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Medical Research Council Human Reproductive Sciences Unit (H.M.F., H.W., K.D.M., I.S.), Centre for Reproductive Biology, Edinburgh EH16 4TJ, United Kingdom; and Regeneron Pharmaceuticals (S.J.W.), Tarrytown, New York 10591
Address all correspondence and requests for reprints to: Hamish M. Fraser, D.Sc., Medical Research Council Human Reproductive Sciences Unit, The Queen’s Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. E-mail: h.fraser{at}hrsu.mrc.ac.uk.
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Abstract |
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Objective: The objective of this study was to determine effects of inhibiting VEGF by a VEGF Trap, a receptor-based inhibitor, administered at the early or midluteal phase, on pituitary-ovarian function.
Design: Effects of a single injection of VEGF Trap at three doses in the early luteal phase or a single dose in the midluteal phase were investigated and compared with control cycles.
Setting: This work was conducted in the Primate Unit.
Participants: Eleven stump-tailed macaques with regular ovulatory cycles participated in this study. VEGF Trap was well tolerated, and all completed the study.
Interventions: A single injection of VEGF Trap at a dose of 4, 1, or 0.25 mg/kg was administered in the early luteal phase or 1 mg/kg in the midluteal phase. Controls received vehicle or the constant region of human IgG.
Main Outcome Measures: Changes in serum concentrations of progesterone, estradiol, LH, FSH, inhibin A, VEGF Trap, and menstrual bleeding were the main outcome measures.
Results: Early luteal treatment caused a significant attenuation of the normal serum progesterone and estradiol concentrations, followed by a marked increase in LH and FSH. Inhibin A was not significantly reduced. After 1- and 4-mg/kg doses, progesterone remained suppressed throughout the luteal phase, and premature menstruation occurred; whereas the response to the 0.25-mg/kg dose was transitory, and menstruation was at the normal time. Midluteal treatment also resulted in a significant suppression of progesterone secretion.
Conclusions: VEGF is essential for both the development and maintenance of luteal function.
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Introduction |
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The small size of the marmoset restricts the number of blood samples to monitor the concomitant effects of VEGF inhibition on pituitary and luteal hormones. In contrast, the stump-tailed macaque is a primate with menstrual cycles similar to the human, whose hormone profiles can be determined in detail from daily blood samples. We used this species previously to investigate the gonadotropic regulation of the corpus luteum using GnRH antagonists (15), the response being similar to that subsequently observed in women (16). Recently, we studied this species to evaluate the pituitary-ovarian response to treatment with the potent VEGF antagonist, VEGF TrapR1R2, during the follicular phase, showing it to exert a profound, reversible inhibition of ovarian function, being effective at doses as low as 0.25 mg/kg (12).
The objectives of the current study were: 1) to determine the acute and longer-term effects of a single injection of the VEGF Trap administered at the early luteal phase, the period of intense angiogenesis, on serum concentrations of progesterone, estradiol, inhibin A, LH, and FSH; 2) to determine the minimal dose of VEGF Trap that would be required to attenuate luteal function, and whether the duration of the subsequent suppression of ovarian function would also be dose-related; and 3) to assess the effects of acute VEGF inhibition during the midluteal phase, when angiogenesis is complete and the luteal vasculature established.
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Materials and Methods |
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Eleven stump-tailed macaques, 5–19 yr old and weighing 10–16 kg, were housed as described previously (17). They were trained so that vaginal swabs could be taken, to detect the pattern of menstrual bleeding, and blood samples collected by femoral venipuncture. All had regular ovulatory cycles as determined from menstrual pattern and serum concentrations of estradiol-17ß and progesterone in blood samples obtained three times per week for two cycles before treatment. The study was approved by the local Primate Ethical Committee and carried out under Project License PPL 60/3250 governed by the United Kingdom Home Office.
Treatments
VEGF TrapR1R2 (Regeneron Pharmaceuticals, Inc.) is a recombinant, chimeric protein comprising Ig domains 2 of human VEGF-R1 and 3 of VEGF-R2, expressed in sequence with the human constant region of human IgG (Fc) (18), provided at a concentration of 24.3 mg/ml in 2-ml aliquots in vehicle (12). Control treatments employed human Fc, at a concentration of 19.7 mg/ml in vehicle, or vehicle alone.
