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The effect of acetylsalicylic acid and captopril on uterine and ovarian blood flow during the estrous cycle in mares
Theriogenology 61 (2004) 301–309
The effect of acetylsalicylic acid and captopril on uterine and ovarian blood flow during the estrous cycle in mares Heinrich Bollwein*, Frank Weber, Stefanie Steffen, R. Stolla Department of Animal Reproduction, Veterinary College, University of Munich, Ko¨niginstr. 12, 80539 Munich, Germany Received 12 March 2003; accepted 10 April 2003
Abstract In previous studies, transrectal color Doppler sonography was used to demonstrate an increase in genital blood flow resistance in subfertile mares. The objectives of the present study were to determine the effects of an anticoagulant (acetylsalicylic acid) and a vasodilator (captopril) on uterine and ovarian perfusion and plasma progesterone concentrations in cycling mares. From Day 1 to 11 of an estrous cycle (Day 0 ¼ day of ovulation following prostaglandin-induced luteolysis), five Trotter mares were given 2500 mg lactose, 2500 mg ASA, or 50 mg captopril twice daily in their feed (one compound per cycle, in random order). Transrectal color Doppler sonography was used to examine both uterine arteries and the ovarian artery ipsilateral to the corpus luteum once daily, immediately prior to administration of the drug. Blood flow resistance was determined semiquantitatively using the pulsatility index (PI) and plasma progesterone concentrations were determined with an enzyme immunoassay. Compared to the placebo, both ASA and captopril decreased mean PI values of both uterine arteries of all mares. On average, ASA decreased the PI of the uterine arteries by 25%; this was more (P < 0:05) than the average decrease (13%) caused by captopril. Both drugs decreased (P < 0:05) blood flow resistance in the ovarian arteries, although there was no difference (P < 0:05) in their efficacy. In addition, both ASA and captopril increased (P < 0:0001) plasma progesterone concentrations (18 and 17%, respectively). In conclusion, either ASA or captopril improved uterine and ovarian perfusion; however the effects on fertility were not determined. # 2003 Elsevier Inc. All rights reserved. Keywords: Mare; Estrous cycle; Color Doppler sonography; Acetylsalicylic acid; Captopril
* Corresponding author. Tel.: þ49-89-2180-2612; fax: þ49-89-2180-2161. E-mail address: [email protected] (H. Bollwein).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0093-691X(03)00214-0
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1. Introduction Color Doppler sonography has demonstrated that poor uterine perfusion is a cause of infertility in women [1–3]; women with an increased pulsatility index (PI) value in their uterine arteries have lower conception rates. Using transrectal color Doppler sonography, Stolla and Bollwein [4] demonstrated that an increase in the severity of endometrial fibrosis was associated with an increase in uterine blood flow resistance in mares. In women with fertility problems and poor uterine circulation, administration of reproductive steroids [1], anticoagulants or drugs causing vasodilation [2,5] improves uterine perfusion and the rate of implantation. However, Bollwein et al. [6] demonstrated that administration of exogenous estrogen or progesterone to cycling mares decreased uterine perfusion. The objectives of the present study were to determine the effects of an anticoagulant (acetylsalicylic acid) and a vasodilator (captopril) on uterine and ovarian perfusion and plasma progesterone concentrations in cycling mares.
2. Materials and methods Five Trotter mares, aged 8–14 years were used. The oldest mare (A) was multiparous and the remaining four (11, 9, 8 and 7 years for mares (B–D), respectively) were nulliparous. Each mare was examined daily during three estrous cycles (all cycles started with an ovulation preceded by prostaglandin-induced luteolysis). Lactose (placebo), acetylsalicylic acid (ASA), or captopril were administered (in a randomised order) in one cycle each. Although mares were not examined in the cycles following treatment, they mares were examined (with B-mode ultrasonography) daily during estrus to detect ovulation. The day of ovulation (Day 0) was considered the day when the ovulatory follicle was no longer detected. In all cycles, uterine swabs were obtained on the first day of behavioral estrus for bacteriological and cytological examination. A placebo (lactose 2500 mg), ASA (2500 mg) or captopril (50 mg) was administered orally every 12 h (06:00–08:0 h and 18:00–20:00 h) from Day 1 to 11 of the cycle; these cycles were denoted as a placebo, ASA or captopril cycle, respectively. After the Doppler sonographic examination on Day 11, luteolysis was induced by the administration of a PGF2a analogue (Tiaprost, 450 mg i.m.). All medications used were supplied as tablets; they were dissolved in water and mixed with bran and grain for oral administration. The uterine and ovarian blood supply were studied by assessment of both the left and right uterine arteries and the ovarian artery ipsilateral to the ovary with the most recent corpus luteum. The uterine and ovarian arteries ipsilateral to the corpus luteum are referred to as dominant arteries and the uterine artery contralateral to the corpus luteum is referred to as non-dominant artery. The Doppler measurements and calculations of uterine and ovarian blood flow were performed as previously published [7]. Briefly, a Toshiba SSH-140 A ultrasound machine (Toshiba Co., Tokyo, Japan) equipped with a 7 MHz microconvex probe was used. The observations were displayed on line and recorded on videotape. Doppler calculations were performed off-line by using two similar consecutive flow velocity waveforms. Blood flow
