FERTILITY AND STERILITY Copyright c 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Angiotensin-converting enzyme inhibition reverses luteal phase steroid production in oocyte donors Randy S. Morris, M.D. Richard J. Paulson, M.D. Steven R. Lindheim, M.D. Richard S. Legro, M.D. Rogerio A. Lobo, M.D. Mark V. Sauer, M.D.t Division of Reproductive Endocrinology and In Vitro Fertilization Program at California Medical Center, University of Southern California, Los Angeles, California Objective: To determine whether angiotensin-converting enzyme (ACE) inhibition would affect ovarian steroid synthesis in the oocyte donors undergoing controlled ovarian hyperstimulation (COH). Setting: The IVF program of the University of Southern California. Design: Prospective matched clinical trial. Patients: Twelve oocyte donors were studied in 28 hyperstimulation cycles. Interventions: Donors underwent a standard COH protocol. Follicle aspiration was performed 34 hours after administration of hcg. After the procedure, seven donors were administered the ACE inhibitor, captopril, 6.25 mg orally twice daily for 4 days. The remaining patients served as controls. Main Outcome Measures: Serum E2, P, plasma prorenin, active renin, and angiotensin II (Ang 11). Results: Angiotensin II increased after aspiration in both groups but was significantly lower in those receiving captopril. Peak P in the captopril group was significantly lower than controls (81.8 ± 27.8 versus 208.5 ± 23.9 ng/ml [conversion factor to SI unit, 3.180]). Peak E2 was significantly higher (2,222.4 ± 875.3 versus 425.6 ± 490.4 [conversion factor to SI unit, 3.671]). Active renin and Ang II correlated with P. Conclusions: In stimulated cycles, inhibition of Ang II production appears to raise serum E2 and lower P levels. Angiotensin II, therefore, may have a role in the regulation of ovarian steroid synthesis. Fertil Steril 1995;63:854-8 Key Words: Ovarian-derived prorenin to angiotensin cascade (ODPAC), angiotensin-converting enzyme (ACE) inhibition, captopril, oocyte donation, ovarian steroid synthesis, ovarian hyperstimulation syndrome (OHSS) Aberrations in the ovarian-derived pro renin to angiotensin cascade have been implicated in the Received May 23, 1994; revised and accepted October 26, 1994. * Presented at the Conjoint Meeting of The American Fertility Society and the Canadian Fertility and Andrology Society, October 11 to 14, 1993, Montreal, Quebec, Canada. t Reprint requests: Mark V. Sauer, M.D., University of Southern California School of Medicine, Los Angeles County Women's and Children's Hospital, Division of Reproductive Endocrinology, 1240 North Mission Road, Room L-1013, Los Angeles, California 90033 (FAX: 213-226-3509). 854 Morris et al. ACE inhibition in oocyte donors pathophysiology of ovarian hyperstimulation syndrome (OHSS) (1). Angiotensin II (Ang II) has been demonstrated to increase vascular permeability, the underlying derangement in the development of OHSS (2). It has been suggested that inhibition of Ang II may be useful in the prevention or treatment of OHSS (3). However, Ang II has also been found to alter other aspects of ovarian physiology such as oocyte maturation and ovulation (4), corpus luteum angiogenesis (5), and steroid production (1). Before inhibition of Ang II can be advocated as a potential treatment, these other possible ovarian effects must be explored more fully.
We sought to determine whether use ofthe angiotensin-converting enzyme (ACE) inhibitor, captopril, would have any effect on ovarian steroid synthesis in a group of hyperstimulated patients. We chose to study oocyte donors because they have a very low risk of severe OHSS (Morris RS, Wong IL, Sauer MV, Lobo RA, Paulson RJ, abstract), receive no luteal phase hormone supplementation, and are not transferred embryos. Patients MATERIALS AND METHODS This protocol was approved in advance by the Institutional Review Board of the University of Southern California School of Medicine. Volunteers were recruited from the oocyte donor program at the University of Southern California. Each patient previously had been through at least one donation cycle and was therefore known to have a satisfactory response to gonadotropin stimulation. All donors were between 18 and 35 years of age (mean ± SEM, 25 ± 4 years). Donors were free of any serious medical problems and were not taking any medications during the study cycle except those required for controlled ovarian hyperstimulation (COH). All patients previously had passed routine screening procedures for donation as described previously (6). Stimulation Protocol Down regulation was achieved either in the luteal phase with 1 mgjd leuprolide acetate (LA) for 14 days or in the follicular phase with LA for 14 days and 5 mgjd norethindrone for 5 days (Paulson RJ, Sauer MV, Francis MM, Macaso TM, Lobo RA, abstract) Follicular-phase down regulation was used to assist in coordination with recipient cycles. One patient in each group underwent follicularphase down regulation. Down regulation was considered successful when serum E2 was <30 pgjml (conversion factor to SI unit, 3.671) in the absence of any ovarian cyst> 1.5 cm on transvaginal ultrasound. Controlled ovarian hyperstimulation was then initiated with either FSH (Metrodin; Serono, Norwell, MA) or hmg (Pergonal; Serono). Daily dosage was determined based on previous response and in most cases was between 150 and 225 IU. Cycle monitoring included serum E2 determinations and transvaginal