VEGF Trap was administered as a single injection (iv) during the early luteal phase at a dose of 4, 1 (n = 4 per group), or 0.25 mg/kg (n = 6). VEGF Trap at the midluteal phase was studied at a dose of 1 mg/kg (n = 4). These doses were chosen because we have previously shown them to be effective when administered during the follicular phase in this species (12). In control cycles, macaques were treated with either 1 mg/kg human Fc (iv) (n = 4) or vehicle (n = 7) during the early phase.
Blood samples were collected at 0, 4, and 8 h, after start of treatment, daily for the duration of the luteal phase, then three times per week until normal ovulatory cycles were reestablished, as evidenced by elevation of progesterone levels consistent with values measured in pretreatment cycles. Because of variation among animals in the magnitude of the progesterone rise, with a lesser variation between cycles, the same animals were used for control and treated cycles. Because of limits in numbers of animals available, most received more than one treatment with VEGF Trap and/or human Fc. Because no differences were noted in the effects of vehicle and Fc, control cycles from both were combined for statistical analyses.
Assays
Estradiol-17ß and progesterone were measured by RIAs as described previously (19), detection limits being 30 pM and 0.7 nM, respectively. LH and FSH were measured by RIAs (12), detection limits being 0.3 µg/liter cynomolgus LH (AFP342994) and 2 µg/liter NICHHD recombinant monkey FSH-RP-1 (AFP-6940A). Inhibin A was measured as described previously and had a detection limit of 5 ng/liter (17).
VEGF Trap was measured by an ELISA, using human VEGF 165 to capture and an antibody to the human Fc region as the reporter (12, 18). Serum samples were diluted in assay buffer and run against standards in assay buffer. Each dilution was assayed, and those that read on the linear part of the standard curve were selected for analysis. If values were below the limit of detection, samples were reassayed neat and the standards spiked with an equivalent volume of macaque serum.
Data analysis
The day of ovulation (luteal d 0) was defined as the day of the LH peak. In normal cycles, the LH peak was followed within 1 d by a rise in progesterone levels, which were sustained for 14–16 d. In pre- and posttreatment cycles where blood samples were obtained three times per week, gonadotropin, as well as ovarian steroid levels, were evaluated to provide a best estimate of the day of ovulation.
Data from the day of ovulation were plotted for the control cycles. Data for ovarian and pituitary hormones, as well as VEGF Trap concentrations for each individual animal, were plotted with reference to the day of treatment (d 0).
Data for effects on hormone concentrations and time to ovulation after treatment were subjected to ANOVA using the Prism program 4 for Macintosh (GraphPad Software, Inc., San Diego, CA) followed by Bonferroni’s multiple-comparison tests. The posttreatment period subjected to statistical analysis for each group was based on the duration of the normal luteal phase. Progesterone area under the curve and estradiol peaks were compared for pre- and posttreatment cycles. Number of days of menstrual bleeding during the 28-d periods before and after were calculated and compared by the paired t test. Differences were considered significant at a level of P < 0.05.
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Results |
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In vehicle- or Fc-treated controls, serum progesterone levels, plotted around the day of ovulation, rose as expected before falling to follicular phase values between d 14 and 16 after ovulation (Fig. 1
). Estradiol and inhibin A levels followed the progesterone profile, although the magnitude of the rise in estradiol was comparatively minor. Serum LH and FSH declined from their peak values on luteal d 0, LH remaining at basal levels for the duration of the luteal phase, whereas FSH levels showed a gradual rise beginning a few days before the end of the luteal phase.
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The exact day of start of treatment with VEGF Trap was determined when results from all hormone assays were available and was subsequently found to range from luteal d 0–4. Because of the acute increases in progesterone taking place at this period, data were divided according to whether treatment commenced on luteal d 0 or 1 (n = 4 at the 4-mg/kg dose and 2 at the 1-mg/kg dose) or between d 2–4 (n = 2 at the 1-mg/kg dose). Effects of 4 and 1 mg/kg VEGF Trap given at luteal d 0 or 1 were similar for the first 2 wk after treatment (the normal duration of the luteal phase) and were therefore combined in Fig. 1
, plotted from the start of treatment.
