdom or Holland and housed in a unit opened in 1996 and designed with an emphasis on environmental enrichment (20). The animals moved freely from their living rooms via a tunnel to cages for their water supply and sleeping area and collection of blood samples.
Vaginal swabs were taken with a cotton-tipped applicator each morning and the pattern of menstrual bleeding recorded. The animals were trained to enable blood sample collection by femoral venipuncture without anesthesia and with minimal or no restraint. All the animals in the study had regular ovulatory menstrual cycles as determined from menstrual pattern and serum concentrations of estradiol-17ß and progesterone in blood samples obtained three times per week before treatment. The study was approved by the local Primate Ethical Committee and carried out under a project license granted by the United Kingdom Home Office.
Treatments
Endogenous VEGF was inhibited by administration of VEGF TrapR1R2, a recombinant, chimeric protein comprising Ig domain 2 of human VEGF-R1 and Ig domain 3 of human VEGF-R2, expressed in sequence with the human Fc. Compared with earlier versions of receptor-based fusion proteins, the VEGF TrapR1R2 exhibits greater affinity for VEGF-A (affinity constant 
1 pM) as well as improved bioavailability and pharmacokinetic properties (21). VEGF TrapR1R2 (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) was provided at a concentration of 24.3 mg/ml in 2-ml aliquots in buffer composed of 5 mM phosphate, 5 mM citrate, 100 mM NaCl (pH 6.0), and 0.1% wt/vol Tween 20, with either 20% glycerol or 20% sucrose. Human Fc, for control treatments, was provided at a concentration of 19.7 mg/ml in buffer composed of 40 mM phosphate and 20 mM NaCl (pH 7.4). VEGF Trap vehicle alone also was employed during some control cycles. The compounds were stored at –20 C until required, at which time they were thawed. Any compound remaining was stored at 4 C and used within 2 wk.
In a pilot study in two macaques, a single iv injection of 12.5 mg/kg, VEGF TrapR1R2 was found to effectively inhibit ovarian function for more than 40 d. Therefore, in the main study, we elected to investigate the response to 4.0, 1.0, and 0.25 mg/kg (n = 4 per group) administered as a single dose (iv) during the midfollicular phase, d 6–8 of the cycle. Late-follicular-phase administration was studied at the intermediate dose of 1 mg/kg (n = 4). In comparable control cycles, macaques were treated with either 1 mg/kg human Fc (iv) during the mid- (n = 3) or late follicular phase (n = 2) or vehicle administered during the mid- (n = 3) or late follicular phase (n = 3).
After treatment with VEGF TrapR1R2, vehicle, or Fc, (d 0), blood samples were collected at 0 and 15 min and 4, 6, and 8 h and then daily for the next 12–14 d. Thereafter, blood samples were collected three times per week until normal ovulatory cycles were reestablished, as evidenced by elevation of progesterone levels consistent with luteal values measured in pretreatment cycles for that macaque. Because of limits in numbers of animals available, five animals received two treatments with VEGF TrapR1R2. In addition, one animal had been treated in the pilot study. In this case, antibodies to the VEGF TrapR1R2 were detected during the second treatment cycle, and the affected cycle was excluded from further analysis (see Results).
Because no differences were noted in the effects of vehicle and Fc administration (neither treatment produced an appreciable affect on ovarian or pituitary hormones), control cycles from both control treatment conditions were combined for statistical analyses.
Assays
Estradiol-17ß and progesterone were measured by RIAs as described previously (22), detection limits being 30 pM and 0.7 nM, respectively. LH and FSH were measured by RIAs based on recombinant cynomolgus monkey LH and FSH supplied by the National Hormone and Pituitary Program (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases). FSH was measured using anticynomolgus FSH with a detection limit of 2 µg/liter (National Institute of Child Health and Human Development, rec-mo-FSH-RP-1, AFP-6940A), and interassay coefficient of variance of 12%. LH was measured using a rabbit antiserum to cynomolgus LH (AFP342994) used at a final dilution of 1:750,000. Rec-mo LH-RP-1 (AFP-6936H) was used for radioiodination as instructed and results expressed as micrograms per liter of the same preparation. Assay sensitivity was 0.3 µg/liter and interassay coefficient of variance 11%. Inhibin B was measured throughout the study period in animals in the VEGF Trap treatment groups only. The assay was as described previously (20) and had a detection limit of 10 ng/liter.
