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The relationship between experimentally determined litter size and maternal blood pressure in spontaneously hypertensive rats
Volume 162 Number 3
12. Steer HW. Implantation of the rabbit blastocyst: the invasive phase. J Anat 1971 ; 110:445-62. 13. Enders AC, Schlafke S. Penetration of the uterine epithelium during implantation in the rabbit. Am J Anat 1971; 132:219-40. 14. Davies J, Hoffman LH. Studies of the progestational endometrium of the rabbit. I. Light microscopy, day 0-13 of gonadotropin-induced pseudopregnancy. Am J Anat 1973; 137:423-45. 15. Der ynck R. Transforming growth factor-alpha: structure and biological activities. J Cell Physiol 1986;32:293-304. 16. Brown MJ, Zogg JL, Schult GS, Hilton FK. Increased binding of epidermal growth factor at preimplantation sites in mouse uteri. Endocrinology 1989;124 :2882-8.
Epidermal growth factor receptor during implantation
17. Chakraborty C, Tawfik OW, Dey SK. Epidermal growth factor binding in rat uterus during the peri-implantation period. Biochem Biophys Res Commun 1988; 153:564-9. 18. Jaffe RB. Protein hormones of the placenta, decidua and fetal membranes. In: Yen SSC, RB Jaffe, eds. Reproductive endocrinology: physiology, pathology, and clinical managements. Philadelphia: WB Saunders, 1986:758-69. 19. Hay DL, Lopata A. Chorionic gonadotropin secretion by human embryos in vitro. J Clin Endocrinol Metab 1988;67: 1322-4. 20. Downward J, Yarden Y, Mayes E. Close similarity of the epidermal growth factor receptor and v-erb-B oncogene protein sequence. Nature 1984;307:521-7.
The relationship between experimentally determined litter size and maternal blood pressure in spontaneously hypertensive rats Robert A. Ahokas, PhD, and Baha M. Sihai, MD M emphis, Tennessee Pregnancy lowers blood pressure in hypertensive rats. To evaluate the role of the conceptus in maternal blood pressure regulation, we measured the changes in systolic blood pressure (by tail-cuff plethysmography) throughout gestation and mean arterial pressure, cardiac output, and organ blood flows (with radioactive microspheres) on postmating day 21 for calculation of total peripheral and organ vascular resistances in spontaneously hypertensive rats with litter size surgically adjusted to 0 to 10 conceptuses on postmating day 7. Blood pressure remained elevated in those rats with zero fetuses but decreased during the last week of pregnancy in those rats with three or more fetuses. The magnitude of the decrease was directly related to litter size. At term, cardiac output was positively correlated (r = 0.61; p < 0.001), whereas mean arterial pressure and total peripheral resistance were negatively correlated (r = - 0.74; p < 0 .001 and r = -0.79; P < 0 .001, respectively) with litter size. Resistances of all the vascular beds in the body, except the kidneys, spleen, and hepatic artery were also negatively correlated with fetal number. Thus pregnancy is characterized by a generalized maternal vasodilation, and the fetal/placental unit may playa significant role in modulating maternal vascular tone. (AM J OBSTET GVNECOL 1990;162:841-7.)
Key words: Blood pressure, vascular resistance, pregnancy, spontaneously hypertensive rat
During the course of normal mammalian pregnancy, maternal blood volume and cardiac output increase to meet the demands of the expanding uteroplacental circulation, whereas systemic vascular resistance decreases to maintain perfusion of other maternal organs.' The From the DivlSlon of Maternal-Fetal Medlcme, Department of Obstetncs and G.~necology, and the Department of Ph.~slOlogy and BlOph}sics, UnIVersIty of Tennessee, MemphIS. Supported by the Tennessee Affihate of the Amencan Heart AssociatIOn, Grant No. TN-87-G-9 . R eceived for publuatlOn July 17. 1989; accepted October 17. 1989. R epnllt requests: Robert A. Ahokas. PhD. Department of Obstetrzes and G}necology, UniversIty of T ennessee. M emphlS. MemphlS , TN
38163. 6/1 /17474
net effect of these opposing cardiovascular changes is a moderate decrease or maintenance of blood pressure constant. Although evidence suggests that a blunting of vascular responsiveness to angiotensin II, and perhaps other vasoconstrictors, may play a role in these adaptations, the precise mechanisms are still not completely understood. In hypertensive animals, such as the spontaneously hypertensive rat, the vasodilator effect of pregnancy has a profound blood pressurelowering effect. During the last week of gestation, blood pressure progressively falls and usually reaches normotensive levels by term.'" Thus the spontaneously hypertensive rat is a unique model in which to study the cardiovascular adaptations to pregnancy.