Progesterone rises were suppressed after treatment, values being significantly different (P < 0.001) on each day between 4 and 11. The small rise in estradiol, also largely a product of the corpus luteum, was also suppressed overall by treatment (P < 0.0001) but was not significantly different on individual days. Although in controls, LH and FSH returned to basal values after the preovulatory surge, they did not return to presurge levels in the treated animals, being significantly increased (P < 0.001) after treatment. Inhibin A values tended to be lower in the treated animals, but the difference was not significant. Similar responses were observed in the two animals receiving 1 mg/kg on d 2 or 4 mg/kg after ovulation (data not shown).
In animals receiving 0.25 mg/kg VEGF Trap, the effect on hormonal profiles was attenuated. To appreciate these more subtle responses, the data from treatment cycles have been superimposed on the control cycle values in Fig. 2. Progesterone was significantly lower than controls overall (P < 0.004) but was not significantly different on individual days. The pattern of progesterone secretion indicated a moderate suppression between d 2–5, followed by a gradual recovery to normal levels during the remainder of the luteal phase. FSH rose in response to treatment and was significantly elevated (P < 0.001) between d 5–7. This pattern was mirrored by LH, but the response was relatively minor and not statistically significant. Inhibin A also tended to be suppressed but was not significantly affected by treatment. There was no evident change in estradiol levels.
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By the midluteal phase, serum progesterone concentrations have reached a plateau before the decline that begins around d 10 after ovulation. In macaques treated with 1 mg/kg VEGF Trap on d 7 or 8, serum progesterone levels began to decline the following day and were significantly lower than controls (P < 0.05) on 2 and 3 d after treatment (Fig. 3). Estradiol concentrations were also significantly suppressed (P < 0.0002) after treatment. Inhibin A levels again tended to be reduced but were not significantly different from controls. There was a gradual rise (P < 0.05) in LH after treatment, whereas FSH rose markedly (P < 0.001).
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Typical examples of individual responses, the duration of response, and recovery cycles in control and treated animals in the early luteal group are shown in Fig. 4 and for the midluteal group in Fig. 5. In controls, the time to ovulation after vehicle or Fc was 29.0 ± 0.4 d. In macaques receiving 0.25 mg/kg VEGF Trap, luteal regression was followed by a follicular phase showing a normal hormone profile with ovulation at 27.8 ± 0.7 d after treatment. In the 1-mg/kg group, elevations in serum LH and FSH were maintained for approximately 3 wk, at which time estradiol began to rise, and the gonadotropin concentrations rapidly normalized, and ovulation occurred at 30.3 ± 1.9 d after treatment in three of the macaques, a time not significantly different from controls. In the remaining animal, recovery of ovarian function, as evidenced by rises in serum estradiol, occurred in a similar time frame, but this was not followed by a rise in progesterone until 51 d after onset of treatment. The estradiol profile suggested that follicular development had been reinitiated, but there was either a failure of ovulation, or maturation of the dominant follicle was delayed. In the group receiving 1 mg/kg VEGF Trap in the midluteal phase, ovulation occurred at 29.2 ± 0.8 d after treatment, similar to the early luteal phase group. In macaques receiving 4 mg/kg VEGF Trap, the effects were more prolonged. Estradiol remained suppressed, whereas LH and FSH were elevated for 30 d after treatment, at which time all three hormones reverted to a characteristic midfollicular profile followed by ovulation 45 ± 1.5 d after treatment. Thus, the 4-mg/kg dose resulted in a significant delay (P < 0.001) in time until ovulation, when compared with all other groups.
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and 5), the time at which VEGF Trap concentrations fell less than 1 mg/liter (Fig. 6). The timing to recovery in the 0.25-mg/kg group was also associated with the point at which VEGF Trap concentrations declined to 1 mg/liter, i.e. by 6 d after treatment (Figs. 2 and 6), but time to ovulation was extended, presumably because of the reinitiation of luteal function exerting a negative feedback effect.