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was characterized by the pulsatility index (PI); this was calculated as the ratio of the difference between peak systolic frequency shift (PSF) and minimum frequency shift (MF) to time-averaged maximum frequency shift (TAMF): PI ¼ ðPSF MFÞ=TAMF. The PI increases if the vascular conditions proximal to the site of measurement remain constant and the vascular bed distal to the site of measurement constricts. Conversely a low PI value indicates decreased impedance to blood flow in the distal vasculature [8]. The PI values of two consecutive pulse waves were averaged. Blood samples were collected after each sonographic examination. Plasma was separated within 1 h and frozen at 20 8C until analysis. Progesterone concentration was measured by enzyme immunoassays, as published earlier [9]. Measurements were performed directly using 1 ml plasma (antigen: progesterone-7m-carboxyethylthioetherBSA; enzyme: progesterone-6b-hemisuccinate-HRP). All intra- and interassay variations were <9.5%. Statistical analyses were carried out using the Staview II þ GraphicsTM statistical software package (Abacus Concepts, Inc., Berkeley, CA, USA). Measurements were subjected to analysis of variance of repeated measurements. In addition, Fisher’s protected LSD test was used to determine differences in measurements between dominant and nondominant uterine arteries, between different mares and between days of estrous cycle and treatments within mares. The relationship between PI values of the dominant and nondominant uterine arteries and the relationship between the decrease in PI values of the ovarian arteries and the increase of plasma progesterone in different mares during treatments was measured using Pearson’s correlation and the significance of correlations was established using Fisher’s r to z transformation.
3. Results The bacteriological and cytological results of all uterine swabs were negative. The PI values of the dominant and non-dominant uterine arteries had a good correlation (r ¼ 0:54; P < 0:0001) and were not different (P > 0:05). Thus, the mean of their values was used for calculations (Fig. 1). In all estrous cycles, uterine blood flow had a characteristic pattern (Fig. 1). Mean PI values were highest on Day 1, then decreased (P < 0:05) in early diestrus (until Days 3–5), remained at a constant low level (until Days 6–7), then increased (P < 0:05) until Days 9– 11. Compared to placebo cycles (mean PI S:E:M:: 2:07 0:06), mean PI values of the uterine arteries in the ASA (mean PI: 1:57 0:05) and captopril (mean PI: 1:80 0:06) cycles decreased significantly (P < 0:05; Table 1). On average, ASA decreased mean PI of the uterine arteries by 25%; this was more (P < 0:05) than the average decrease of 13% caused by captopril. A decrease (P < 0:05) in uterine PI values was observed in three of five captopril-treated mares and in all ASA-treated mares. There was no association (P > 0:05) between the level of the PI values of individual mares and the PI decrease after administration of ASA or captopril; the relative decrease was similar in mares with high values (A, E) and in those with low values (B, C, D). In all placebo and treated cycles, the dominant ovarian artery had a characteristic blood flow pattern similar to that of the uterine arteries (Fig. 2). The PI values decreased
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Fig. 1. Mean S:E:M: pulsatility index of both uterine arteries during an estrous cycle in five mares given a placebo, acetylsalicylic acid or captopril twice daily on Days 1–11. Values with letters (a, c, p) are different (P < 0:05) from the corresponding value on the previous day of the estrous cycle.
(P < 0:05) from Days 1 to 3 and then remained at a low level until Day 11. Compared to placebo cycles (mean PI: 2:07 0:06), administration of either ASA (mean PI: 1:57 0:05) or captopril (mean PI: 1:80 0:06) decreased (P < 0:05) blood flow resistance in the dominant ovarian artery (with no significant difference in efficacy between the two compounds). On average, ASA and captopril decreased ovarian PI values 18 and 17%, respectively. Ovarian PI values did not decrease (P > 0:05) after the administration of captopril in two mares (A, B) and of ASA in two mares (A, D) compared to placebo cycles. There was no association (P > 0:05) between the level of the ovarian PI values in the placebo cycle and the PI decrease after administration of ASA or captopril. Plasma progesterone profiles had a normal course in placebo cycles and treated cycles (Fig. 3). Plasma progesterone concentrations increased markedly (P < 0:05) from Days 1 Table 1 Pulsatility index of uterine arteries of five mares (A–E) treated with a placebo, acetylsalicylic acid or captopril during on Days 1 to 11 of estrous cycles Treatment Placebo ASA Captopril
A
B a
2.43 0.19 2.03 0.17b 2.41 0.17a
C a
1.79 0.06 1.22 0.06b 1.41 0.05c
D a
1.97 0.12 1.37 0.07b 1.64 0.07c
E a
Mean a
1.90 0.07 2.25 0.09 1.56 0.10b 1.67 0.06b 1.71 0.10a,b 1.84 0.09c
2.07 0.06a 1.57 0.05b 1.80 0.06c
Within columns, values with different superscripts (a–c) are different (P < 0:05). Values are means S:E:M: of both uterine arteries over 10 days (Days 2–11).