VEGF TrapR1R2 was measured by an ELISA, using human VEGF 165 to capture and an antibody to the human Fc region as the reporter (21). Capture of the VEGF TrapR1R2 by VEGF coated on the microplate requires a vacant VEGF binding site. Consequently, this ELISA specifically detects only VEGF Trap that is not already bound to endogenous VEGF. Serum samples were diluted in assay buffer and run against standards also prepared in assay buffer. Each dilution level was assayed, and those that read on the linear part of the standard curve, in which the samples ran parallel to that of the reference standard, were selected for analysis. If in the initial assay, values were below the limit of detection, samples were reassayed neat and the standards spiked with an equivalent volume of mouse serum. Assay sensitivity was 0.14 µg/liter, and interassay variation based on low-, medium-, and high-quality controls were less than 10%. Concentration vs. time curves were constructed from ELISA-generated VEGF Trap values obtained from individual animals. The pharmacokinetic parameter estimates were determined by fitting the serum concentration vs. time profile to a noncompartmental model (WinNonLin, version 2.0, Pharsight Corp., Mountain View, CA).
Data analysis and statistics
The day of ovulation was defined as the day of the LH peak. Peak levels of estradiol were typically noted on the previous day or in some cycles on the same day as the LH peak. In normal cycles, the LH peak was followed within 1 d by a rise in progesterone levels, which were sustained for 2 wk. In pre- and posttreatment cycles in which blood samples were obtained three times per week, gonadotropin and ovarian steroid levels were evaluated to provide a best estimate of the day of ovulation.
Data for ovarian and pituitary hormones as well as VEGF TrapR1R2 concentrations for each individual animal were plotted with reference to the day of treatment (d 0). Data around d 0 were also plotted as mean ± SEM values for each treatment group: beyond the point at which daily blood samples were available (after 12–14 d), mean values were obtained by averaging samples taken from all animals at the nearest equivalent times (e.g. in a group of four, collections of n = 1 at d 20, n = 2 at d 21, and n = 1 at d 22 were averaged and plotted as n = 4 at d 21).
Data for time to ovulation after treatment and effects of treatment on hormone concentrations were subjected to ANOVA using the Prism program 4 for Macintosh (GraphPad Prism, San Diego, CA) followed by Bonferroni’s multiple comparison tests. To determine effects of treatment within an animal, the mean of the three pretreatment values for each hormone was used. The posttreatment period subjected to statistical analysis for each group was based on the observed duration of response to treatment defined as the average period of suppression of estradiol below pretreatment value. The final day of response was the one that preceded three consecutive values above pretreatment value. According to this definition, average duration of response was 30, 17, and 10 d for the 4, 1, and 0.25 mg/kg doses, respectively. Similar estimates of the duration of response were obtained using suppression of inhibin B as an end point. Area under the curve for progesterone and estradiol peaks was compared for pre- and posttreatment cycles. Differences were considered significant at a level of P < 0.05.
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Results |
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A representative example of the hormone profile for vehicle-treated or Fc-treated control cycles is shown in Fig. 1
. After control injections, plasma estradiol levels continued to rise, increasing sharply 4–6 d later, before ovulation. The ovulatory surge in LH/FSH took place 7.2 ± 0.4 d (mean ± SEM) after treatment and was followed by a sustained elevation in plasma progesterone, which reached a plateau 8–12 d post ovulation before falling to follicular phase values around luteal d 14–16. Of the six midfollicular control cycles studied, ovulation was not observed at the anticipated time in one vehicle-treated animal, and this cycle was excluded from further evaluation. This animal had an extended follicular phase of 20 d, but this was distinguished from the response to VEGF Trap treatment in that the delay in ovulation was not accompanied by a suppression of estradiol levels or a prolonged rise in LH and FSH (see below).