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Effects on menstrual pattern
The number of days of menstrual bleeding was not different between the 28-d intervals pre- and posttreatment in any group. Days of menses pre- and posttreatment were 12 ± 2 vs. 10 ± 4 for 4 mg/kg, 8 ± 1 vs. 12 ± 2 for 1 mg/kg, and 7 ± 1 vs. 5 ± 1 for 0.25 mg/kg doses given at the early luteal phase and 5 ± 1 vs. 5 ± 1 for the 1 mg/kg dose given at the midluteal phase. With respect to timing of menstruation, in eight of the 11 vehicle- or Fc-treated cycles, menstruation occurred on schedule between d 13 and 17 after ovulation; whereas in the remainder, bleeding also occurred during the period of elevated progesterone. In both the 1- and 4-mg/kg VEGF Trap groups, treatment in the early luteal phase resulted in bleeding at various times before d 13. The incidence of bleeding before d 13 was significantly higher in both treatment groups when compared with the controls (P < 0.05; Fisher’s exact test). In the 0.25-mg/kg group, none of the animals bled prematurely. In the group receiving 1 mg/kg at the midluteal phase, two of four animals menstruated early, on d 11 after ovulation.
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Discussion |
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The findings of the effects of inhibition of VEGF during the early luteal phase in macaques confirm and extend our previous observations on effects of VEGF inhibition on cellular and molecular changes related to luteal angiogenesis in the marmoset (13). The inhibition of the normal progressive increase in progesterone levels observed in macaques after single injections of the VEGF Trap at the early luteal phase are consistent with the morphological changes observed in the marmoset corpus luteum after inhibition of VEGF.
The response to the lowest dose studied, 0.25 mg/kg, was of special interest in that, whereas the attenuation of progesterone secretion was initially similar to that seen after the higher doses, the subsequent recovery of serum progesterone levels indicated that effective VEGF inhibition did not extend beyond the first week of treatment and that the corpus luteum was able to recover normal function during the second half of the luteal phase. Although the inhibitory responses on ovarian function agree with our previous report on the effects of these same doses of VEGF Trap administered at the midfollicular phase (12), the comparisons of the response to the 0.25-mg/kg dose reveal a difference in the response of antral follicles and the corpus luteum to transient VEGF inhibition. The recovery of the corpus luteum of the cycle contrasts with the termination of the follicular phase observed after the 0.25-mg/kg dose administered at the midfollicular phase that was indicative of atresia of the most developed follicles. This was followed by a dose-dependent period of ovarian quiescence, leading to a characteristic follicular phase reflecting the recruitment of a new wave of antral follicles and maturation of a dominant follicle (12, 20). Thus, the 1-mg/kg dose suppressed ovulation for a similar period, whether given in the midfollicular or early or midluteal phase (approximately 28 d). However, the 0.25-mg/kg dose resulted in an earlier return to ovulation when given in the midfollicular phase (23 ± 0.8 d) (12) than in the early luteal phase (27.8 ± 0.7 d), a statistically significant difference (P < 0.005). The longer period required for reinitiation of ovulation in the early luteal phase group is attributable to the recovery of luteal function by the midluteal phase, resulting in a suppression of follicular development as in untreated macaques. For all groups, recovery of ovarian function occurred when serum concentrations had declined to 1 mg/liter, as had been observed for the follicular phase study (12).
The response of the pituitary gland after VEGF Trap-induced suppression of the corpus luteum was characterized by a rapid and sustained rise in serum LH and FSH, which persisted until reactivation of follicular estradiol secretion. The trigger for this rise is presumed to be the reduction in negative feedback of secreted products of the corpus luteum. The rise in LH after treatment is a reflection of suppression of estradiol as well as progesterone (21). VEGF inhibition during the follicular phase had been followed by a rapid fall in serum inhibin B that, together with the decline in estradiol, would explain the rise in FSH (11, 12). Although inhibin B is the product of the recruited antral follicles, inhibin A is derived from the corpus luteum in primates and is the principal form of inhibin in the circulation during the luteal phase (17, 22). Like progesterone, it is a product of the hormone-producing cells (2). Therefore, we had anticipated that a decline in inhibin A would be observed in the current experiment preceding an increase in FSH levels. However, only a marginal suppression of inhibin A was observed. How then can the rise in FSH after luteal phase inhibition of VEGF be explained? Although direct evidence for a role of inhibin A in suppressing FSH secretion in primates is controversial (23, 24, 25), estradiol does clearly act to inhibit FSH secretion during the luteal phase (26, 27, 28). Although not showing a marked decline, estradiol remained low during the period of treatment. Finally, although inhibin B levels are comparatively low during the course of the luteal phase, compared with the follicular phase (29), it nevertheless continues to be synthesized by remaining healthy small antral follicles (30, 31). Because inhibin B from this source is highly sensitive to inhibition of VEGF, a fall in inhibin B could also contribute to the observed posttreatment rise in circulating FSH.