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It has been reported that the number of live-born pups is directly related to the magnitude of the reduction in blood pressure and the perinatal mortality rate is greater in spontaneously hypertensive rats than in normotensive control rats, suggesting that abnormal blood pressure regulation during pregnancy adversely affects reproductive performance. 5 Recently we also reported that litter size was negatively correlated with mean arterial pressure at term in spontaneously hypertensive rats fed a high-carbohydrate, low-protein diet. 6 Furthermore we observed that maternal placental blood flow was also negatively correlated with mean arterial pressure, suggesting that maintenance of hypertension during pregnan~y could adversely affect the fetus. An alternative explanation for the inverse relationship of maternal blood pressure with litter size could be that the conceptus regulates maternal vascular tone. More than two decades ago it was reported that the magnitude of the antihypertensive effect of pregnancy was reduced in renal hypertensive rats by fetal ablation at mid pregnancy with maintenance of viable placentas until term. 7 In addition, extracts of newborn rat kidney homogenate induced a rapid, transient fall in blood pressure when administered intravenously into hypertensive rats." Thus there is evidence to support a role by the conceptus in the regulation of maternal vascular tone during pregnancy. To further test the hypothesis that the fetal! placental unit plays a role in the regulation of maternal blood pressure, this study was undertaken to determine the effect of experimental reduction of litter size shortly after implantation on the changes in maternal blood pressure during the last 2 weeks of pregnancy. In ad-
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Material and methods This project confOl'ms with the "Guiding PrinCiples in the Care and Use of Laboratory Animals" approved hy the Council of the American Physiological SOCiety and with federal laws and regulations. The protocol used was approved by the Unive rsity of Tennessee. Memphis. Animal Care and Use Committee. Virgin female spontaneously hypertensive rats (10 to 12 weeks old) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, Ind .). The animals were housed four per cage in a room with temperature controlled at 22° ± 2° C with the lights on from 7 AM to 7 PM and were fed standard laborato"y rodent chow (Ralston Purina. St. Louis) with tap water to drink as desired. FOI' breeding. females were caged 1: I with mature spontaneously hypertens ive rat male breeders and vaginal smears were checked each morning for the presence of spermatozoa. The day vaginal smears were sperm po~itive was designated as day 0 of gestation and the pregnant rats were sub,equently housed singly in stainless steel wire-bottom cages thnmghout gestation. Systolic blood pressure was measured on days O. 7, and 14 and semidaily during the last week of pregnancy by tail-cuff plethys mograph y after pl'ewarming the rats at 300 C for 15 minutes. On day 7 the rats (n = 40) were light!} anesthetized with methoxyflurane and a midline laparotomy was perf'lrmed. Litter size wa~ adjusted variably to O. 2, 4. G, 8. or 10 conceptuses by aspirating embryos throug-h small inciSIOns in the antime some trial uterine wall.O The uterine wall and abdominal incisions were sutured closed and the rats were injected with penicillin G (30,000 I UI rat, administered intramuscu larl y) a nd returned to their cages. On day 2 I, the day before delivery. 'he ,'ats were ag-ain anesthetized with methox yHut'ane and polyethylene catheters were inserted into the left ventricle of the hearl , through the right carotid artery. a nd into the left fem oral artery. The catheters. filled with heparinized 0.9 % saline solution. were tunneled subcutaneow.ly to the
back of the neck and led out through a small incision , The rats were placed in a Plexiglas restrainer to recover from anesthesia. and the arterial catheter was connected to a Statham P23Gb pressure transducer (Gould Inc., Cleveland, Ohio) for continuous recording of blood pressure on a Gould 22005 physiologic recorder (Gould Inc.. Cleveland). After a recovery period of 2 to 3 hours, cardiac output and organ blood flows were measured according to the radioactive-labeled microsphere technique." Briefly, approximately 1 x 10' microspheres ( 15 ± 3 f.Lm in diameter suspended in 0.9% saline solution With 0.01 % Tween HO added and labeled with tin 11 301' gadolinium 153) were infused into the left ventricle of the heart during a 30-second period while simultaneously withdrawing an arterial blood reference sample at 0.5 ml /min . The arterial blood reference withdrawal was continued for 1 minute after microsphere infusion to ensure that all microspheres in transit were collected. The a rterial blood reference sam pie was transferred to a -v-counting vial. The rat \,'as killed with an overdose of anesthetic and the