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. Pituitary-ovarian function was clearly altered in every case at each dose tested, and the observed changes in hormonal profiles followed a consistent and distinctive pattern. Treatment with VEGF TrapR1R2 not only inhibited the late follicular phase rise in plasma estradiol but also resulted in a rapid decline in estradiol concentrations to early follicular phase levels. Within 2–3 d of injection, a progressive increase in plasma LH and FSH was observed, and the FSH to LH ratio was increased in all animals relative to those normally observed during the preovulatory surge of LH and FSH (Fig. 1).
Comparison of mean data for control and treated cycles is shown in Fig. 2. In the posttreatment period, serum estradiol levels in VEGF Trap-treated cycles were significantly lower than in control cycles for all doses tested (P < 0.0001). A sustained reduction of estradiol levels below pretreatment follicular values was observed in the 4 mg/kg group (P < 0.0001), in which estradiol levels remained significantly below normal follicular levels between d 6 and 28 post treatment. Conversely, VEGF Trap treatment resulted in a significant stimulatory effect (P < 0.01) on LH concentrations at all three dose levels. Serum FSH levels also were significantly higher (P < 0.0001) than control cycle values in all three treatment groups. FSH appeared to rise and reach a plateau slightly more rapidly than LH. The rate of the rise in serum gonadotropins appeared unaffected by dose of VEGF Trap, although peak values seen between 15–20 in the higher dose groups may not have been obtained in the 0.25 mg group as a result of the more rapid recovery.
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Mean peak concentrations of free VEGF TrapR1R2 measured in the first postinjection blood sample at 15 min were 10.8, 34, and 140 mg/liter for the 0.25, 1, and 4 mg/kg groups, respectively. The clearance at the different doses ranged from 5.8 to 8.7 ml/d/kg, and the steady-state volume of distribution was approximately 34.8 ml/kg.
The differential duration of suppression in ovarian function observed at each dose of VEGF TrapR1R2 was temporally correlated with clearance of unbound VEGF Trap from the circulation. The mean estradiol and inhibin B values for each of the three dose groups are plotted in relation to levels of VEGF Trap in Fig. 4. At each dose, inhibin B was suppressed to the detection limit of the assay, whereas estradiol was maintained around early follicular levels until plasma concentrations of VEGF TrapR1R2 fell below approximately 1 mg/liter.
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Characteristics of posttreatment recovery of ovarian cycles
Evaluation of the hormonal profiles on recovery of ovarian function suggested that posttreatment menstrual cycles were characteristically normal in terms of length as well as the pattern and magnitude of pituitary gonadotrophin and ovarian steroid levels. This impression was confirmed by quantitative analyses, which showed that peak preovulatory estradiol levels and area under the curve for progesterone during subsequent luteal phase were not statistically different in the immediate pre- and posttreatment cycles (Table 1).
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In control cycles, bleeding was detected during the first week post treatment in one of six cases. Nine of the 12 macaques treated with VEGF TrapR1R2 during the midfollicular phase exhibited bleeding during this period, likely reflecting the abrupt and sustained reduction in estradiol levels.
Late follicular phase treatment
Where control injections were given during the late follicular phase, serum estradiol levels continued to rise and a distinct LH/FSH surge occurred 1–5 d later, followed by a sustained rise in serum progesterone. In marked contrast, the anticipated preovulatory rise in estradiol and ovulatory progesterone were blocked in all macaques treated with VEGF Trap (1 mg/kg); rather there was a rapid and sustained decrease in plasma estradiol levels (Figs. 6 and 7), which persisted for an average of 19 d in three of the four treated macaques. Treatment was followed within 1 d by a marked increase in LH and FSH secretion (Figs. 6 and 7). In contrast to the midcycle gonadotrophin surge seen in normal and control cycles, the LH/FSH increase produced by administration of VEGF TrapR1R2 was characterized by a marked increase in the FSH to LH ratio. In the remaining animal, the suppression in estradiol was maintained for only 8 d, and the duration of the LH/FSH rise was similarly abbreviated.
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In all four cases, the luteal phase progesterone rise failed to occur at the anticipated time, and progesterone remained at follicular phase levels until after the first posttreatment ovulation, which occurred 32 ± 0.9 d after treatment, a significant delay, compared with control cycles (P < 0.001) in which ovulation occurred 3.4 ± 0.7 d after injection of vehicle or hFc (Fig. 7).