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Fig. 2. Mean S:E:M: pulsatility index of the dominant ovarian artery during an estrous cycle in five mares given a placebo, acetylsalicylic acid or captopril twice daily on Days 1–11. Values with letters (a, c, p) are different (P < 0:05) from the corresponding value on the previous day of the estrous cycle.
Fig. 3. Mean S:E:M: plasma progesterone concentrations during an estrous cycle in five mares given a placebo, acetylsalicylic acid or captopril twice daily on Days 1–11. Values with letters (a, c, p) are different from the corresponding value on the previous day of the estrous cycle (P < 0:05).
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Table 2 Pulsatility index of dominant ovarian arteries of five mares (A–E) treated with a placebo, acetylsalicylic acid or captopril on Days 1–11 of estrous cycles Treatment
A
B a
2.37 0.13 2.33 0.33 2.25 0.23
Placebo ASA Captopril
a a
C a
2.68 0.14 1.88 0.11b 2.64 0.16a
D a
2.69 0.13 1.81 0.09b 2.37 0.10c
E a
3.18 0.18 2.93 0.28a 2.25 0.13b
Mean a
3.38 0.21 2.80 0.11b 2.32 0.12b
2.86 0.05a 2.35 0.11b 2.36 0.05b
Within columns, values with different superscripts (a–c) are different (P < 0:05). Values are means S:E:M: of both uterine arteries over 10 days (Days 2–11).
Table 3 Plasma progesterone concentrations (pmol/l) of five mares (A–E) treated with a placebo, acetylsalicylic acid or captopril on Days 1–11 of estrous cycles Treatment
A
B
C
D
E
Placebo ASA Captopril
68.9 10.6a 91.6 14.1b 81.0 10.8a,b
46.1 4.4a 81.2 7.9b 67.3 8.2c
48.7 5.3a 80.9 9.8b 83.1 8.3b
39.4 5.3a 68.4 9.4a 107.3 11.1b 71.8 6.9a 96.5 9.8b 123.4 15.0b
Mean 54.3 2.1a 86.5 4.7b 90.2 3.1b
Within columns, values with different superscripts (a–c) are different (P < 0:05). Values are means S:E:M: of both uterine arteries over 10 days (Days 2–11).
to 4 and remained at a relatively constant (P > 0:05) and high level for the next few days. Compared to the placebo cycle (mean plasma progesterone concentration: 54:3 2:1 pmol/l) the mean concentration of plasma progesterone increased (P < 0:0001) after administration of ASA (mean plasma progesterone concentration: 86:5 2:1 pmol/l) and captopril (mean plasma progesterone concentration: 90:2 3:1 pmol/l) by an average of 59 and 66%, respectively. There was no difference (P > 0:05) in the effect of ASA and captopril on plasma progesterone concentrations. There was no significant increase in plasma progesterone concentration in one mare (E) treated with ASA and another mare (A) treated with captopril. There was no correlation (P > 0:05) between the increase in plasma progesterone concentration and the decrease in ovarian blood flow resistance in individual mares. For example, in mare (D), the ovarian blood flow resistance did not change (P > 0:05) after administration of ASA; however in the same cycle, plasma progesterone concentrations increased by 272% from 39:4 5:3 to 107:3 1:1 pmol/l (Tables 2 and 3).
4. Discussion To our knowledge, the effect of hyperemia-inducing drugs on uterine and ovarian perfusion has not been investigated in mares. Thus, we tested ASA and captopril, compounds that have been shown to increase blood flow in the genital tract of humans and several other species. The doses of ASA and captopril used in the present study were those used to treat inflammatory conditions and cardiac insufficiency, respectively, in the horse [10,11].