The most intriguing observation in the current study concerns the suppressive effects of the VEGF Trap when administered during the midluteal phase. The 1-mg/kg dose led to a significant suppression of serum progesterone levels, albeit not to follicular phase values. This was accompanied by a corresponding compensatory response in FSH secretion. This outcome was not entirely unexpected, because we previously reported a suppression of plasma progesterone in marmosets that received a series of three daily injections of an antibody to VEGF at the midluteal phase (32). However, in that study, the degree of suppression was not as marked and it was also transient, progesterone levels returning to normal before the end of the treatment cycle (Fraser, H. M., unpublished observations). The current results are of added importance because they are in an old-world primate. Although angiogenesis is pronounced in the early luteal phase, by the midluteal phase the luteal microvascular tree is essentially complete (4). Thus, inhibition of angiogenesis is unlikely to be a major contributor to the suppression in progesterone secretion when VEGF inhibition is initiated during the second half of the luteal phase. LH is known to be the major stimulator of progesterone secretion (15), but LH levels do not fall, but rather rise, with VEGF Trap treatment. One possible explanation is that VEGF also is required as a survival factor for the newly formed blood vessels. Alternatively, because VEGF is also a permeability factor, inhibition could lead to decreased ovarian vascular permeability, such that access of circulating LH and LDL cholesterol to hormone-producing cells for progesterone synthesis is impaired.
Because we have shown that inhibition of VEGF is capable of suppressing luteal function at all stages, the question is raised as to the role of VEGF during a fertile cycle where luteal progesterone is required for establishment of pregnancy. In the rodent, blockade of VEGF action by administration of antagonist before and after implantation blocks pregnancy, either by blocking progesterone secretion (33) or a direct effect on the uterus (34, 35). Although administration of antibodies to VEGF during the early luteal phase in the marmoset did not block pregnancy (36), the VEGF Trap may be effective. The possible use of inhibition of angiogenesis and/or vascular permeability as a method of postovulatory fertility control has been considered a potentially useful addition to existing methods (37), and studies should now explore these effects of inhibition of VEGF in higher primates.
Effects on menstrual bleeding showed that the 1- and 4-mg/kg doses administered at the early luteal phase result in premature bleeding, whereas the 0.25-mg/kg dose did not. This indicates that the occurrence of bleeding is a reflection of the decline in progesterone secretion. Little is known about possible direct effects of VEGF antagonists on the primate endometrium, but the duration of posttreatment bleeding was not extended beyond that normally observed during menses, though the VEGF Trap was present in the circulation at levels that effectively suppressed ovarian function up to a month at the 4-mg/kg dose.
In conclusion, a single injection of VEGF Trap in the early or midluteal phase in the macaque leads to a rapid suppression of progesterone secretion, with the interval required for recovery of ovarian function being dose dependent. Taken together with previous studies showing that inhibition of VEGF in macaques prevents follicular development when antagonists are administered in the early (11), mid- (12), or late follicular phase (12, 38), it is now clear that VEGF plays a critical role throughout the ovarian cycle. Continued advances in our ability to stimulate or inhibit angiogenesis should lead to improved treatments for ovarian dysfunction and manipulation of fertility.
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Acknowledgments |
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Footnotes |
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Abbreviations: Fc, Constant region of human IgG; VEGF, vascular endothelial growth factor.
Received May 31, 2005.
Accepted July 14, 2005.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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