Comparison of mean hormonal data for late follicular phase control and treated cycles is shown in Fig. 7. Serum estradiol levels were significantly reduced, compared with pretreatment values (P < 0.0001) after administration of VEGF TrapR1R2. Progesterone concentrations also were significantly suppressed (P < 0.0001) in the treated cycles relative to controls beyond d 6 (i.e. coincident with the luteal phase rise in progesterone in vehicle and Fc control cycles).
Similar to the observations after treatment in the midfollicular phase, three of the four treated animals exhibited vaginal bleeding during the first week, compared with one of five controls.
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Discussion |
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The present findings in macaques confirm and extend our previous observations on effects of VEGF inhibition on ovarian structure and function in the marmoset, which focused on molecular and cellular changes (16, 17). In marmosets, VEGF inhibition throughout the follicular phase severely inhibits thecal angiogenesis and inhibits the growth of secondary follicles beyond the early antral stage (17). The inhibition of the normal, progressive increase in estradiol levels and blockade of ovulatory progesterone rises observed in the present study after single injections of the VEGF Trap in macaques are consistent with the anatomical changes observed in the marmoset ovary after Trap treatment. Somewhat surprisingly, macaques treated during the late follicular phase also exhibited a marked and persistent decline in estradiol levels. Because the dominant follicle has been selected by this time, this finding indicates that it too is susceptible to VEGF inhibition. Because much of the thecal vasculature of the preovulatory follicle has already been elaborated (23, 24), diminished vascular permeability may play a particularly significant role in the apparent ovulatory failure observed after VEGF inhibition in the late follicular phase. Taken together with the marmoset data, the above observations indicate that the inhibition of follicular maturation produced by administration of VEGF TrapR1R2 is secondary to attenuation of the follicular vascular density and/or permeability, which in turn reduces the availability of growth factors, hormones, lipoproteins for steroid production, nutrients, and oxygen to the growing follicles.
The rapid and persistent rise in LH and FSH levels consistently observed after administration of the VEGF TrapR1R2 is likely a direct consequence of the marked and abrupt attenuation of serum estradiol and inhibin B concentrations produced by VEGF inhibition. A similar scenario was described after administration of anti-VEGFR-2 in the rhesus monkey starting at the early follicular phase (10). This is the expected response to withdrawal of the negative feedback effect of the ovarian steroids and inhibin B on pituitary LH/FSH secretion. In the present study, the rise in gonadotrophin levels was most prompt when VEGF inhibition was initiated in the late follicular phase, perhaps reflecting increased sensitivity to withdrawal of negative feedback at this time. Irrespective of the timing of VEGF Trap administration, the magnitude of the FSH increase was relatively greater than that observed for LH and was similar in magnitude to that observed in the same species after specific inhibition of estradiol (20). However, the tonic elevation in pituitary gonadotrophin levels observed after administration of VEGF TrapR1R2 was ineffective in promoting follicular development in the face of ongoing VEGF inhibition. Rather, restitution of follicular maturation, as evidenced by increasing serum estradiol and inhibin B levels, was evident only when circulating levels of free VEGF TrapR1R2 fell less than 1 mg/liter. Similarly, administration of exogenous gonadotropins failed to stimulate ovarian follicular angiogenesis and growth in mice when endogenous VEGF signaling was inhibited by administration of antibodies against VEGFR-2 (11). These results, together with similar observations in the rhesus macaque, have led to the proposal that the access of FSH to the ovary is impeded when VEGF is inhibited (10). The rapid nature of the decline in inhibin B after inhibition of VEGF observed in the current study and VEGFR-2 in the early follicular phase (10) also suggests that the secretion of proteins from the developing follicle may also be impaired. The more gradual decline in serum estradiol could indicate that steroid secretion is less affected by the most immediate changes in the follicular vasculature produced by VEGF inhibition.