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The positive effects of ASA on uterine and ovarian blood flow observed in this study were in agreement with the results of Wada et al. [5] and Rubinstein et al. [2], who administered ASA just before embryo transfer to women with poor genital perfusion. They attributed the increase in blood flow to inhibition of platelet cyclooxygenase by ASA; this results in inhibition of the synthesis of thromboxane A2, which facilitates platelet aggregation and vasoconstriction. Increased ovarian and uterine blood flow resulted in higher embryo implantation rates in women [2,5]. Furthermore, administration of a cyclooxygenase inhibitor (ibuprofen) improved pregnancy rates in cows that were the recipient of a transferred embryo [12]. Similar to ASA, captopril increased both uterine and ovarian perfusion. Captopril is an angiotensin-converting enzyme (ACE) inhibitor; ACE promotes synthesis of the vasoconstrictor angiotensin II and the production of bradykinin, which enhances contractility of uterine smooth muscle [13]. Because ACE occurs in the equine endometrium [14], it is plausible that captopril also acts as a vasodilator in the uterus and inhibits contractility of the uterine musculature. We know of no other studies of the effects of captopril on genital perfusion during the estrous cycle. In contrast to the results of the present study, experiments using pregnant laboratory animals showed that captopril decreased uterine and uteroplacental circulation [15–17]. These conflicting findings may be attributable to changes in the renin–angiotensin system that occur during pregnancy. Abdul-Karim and Assali [18] found that in pregnant women, the blood pressure system becomes less sensitive to angiotensin II. Furthermore, Ferris and Weir [15] postulated that during pregnancy, angiotensin II causes increased synthesis of prostaglandin E (that increases uterine blood flow). According to Hayashi and Miyamoto [19] and Yoshimura [20], who identified an intrinsic renin–angiotensin system in bovine and rat ovaries, captopril could account for the increase in ovarian blood flow in the dominant ovarian artery. In vitro studies established that renin stimulated the growth of blood vessels in bovine and rat luteal tissues [21–23]. Administration of ASA and captopril also increased plasma progesterone concentrations. Earlier studies using color Doppler sonography [7,24] have shown a positive relationship between ovarian or luteal perfusion and plasma progesterone concentration in cycling mares. Thus, the increase in plasma progesterone concentration observed in the present study may have been due to increased luteal blood flow. However, comparison of the effects of the drugs on ovarian blood flow and their effects on plasma progesterone concentration in individual mares failed to show a correlation between the changes in these parameters. Acetylsalicylic acid and captopril inhibit the synthesis of PGF2a and angiotensin II, respectively, which not inhibits coagulation and causes vasodilation, respectively, but also may stimulate the production of progesterone. Since both PGF2a and angiotensin II directly inhibit progesterone synthesis in luteal cells [19,22,25,26], it is plausible that the positive effects of ASA and captopril were not only due to increased ovarian perfusion. Comparison of the efficacy of ASA and captopril among mares indicated that an increase in uterine perfusion was more reliably caused by ASA than captopril. Acetylsalicylic acid decreased uterine blood flow resistance in all mares, whereas captopril did not cause an increase in uterine perfusion in one mare. Within mares, there was no difference in the effects of ASA and captopril on ovarian blood flow and plasma progesterone concentration. Administration of ASA and captopril resulted in no decrease in the ovarian PI values in two
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mares each and no increase in the plasma progesterone concentration in one mare each. Interestingly, the efficacy of both drugs on uterine and ovarian perfusion was not related to the level of PI values during the placebo cycle; improvement in genital perfusion was observed in treated mares with high as well as those with low uterine and ovarian blood flow resistance values in the placebo cycle. In conclusion although both ASA and captopril increased both uterine and ovarian perfusion in cycling mares, the effects of ASA were more pronounced and consistent than those of captopril. Furthermore, both drugs significantly increased plasma progesterone concentration. Additional studies are underway to investigate whether the fertility of subfertile mares can be improved with ASA.
Acknowledgements The authors thank Heidrun Mayrhofer for expert technical assistance.
References [1] Goswamy RK, Williams G, Steptoe PC. Decreased uterine perfusion—a cause of infertility. Hum Reprod 1988;3:955–9. [2] Rubinstein M, Marazzi A, Polak de Fried E. Low-dose aspirin treatment improves ovarian responsiveness, uterine and ovarian blood flow velocity, implantation, and pregnancy rates in patients undergoing in vitro fertilization: a prospective, randomized, double-blind placebo-controlled assay. Fertil Steril 1999;5: 825–9. [3] Steer CV, Tan SL, Dillon D, Mason BA, Campbell S. Vaginal color Doppler assessment of uterine artery impedance correlates with immunohistochemical markers of endometrial receptivity required for the implantation of an embryo. Fertil Steril 1995;63:101–8. [4] Stolla R, Bollwein H. Color Doppler sonography of the uterine artery in subfertile mares. Pferdeheilkunde 1997;13:547. [5] Wada I, Hsu CC, Williams G, Macnamee MC, Brinsden PR. The benefits of low-dose aspirin therapy in women with impaired uterine perfusion during assisted conception. Hum Reprod 1994;9:1954–7. [6] Bollwein H, Kolberg B, Stolla R. The effect of altrenogest and estradiol benzoate on uterine blood flow in mares. Reprod Dom Anim 2000;35:15 (abstract). [7] Bollwein H, Weber F, Kolberg B, Stolla R. Uterine and ovarian blood flow during the estrous cycle in mares. Theriogenology 2002;57:2129–38. [8] Thompson RS, Trudinger BJ, Colleen CM. Doppler ultrasound waveform indices: A/B ratio, pulsatility index and Pourcelot ratio. Br J Obstet Gynaecol 1988;95:581–8. [9] Prakash BS, Meyer HHD, Schallenberger E, van de Wiel DFM. Development of a sensitive enzymeimmunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique. J Steroid Biochem 1987;28:623–7. [10] Cambridge H, Lees P, Hooke RE, Russell CS. Antithromotic actions of aspirin in the horse. Equine Vet J 1991;23:123–7. [11] Vieth J. Evaluation of the effect of ACE-inhibitors in horses with mitral valve insufficiency. Tiera¨ rztliche Hochschule Hannover, Germany; 2001. [12] Elli M, Gaffuri B, Frigerio A, Zanardelli M, Covini D, Candiani M, et al. Effect of a single dose of ibuprofen lysinate before embryo transfer on pregnancy in cows. Reproduction 2001;121:151–4. [13] Murone C, Chai SY, Mueller Esterl W, Mendelsohn FA, Clements J. Localization of bradikinin B2 receptors in the endometrium of rat uterus and effects of estrogen and progesterone. Endocrinology 1999; 140:3372–82.