ultrasound. Human chorionic gonadotropin (10,000 IU) was administered after appro- priate follicular development. The day of hcg administration is designated as day o. Follicles were aspirated 34 hours later. ACE Inhibition Seven patients were chosen randomly to receive the ACE inhibitor, captopril (Capoten; Squibb, Princeton, NJ), 6.25 mg orally twice daily starting on the day of aspiration. This low dose was chosen to avoid any possibility of precipitous blood pressure decrements. Treatment was continued for 4 days. An additional five patients were studied who received no medications during this time. Compliance was monitored by pill counting and frequent telephone contact. Collections Blood samples were obtained on the day of hcg administration, the day of aspiration, and again 2 and 4 days later. Venipuncture was performed on the antecubital vein with the patient in a sitting position for ~10 minutes. All blood was collected between 8:00 and 10:00 A.M. Samples for plasma prorenin and active renin were collected into ethylenediaminetetraacetic acid (EDT A) containing tubes at room temperature. Within 30 minutes samples were centrifuged at room temperature at 3,000 X g for 15 minutes duration. Plasma was pipetted into 2-mL cryotubes and immediately plunged and stored in liquid nitrogen until assay. Samples for Ang II were collected into prechilled EDT A containing tubes. Samples were kept on ice until centrifugation in. a refrigerated centrifuge (4 C). Plasma was pipetted into 2-mL cryotubes and immediately plunged and stored in liquid nitrogen until assay. The remaining samples were prepared in the same manner as the plasma prorenin. Assays Plasma active renin levels were determined directly using a commercially available immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). Plasma prorenin was determined after complete activation of the plasma prorenin in the specimen and then finding the difference (plasmaprorenin = total renin - active renin). The interassay and intra -assay coefficients of variation are 9.9% and 2.5%. Angiotensin II was measured using an extraction RIA kit (Nichols Institute Diagnostics). The interassay and intra-assay coefficients of variation are 9.3% and 4.0%. Estra- Morris et al. ACE inhibition in oocyte donors 855
ANGII 100r-------------------------~ CONTROL -CAPTOPRIL 80 60 40 n-----" 20 --- ------"-.--- 2L--------------4------------~6 DAY Figure 1 Angiotensin II levels in the luteal phase. Day 0 = day of hcg administration (*P < 0.05) (conversion factor to SI unit, 0.9560). diol and P were measured by standard RIA methods. The interassay and intra-assay coefficients of variation did not exceed 10% and 5% in any assay. Statistics All data are presented as the mean ± SEM. Changes from baseline within each group were determined using analysis of variance (ANOVA) with the Bonferroni correction for multiple time points. Differences between groups were determined by single-measure ANOVA. Linear regression analysis was used when correlating continuous variables. RESULTS Figure 1 shows the Ang II levels in both groups. Baseline Ang II levels were similar. Although Ang II increased in both groups, levels were significantly lower in those who received captopril (P < 0.05). Levels of plasma prorenin, active renin, and Ang II on day 0 were elevated compared with the luteal phase of unstimulated cycles (Morris RS, Paulson RJ, Lindheim SR, Legro RS, Lobo RA, Sauer MV, unpublished observations). Levels of plasma prorenin and active renin can be seen in Table 1. Estradiol levels were not different between the two groups on either the day of hcg (day 0) or the day of aspiration (day 2). By day 6, however, E2 in the captopril group was significantly higher than controls (P = 0.004, Fig. 2A). By contrast, P levels, although initially similar (8.14 ± 1.1 versus 8.2 ± 1.2 ngjml; conversion factor to SI unit, 3.180; not significant), increased at a slower rate in the captopril group so that by day 6 levels in the captopril group were significantly lower compared with 856 Morris et al. ACE inhibition in oocyte donors Table 1 Levels of Plasma Prorenin and Active Renin After Aspiration* Plasma prorenint:j: Active renint:j: Day Control Captopril Control Captopril 2 847 ± 167 1176 ± 141 27 ± 6 32 ± 6 4 853 ± 212 1232 ± 240 31 ± 6 32 ± 2 6 743 ± 216 1285 ± 325 34 ± 6 43 ± 7 * Values are means ± SEM. t Conversion factor to SI units, 1.667. :j: No significant differences between control and captopril groups by unpaired t-test. the untreated controls (P < 0.05, Fig. 2B). A significant correlation was found to exist between active renin and P (r = 0.473, P = 0.017) and Ang II and P (r = 0.734, P < 0.001). DISCUSSION 2 4 6 DAY I-CAPTOPAIL The importance of ovarian-derived prorenin to angiotensin cascade is uncertain but it has been suggested that it plays a role in steroid modulation. Angiotensin II produced from the kidney renin-angiotensin system has as one of its principle functions the stimulation of aldosterone secretion from the adrenal cortex (7). This is accomplished via Ang II interaction with the cytochrome P-450 sidechain cleavage (Cyt-P450scc) enzyme, which converts cholesterol into pregnenolone (8). Because this enzyme serves as the rate-limiting step in step ng/ml B CONTROL Figure 2 (A), Estradiol levels in the lutealphase (* P = 0.004). (B), Progesterone levels in the luteal phase (*P < 0.05) 0 = day of hcg administration; 2 = day of aspiration (conversion factor to SI units, P, 3.180; E 2, 3.671).