Once unbound VEGF TrapR1R2 levels in the blood fell below efficacious concentrations, normal ovarian function was rapidly restored in all cases. The time required for the recovery of follicular activity was dose dependent, but remarkably consistent within doses, and was followed within 2 wk by ovulation such that the first posttreatment ovulations occurred on average 23, 30, and 43 d after injection of 0.25, 1, or 4 mg/kg of VEGF Trap R1R2, respectively. Once reestablished, posttreatment ovarian cycles were comprised of characteristic follicular and luteal phases of normal length, and hormonal measurements confirmed that the magnitude as well as duration of follicular estradiol and luteal progesterone secretion was not significantly different between pre-and posttreatment cycles. Thus, there was no evidence of a deleterious effect of transient VEGF inhibition on subsequent ovarian cycles. Immature follicles are not likely to be adversely influenced by transient VEGF inhibition, or the resultant increases in serum LH/FSH levels, because little or no angiogenesis is taking place in the theca of small follicles, and their growth and survival is thought to be gonadotropin independent at this stage (24, 25). However, longer-term effects of VEGF inhibition on pituitary-ovarian function cannot be excluded, particularly in the context of chronic treatment.
Intravenous administration of VEGF TrapR1R2 was well tolerated in all cases. Only one macaque produced antibodies to the protein on repeated treatment. This animal had initially received a dose of 12.5 mg/kg. After a second injection of 4 mg/kg, the posttreatment recovery of ovarian function occurred earlier than usual, coincident with a sudden decline in serum levels of free Trap due to the development of antibodies. There was no evidence of concomitant morbidity, and further observation confirmed that there was no appreciable effect on subsequent ovulatory cycles.
An increased incidence of vaginal bleeding was observed during the first week of treatment in all VEGF Trap-treated groups, presumably reflecting the response of the endometrium to suppression of ovarian steroids. Possible direct effects of inhibition of VEGF at the endometrial level have yet to be determined in a menstruating species.
The development of effective inhibitors of angiogenesis, and in particular those that block the actions of VEGF, may open new avenues for the treatment of reproductive disorders characterized by pathological angiogenesis, inflammation, and increased vascular permeability. For example, polycystic ovarian syndrome (PCOS) is characterized by the formation of multiple follicular cysts in which the theca is hyperplastic and hypervascularized and stromal blood flow is increased (26, 27). We elected to study the effects of VEGF inhibition at the midfollicular phase because it is this stage of the cycle that normal follicular size and estradiol production are closest to that observed in the ovaries of women with PCOS. In the normal cycle, administration of the VEGF TrapR1R2 blocks the expected progressive rise in estradiol that ordinarily occurs in the second half of the follicular phase, indicating that VEGF inhibition induces atresia of the recruited follicles at the antral stage of development. Thus, short-term administration of VEGF inhibitors to women with PCOS may cause atresia of accumulated antral follicles and also reduce ovarian vascular permeability, thereby producing benefits similar to those currently derived from ovarian cauterization (28).
Antiangiogenic therapies, particularly those that target VEGF signaling, also have potential applications to other reproductive disorders. For example, ovarian hyperstimulation syndrome (OHSS) is an infrequent but severe complication of the hormonal regimen used to induce follicular growth for in vitro fertilization (12, 29, 30). It is characterized by massive follicular growth and luteinization, associated with a dramatic and global increase in vascular permeability. VEGF is thought to play an important role in the pathogenesis of OHSS (29, 30), and its inhibition may be a valuable therapeutic option (12). Administration of antiangiogenic agents may also find applications in reproductive medicine beyond syndromes characterized by a primary ovarian pathology. For example, uterine conditions, such as endometriosis, fibroids, and menorrhagia, are influenced by ovarian steroids and also involve an angiogenic component. In these situations, administration of an antiangiogenic agent could have dual benefits, as a consequence of reduction in ovarian steroids coupled with a direct antiangiogenic effect at the pathological site.
In conclusion, it has been demonstrated that a single injection of VEGF Trap in the mid- or late follicular phase in the macaque produces a prolonged, dose-related suppression of ovarian function. These results suggest that VEGF Trap affords the opportunity for development of novel treatments for some types of ovarian dysfunction and infertility, particularly those conditions characterized by pathological angiogenesis and excessive vascular permeability, such as PCOS, OHSS, and endometriosis.
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Acknowledgments |
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Footnotes |
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Abbreviations: OHSS, Ovarian hyperstimulation syndrome; PCOS, polycystic ovarian syndrome; VEGF, vascular endothelial growth factor.
Received August 10, 2004.
Accepted November 11, 2004.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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