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[14] Evans TJ, Ganjam VK, Miller MA, Niswender KD, Krause WJ, Youngquist RS. Autoradiographic and ultrastructural analyses of the role of ACE in the pathogenesis of equine endometrial periglandular fibrosis. In: Proceedings of the Seventh International Symposium on Equine Reproduction, Pretoria, South Africa. p. 105–6. [15] Ferris TF, Weir EK. Effect of captopril on uterine blood flow and prostaglandin E synthesis in pregnant rabbit. J Clin Invest 1983;71:809–15. [16] Mabie WC, Ahokas RA, Sibai BM. Maternal and uteroplacental hemodynamic effects of chronic captopril in the hypertensive, term-pregnant rat. Am J Obstet Gynecol 1990;163:1861–7. [17] Binder ND, Faber JJ. Effects of captopril on bloodpressure, placental blood flow and uterine oxygen consumption in pregnant rabbits. J Pharm Exp Ther 1992;260:294–9. [18] Abdul-Karim R, Assali JG. Pressor response to angiotensin in pregnant and non-pregnant women. J Obstet Gynecol 1961;82:246. [19] Hayashi K, Miyamoto A. Angiotensin II interacts with prostaglandin F2a and endothelin-1 as a local luteolytic factor in the bovine corpus luteum in vitro. Biol Reprod 1999;60:1104–9. [20] Yoshimura Y. The ovarian renin–angiotensin system in reproductive physiology. Front Neuroendocrinol 1997;18:247–91. [21] Fernandez LA, Tarlatzis BC, Rzasa PJ, et al. Renin-like activity in ovarian follicular fluid. Fertil Steril 1985;44:219–23. [22] Stirling D, Magness RR, Stone R, Waterman MR, Simpson ER. Angiotensin II inhibits luteinizing hormone-stimulated cholesterol side chain cleavage expression and stimulates basic fibroblast growth factor expression in bovine luteal cells in primary culture. J Biol Chem 1990;265:5–8. [23] DeVito E, Guardia DC, Cabrera RR. A renin-like enzyme in rat luteal tissue: evidence of local synthesis and its regulation during pregnancy. J Endocrinol 1991;128:43–9. [24] Bollwein H, Mayer R, Weber F, Stolla R. Luteal blood flow during the estrous cycle in mares. Theriogenology 2002;57:2043–51. [25] Niswender GD, Juengel JL, Silva PJ, Rollyson MK, McIntush EW. Mechanisms controlling the function and life span of the corpus luteum. Physiol Rev 2000;80:1–29. [26] Wiltbank MC, Diskin MG, Niswender GD. Differential actions of second messenger systems in the corpus luteum. J Reprod Fertil Suppl 1991;43:65–75.
The effect of acetylsalicylic acid and captopril on uterine and ovarian blood flow during the estrous cycle in mares Heinrich Bollwein*, Frank Weber, Stefanie Steffen, R. Stolla Department of Animal Reproduction, Veterinary College, University of Munich, Ko¨niginstr. 12, 80539 Munich, Germany Received 12 March 2003; accepted 10 April 2003
Abstract In previous studies, transrectal color Doppler sonography was used to demonstrate an increase in genital blood flow resistance in subfertile mares. The objectives of the present study were to determine the effects of an anticoagulant (acetylsalicylic acid) and a vasodilator (captopril) on uterine and ovarian perfusion and plasma progesterone concentrations in cycling mares. From Day 1 to 11 of an estrous cycle (Day 0 ¼ day of ovulation following prostaglandin-induced luteolysis), five Trotter mares were given 2500 mg lactose, 2500 mg ASA, or 50 mg captopril twice daily in their feed (one compound per cycle, in random order). Transrectal color Doppler sonography was used to examine both uterine arteries and the ovarian artery ipsilateral to the corpus luteum once daily, immediately prior to administration of the drug. Blood flow resistance was determined semiquantitatively using the pulsatility index (PI) and plasma progesterone concentrations were determined with an enzyme immunoassay. Compared to the placebo, both ASA and captopril decreased mean PI values of both uterine arteries of all mares. On average, ASA decreased the PI of the uterine arteries by 25%; this was more (P < 0:05) than the average decrease (13%) caused by captopril. Both drugs decreased (P < 0:05) blood flow resistance in the ovarian arteries, although there was no difference (P < 0:05) in their efficacy. In addition, both ASA and captopril increased (P < 0:0001) plasma progesterone concentrations (18 and 17%, respectively). In conclusion, either ASA or captopril improved uterine and ovarian perfusion; however the effects on fertility were not determined. # 2003 Elsevier Inc. All rights reserved. Keywords: Mare; Estrous cycle; Color Doppler sonography; Acetylsalicylic acid; Captopril
* Corresponding author. Tel.: þ49-89-2180-2612; fax: þ49-89-2180-2161. E-mail address: [email protected] (H. Bollwein).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0093-691X(03)00214-0
302
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1. Introduction Color Doppler sonography has demonstrated that poor uterine perfusion is a cause of infertility in women [1–3]; women with an increased pulsatility index (PI) value in their uterine arteries have lower conception rates. Using transrectal color Doppler sonography, Stolla and Bollwein [4] demonstrated that an increase in the severity of endometrial fibrosis was associated with an increase in uterine blood flow resistance in mares. In women with fertility problems and poor uterine circulation, administration of reproductive steroids [1], anticoagulants or drugs causing vasodilation [2,5] improves uterine perfusion and the rate of implantation. However, Bollwein et al. [6] demonstrated that administration of exogenous estrogen or progesterone to cycling mares decreased uterine perfusion. The objectives of the present study were to determine the effects of an anticoagulant (acetylsalicylic acid) and a vasodilator (captopril) on uterine and ovarian perfusion and plasma progesterone concentrations in cycling mares.