roid synthesis in the adrenal cortex and the ovary, it is feasible that Ang II has a similar function in both. In nonhuman mammals, a number of investigators have shown alterations in steroid synthesis in response to Ang II. Pucell et al. (9) demonstrated an increase in P in cultured rat granulosa cells but the effect was not concentration dependent. On the other hand, Aguilera et al. (10) found decreased P and increased E2 production when Ang II was administered concomitantly with gonadotropins in the rat. When captopril was added, the effect was reversed. The bovine corpus luteum responded by decreasing P secretion with Ang II and increasing it with saralasin (11). Morris and Paulson (1) reported on the effects of Ang II on E2 and P production by luteinized human granulosa cells in culture. In the presence of hcg, Ang II caused a dose-dependent increase in the amount of P produced. This effect was blocked by the addition of saralasin to the cultures. Angiotensin II seemed to have no effect on E2 production. However, when saralasin was added, a substantial increase in E2 was seen, suggesting that Ang II may serve to tonically inhibit E2 secretion. Although this effect was felt to be caused most likely by an inhibition of aromatase, no consistent effect was seen on T levels. Palumbo et al. (12), using a similar model, showed increased levels of all three steroids (E2, P, and T). By contrast, Rainey et al. (13) found that Ang II, either alone or in combination with FSH or LH, did not alter the release of P from fresh or cultured granulosa cells. The results of the present in vivo experiment are parallel to the in vitro results of Morris and Paulson (1). That is, Ang II inhibition resulted in an increase in E2 and a decrease in P levels. It is possible that Ang II has a stimulatory effect on Cyt P450scc just as in the adrenal but in addition has an inhibitory effect on aromatase. Severe OHSS has been associated with elevations in various components of ovarian -derived prorenin to angiotensin cascade. Navot et al. (14) showed that patients with severe OHSS demonstrated enhanced renin activity compared with controls. Ong et al. (15) noted elevations in plasma prorenin, active renin, and aldosterone. In gonadotropin-stimulated cycles, plasma prorenin increases markedly 8 to 16 hours after administration of hcg and peaks in 4 to 6 days (16). Higher levels of renin activity are detectable by 8 days after hcg in those patients who subsequently develop severe OHSS as opposed to those with mild hyperstimulation or controls (15). If ovarian-derived pro renin to angiotensin cascade is involved in the pathogenesis of OHSS, inhibition before this time would seem preferable. The present study examined this time period and reveals that significant changes in the hormonal milieu may be expected if ACE inhibitors are used to attempt prevention of OHSS. Angiotensin-converting enzyme inhibitors have been used successfully to treat other syndromes of increased vascular permeability such as retinopathy and nephropathy (17, 18). However, in the present study none of the patients in either the captopril or the control group developed severe OHSS. Therefore, this study cannot adequately test the efficacy of captopril as a treatment for established OHSS. In conclusion, ACE inhibition is capable of altering ovarian steroid synthesis in the luteal phase of hyperstimulated cycles. Specifically, E2 levels are higher and P levels are lower than in comparable controls. This effect may be of importance if ACE inhibitors are used in the luteal phase of stimulation cycles for IVF or ovulation induction for the treatment or prevention of OHSS. Acknowledgments. Special thanks to Rachel A. McConnell, M.D. (University of Southern California, Division of Reproductive Endocrinology, Los Angeles, CAl for her assistance in data collection. REFERENCES 1. Morris RS, Paulson RJ. Ovarian derived prorenin to angiotensin cascade in human reproduction. Fertil Steril 1994;62:1105-14. 2. Robertson AL, Khairallah P A. Effects of angiotensin II and some analogues on vascular permeability in the rabbit. Circ Res 1972;31:923-7. 3. Navot D, Bergh PA, Laufer N. Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment. Fertil Steril 1992;58:249-61. 4. Pellicer A, Palumbo A, DeCherney AH, Naftolin F. Blockage of ovulation by an angiotensin antagonist. Science 1988;240:1660-1. 5. Fernandez CA, Twickler J, Mead A. Neovascularization produced by angiotensin II. J Lab Clin Med 1985;105:141-5. 6. Sauer MV, Paulson RJ, Macaso TM, Francis-Hernandez M, Lobo RA. Establishment of a nonanonymous donor oocyte program: preliminary experience at the University of Southern California. Fertil Steril 1989;52:433;-6. 7. Peach MJ. Renin-angiotensin system: biochemistry and mechanism of action. Physiol Rev 1977;57:313-70. 8. Kramer RE, Gallant S, Brownie AC. Actions of angiotensin Morris et al. ACE inhibition in oocyte donors 857
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