2. Materials and methods Five Trotter mares, aged 8–14 years were used. The oldest mare (A) was multiparous and the remaining four (11, 9, 8 and 7 years for mares (B–D), respectively) were nulliparous. Each mare was examined daily during three estrous cycles (all cycles started with an ovulation preceded by prostaglandin-induced luteolysis). Lactose (placebo), acetylsalicylic acid (ASA), or captopril were administered (in a randomised order) in one cycle each. Although mares were not examined in the cycles following treatment, they mares were examined (with B-mode ultrasonography) daily during estrus to detect ovulation. The day of ovulation (Day 0) was considered the day when the ovulatory follicle was no longer detected. In all cycles, uterine swabs were obtained on the first day of behavioral estrus for bacteriological and cytological examination. A placebo (lactose 2500 mg), ASA (2500 mg) or captopril (50 mg) was administered orally every 12 h (06:00–08:0 h and 18:00–20:00 h) from Day 1 to 11 of the cycle; these cycles were denoted as a placebo, ASA or captopril cycle, respectively. After the Doppler sonographic examination on Day 11, luteolysis was induced by the administration of a PGF2a analogue (Tiaprost, 450 mg i.m.). All medications used were supplied as tablets; they were dissolved in water and mixed with bran and grain for oral administration. The uterine and ovarian blood supply were studied by assessment of both the left and right uterine arteries and the ovarian artery ipsilateral to the ovary with the most recent corpus luteum. The uterine and ovarian arteries ipsilateral to the corpus luteum are referred to as dominant arteries and the uterine artery contralateral to the corpus luteum is referred to as non-dominant artery. The Doppler measurements and calculations of uterine and ovarian blood flow were performed as previously published [7]. Briefly, a Toshiba SSH-140 A ultrasound machine (Toshiba Co., Tokyo, Japan) equipped with a 7 MHz microconvex probe was used. The observations were displayed on line and recorded on videotape. Doppler calculations were performed off-line by using two similar consecutive flow velocity waveforms. Blood flow
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was characterized by the pulsatility index (PI); this was calculated as the ratio of the difference between peak systolic frequency shift (PSF) and minimum frequency shift (MF) to time-averaged maximum frequency shift (TAMF): PI ¼ ðPSF MFÞ=TAMF. The PI increases if the vascular conditions proximal to the site of measurement remain constant and the vascular bed distal to the site of measurement constricts. Conversely a low PI value indicates decreased impedance to blood flow in the distal vasculature [8]. The PI values of two consecutive pulse waves were averaged. Blood samples were collected after each sonographic examination. Plasma was separated within 1 h and frozen at 20 8C until analysis. Progesterone concentration was measured by enzyme immunoassays, as published earlier [9]. Measurements were performed directly using 1 ml plasma (antigen: progesterone-7m-carboxyethylthioetherBSA; enzyme: progesterone-6b-hemisuccinate-HRP). All intra- and interassay variations were <9.5%. Statistical analyses were carried out using the Staview II þ GraphicsTM statistical software package (Abacus Concepts, Inc., Berkeley, CA, USA). Measurements were subjected to analysis of variance of repeated measurements. In addition, Fisher’s protected LSD test was used to determine differences in measurements between dominant and nondominant uterine arteries, between different mares and between days of estrous cycle and treatments within mares. The relationship between PI values of the dominant and nondominant uterine arteries and the relationship between the decrease in PI values of the ovarian arteries and the increase of plasma progesterone in different mares during treatments was measured using Pearson’s correlation and the significance of correlations was established using Fisher’s r to z transformation.
3. Results The bacteriological and cytological results of all uterine swabs were negative. The PI values of the dominant and non-dominant uterine arteries had a good correlation (r ¼ 0:54; P < 0:0001) and were not different (P > 0:05). Thus, the mean of their values was used for calculations (Fig. 1). In all estrous cycles, uterine blood flow had a characteristic pattern (Fig. 1). Mean PI values were highest on Day 1, then decreased (P < 0:05) in early diestrus (until Days 3–5), remained at a constant low level (until Days 6–7), then increased (P < 0:05) until Days 9– 11. Compared to placebo cycles (mean PI S:E:M:: 2:07 0:06), mean PI values of the uterine arteries in the ASA (mean PI: 1:57 0:05) and captopril (mean PI: 1:80 0:06) cycles decreased significantly (P < 0:05; Table 1). On average, ASA decreased mean PI of the uterine arteries by 25%; this was more (P < 0:05) than the average decrease of 13% caused by captopril. A decrease (P < 0:05) in uterine PI values was observed in three of five captopril-treated mares and in all ASA-treated mares. There was no association (P > 0:05) between the level of the PI values of individual mares and the PI decrease after administration of ASA or captopril; the relative decrease was similar in mares with high values (A, E) and in those with low values (B, C, D). In all placebo and treated cycles, the dominant ovarian artery had a characteristic blood flow pattern similar to that of the uterine arteries (Fig. 2). The PI values decreased
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Fig. 1. Mean S:E:M: pulsatility index of both uterine arteries during an estrous cycle in five mares given a placebo, acetylsalicylic acid or captopril twice daily on Days 1–11. Values with letters (a, c, p) are different (P < 0:05) from the corresponding value on the previous day of the estrous cycle.
(P < 0:05) from Days 1 to 3 and then remained at a low level until Day 11. Compared to placebo cycles (mean PI: 2:07 0:06), administration of either ASA (mean PI: 1:57 0:05) or captopril (mean PI: 1:80 0:06) decreased (P < 0:05) blood flow resistance in the dominant ovarian artery (with no significant difference in efficacy between the two compounds). On average, ASA and captopril decreased ovarian PI values 18 and 17%, respectively. Ovarian PI values did not decrease (P > 0:05) after the administration of captopril in two mares (A, B) and of ASA in two mares (A, D) compared to placebo cycles. There was no association (P > 0:05) between the level of the ovarian PI values in the placebo cycle and the PI decrease after administration of ASA or captopril. Plasma progesterone profiles had a normal course in placebo cycles and treated cycles (Fig. 3). Plasma progesterone concentrations increased markedly (P < 0:05) from Days 1 Table 1 Pulsatility index of uterine arteries of five mares (A–E) treated with a placebo, acetylsalicylic acid or captopril during on Days 1 to 11 of estrous cycles Treatment Placebo ASA Captopril
A
B a
2.43 0.19 2.03 0.17b 2.41 0.17a
C a
1.79 0.06 1.22 0.06b 1.41 0.05c
D a
1.97 0.12 1.37 0.07b 1.64 0.07c
E a
Mean a
1.90 0.07 2.25 0.09 1.56 0.10b 1.67 0.06b 1.71 0.10a,b 1.84 0.09c
2.07 0.06a 1.57 0.05b 1.80 0.06c
Within columns, values with different superscripts (a–c) are different (P < 0:05). Values are means S:E:M: of both uterine arteries over 10 days (Days 2–11).
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Fig. 2. Mean S:E:M: pulsatility index of the dominant ovarian artery during an estrous cycle in five mares given a placebo, acetylsalicylic acid or captopril twice daily on Days 1–11. Values with letters (a, c, p) are different (P < 0:05) from the corresponding value on the previous day of the estrous cycle.
Fig. 3. Mean S:E:M: plasma progesterone concentrations during an estrous cycle in five mares given a placebo, acetylsalicylic acid or captopril twice daily on Days 1–11. Values with letters (a, c, p) are different from the corresponding value on the previous day of the estrous cycle (P < 0:05).
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Table 2 Pulsatility index of dominant ovarian arteries of five mares (A–E) treated with a placebo, acetylsalicylic acid or captopril on Days 1–11 of estrous cycles Treatment
A
B a
2.37 0.13 2.33 0.33 2.25 0.23
Placebo ASA Captopril
a a
C a
2.68 0.14 1.88 0.11b 2.64 0.16a
D a
2.69 0.13 1.81 0.09b 2.37 0.10c
E a
3.18 0.18 2.93 0.28a 2.25 0.13b
Mean a
3.38 0.21 2.80 0.11b 2.32 0.12b
2.86 0.05a 2.35 0.11b 2.36 0.05b
Within columns, values with different superscripts (a–c) are different (P < 0:05). Values are means S:E:M: of both uterine arteries over 10 days (Days 2–11).
Table 3 Plasma progesterone concentrations (pmol/l) of five mares (A–E) treated with a placebo, acetylsalicylic acid or captopril on Days 1–11 of estrous cycles Treatment
A
B
C
D
E
Placebo ASA Captopril
68.9 10.6a 91.6 14.1b 81.0 10.8a,b
46.1 4.4a 81.2 7.9b 67.3 8.2c
48.7 5.3a 80.9 9.8b 83.1 8.3b
39.4 5.3a 68.4 9.4a 107.3 11.1b 71.8 6.9a 96.5 9.8b 123.4 15.0b
Mean 54.3 2.1a 86.5 4.7b 90.2 3.1b
Within columns, values with different superscripts (a–c) are different (P < 0:05). Values are means S:E:M: of both uterine arteries over 10 days (Days 2–11).
to 4 and remained at a relatively constant (P > 0:05) and high level for the next few days. Compared to the placebo cycle (mean plasma progesterone concentration: 54:3 2:1 pmol/l) the mean concentration of plasma progesterone increased (P < 0:0001) after administration of ASA (mean plasma progesterone concentration: 86:5 2:1 pmol/l) and captopril (mean plasma progesterone concentration: 90:2 3:1 pmol/l) by an average of 59 and 66%, respectively. There was no difference (P > 0:05) in the effect of ASA and captopril on plasma progesterone concentrations. There was no significant increase in plasma progesterone concentration in one mare (E) treated with ASA and another mare (A) treated with captopril. There was no correlation (P > 0:05) between the increase in plasma progesterone concentration and the decrease in ovarian blood flow resistance in individual mares. For example, in mare (D), the ovarian blood flow resistance did not change (P > 0:05) after administration of ASA; however in the same cycle, plasma progesterone concentrations increased by 272% from 39:4 5:3 to 107:3 1:1 pmol/l (Tables 2 and 3).
4. Discussion To our knowledge, the effect of hyperemia-inducing drugs on uterine and ovarian perfusion has not been investigated in mares. Thus, we tested ASA and captopril, compounds that have been shown to increase blood flow in the genital tract of humans and several other species. The doses of ASA and captopril used in the present study were those used to treat inflammatory conditions and cardiac insufficiency, respectively, in the horse [10,11].
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The positive effects of ASA on uterine and ovarian blood flow observed in this study were in agreement with the results of Wada et al. [5] and Rubinstein et al. [2], who administered ASA just before embryo transfer to women with poor genital perfusion. They attributed the increase in blood flow to inhibition of platelet cyclooxygenase by ASA; this results in inhibition of the synthesis of thromboxane A2, which facilitates platelet aggregation and vasoconstriction. Increased ovarian and uterine blood flow resulted in higher embryo implantation rates in women [2,5]. Furthermore, administration of a cyclooxygenase inhibitor (ibuprofen) improved pregnancy rates in cows that were the recipient of a transferred embryo [12]. Similar to ASA, captopril increased both uterine and ovarian perfusion. Captopril is an angiotensin-converting enzyme (ACE) inhibitor; ACE promotes synthesis of the vasoconstrictor angiotensin II and the production of bradykinin, which enhances contractility of uterine smooth muscle [13]. Because ACE occurs in the equine endometrium [14], it is plausible that captopril also acts as a vasodilator in the uterus and inhibits contractility of the uterine musculature. We know of no other studies of the effects of captopril on genital perfusion during the estrous cycle. In contrast to the results of the present study, experiments using pregnant laboratory animals showed that captopril decreased uterine and uteroplacental circulation [15–17]. These conflicting findings may be attributable to changes in the renin–angiotensin system that occur during pregnancy. Abdul-Karim and Assali [18] found that in pregnant women, the blood pressure system becomes less sensitive to angiotensin II. Furthermore, Ferris and Weir [15] postulated that during pregnancy, angiotensin II causes increased synthesis of prostaglandin E (that increases uterine blood flow). According to Hayashi and Miyamoto [19] and Yoshimura [20], who identified an intrinsic renin–angiotensin system in bovine and rat ovaries, captopril could account for the increase in ovarian blood flow in the dominant ovarian artery. In vitro studies established that renin stimulated the growth of blood vessels in bovine and rat luteal tissues [21–23]. Administration of ASA and captopril also increased plasma progesterone concentrations. Earlier studies using color Doppler sonography [7,24] have shown a positive relationship between ovarian or luteal perfusion and plasma progesterone concentration in cycling mares. Thus, the increase in plasma progesterone concentration observed in the present study may have been due to increased luteal blood flow. However, comparison of the effects of the drugs on ovarian blood flow and their effects on plasma progesterone concentration in individual mares failed to show a correlation between the changes in these parameters. Acetylsalicylic acid and captopril inhibit the synthesis of PGF2a and angiotensin II, respectively, which not inhibits coagulation and causes vasodilation, respectively, but also may stimulate the production of progesterone. Since both PGF2a and angiotensin II directly inhibit progesterone synthesis in luteal cells [19,22,25,26], it is plausible that the positive effects of ASA and captopril were not only due to increased ovarian perfusion. Comparison of the efficacy of ASA and captopril among mares indicated that an increase in uterine perfusion was more reliably caused by ASA than captopril. Acetylsalicylic acid decreased uterine blood flow resistance in all mares, whereas captopril did not cause an increase in uterine perfusion in one mare. Within mares, there was no difference in the effects of ASA and captopril on ovarian blood flow and plasma progesterone concentration. Administration of ASA and captopril resulted in no decrease in the ovarian PI values in two
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mares each and no increase in the plasma progesterone concentration in one mare each. Interestingly, the efficacy of both drugs on uterine and ovarian perfusion was not related to the level of PI values during the placebo cycle; improvement in genital perfusion was observed in treated mares with high as well as those with low uterine and ovarian blood flow resistance values in the placebo cycle. In conclusion although both ASA and captopril increased both uterine and ovarian perfusion in cycling mares, the effects of ASA were more pronounced and consistent than those of captopril. Furthermore, both drugs significantly increased plasma progesterone concentration. Additional studies are underway to investigate whether the fertility of subfertile mares can be improved with ASA.
Acknowledgements The authors thank Heidrun Mayrhofer for expert technical assistance.
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