organs were removed by dissection, weighed, and placed in individual -v-counting vials. The remaining carcass was skinned. weighed, and cut into small pieces. The skin and carcass sections were distributed among several -v-counting vials. The arterial blood reference sample and all organs and tissues were counted in a -v-well counter (Minaxi Gamma Counter A5530; Packard Instrument Co., Inc.. Downers Grove, III.) set at the photopeak of the isotope used. The counts per minute were transferred directly to a DEC PDP 1170 computer (Digital Equipment Corp ., Marlboro, Mass .) for background correction and calculation of cardiac output and organ blood flows by the following formulas: Cardiac output (mil min) = (cpm in the whole rat x 0.5 ml / min)/cpm in the arterial reference ; organ blood How (mIl min) = (cpm in organ x 0.5 ml /min)/cp m in arterial reference. Total peripheral resistance and organ vascular resistances were calculated by dividing mean arterial
844
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pressure by cardiac output and organ blood flows, respectively. The data are presented as mean ± SEM. The systolic blood pressure data were analyzed by two-way analysis of variance, where factor 1 was group (litter size) and factor 2 was gestational age. Differences between means were determined by the least-squares means method. The relationships between litter size and mean arterial pressure, cardiac output, total peripheral resistance, organ blood flows, and organ vascular resistances were analyzed by linear regression analysis. The 95% confidence interval was accepted as statistical significance in all analyses.
Results Fig. 1 illustrates the changes in systolic blood pressure throughout gestation in rats with experimentally adjusted litter size. There was a significant interaction between group and gestational age (F = 7.42; p ~ 0.001). There were no significant differences between groups in systolic blood pressure on the day of conception (day 0). Blood pressure remained high through pbstmating day 21 in those rats from which all the embryos were removed on postmating day 7. Similarly, blood pressure also remained high throughout pregnancy in those rats in which one or two embryos remained and blood pressure was not significantly lower than that of the rats with zero fetuses on postmating day 21. In the rats with litters of three or more fetuses, however, blood pressure fell progressively during the last 7 days of pregnancy. The magnitude of this decrease was directly related to litter size and was greatest in those rats carrying 8 to 10 fetuses. Fig. 2 illustrates the relationship between litter size and maternal hemodynamics on postmating day 21 for the 36 rats from which we were able to obtain cardiac output and organ blood flow data. Mean arterial blood pressure was inversely and significantly (r = -0.74; p ~ 0.001) correlated with litter size, whereas cardiac output was significantly positively
(r = 0.61; p ~ 0.001) correlated with litter size. Therefore total peripheral vascular resistance was negatively (r = -0.79; P ~ 0.001) correlated to conceptus number. Blood flows and resistances of the gastrointestinal tract, the major fraction of the splanchnic circulation, are illustrated in Fig. 3. Blood flow was not correlated with conceptus number in the stomach and large intestine, but blood flow to the small intestine was positively correlated with litter size. The calculated vascular resistances of the gastrointestinal organs were significantly negatively correlated with litter size. Hepatic artery and spleen blood flows and resistances were not significantly related to litter size (data not shown). Cardiac, but not brain, perfusion was borderline significantly positively correlated to litter size, whereas both cardiac and brain vascular resistances were significantly negatively correlated with litter size (Fig. 4). Neither renal blood flow nor resistance was significantly correlated with litter size. Blood flows to the skin and carcass (skeletal muscle and bone) were both significantly positively correlated to conceptus number and their vascular resistances were negatively correlated to conceptus number (Fig. 5). The relationships between reproductive organ hemodynamics and litter size are illustrated in Fig. 6. Ovarian and uterine wall blood flows were not significantly related to conceptus number, but there was a significant positive correlation between placental perfusion and litter size. There was a significant negative correlation between the vascular resistances of all three fractions of reproductive organ blood flow and litter size.
Comment The antihypertensive effect of pregnancy in various models of experimental hypertension, including genetic hypertension, in rats has been well documented."" 10 " The reduction in blood pressure is manifest in the second half of pregnancy and reaches a maximum 1 to 2 days before delivery. Although the
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factors involved are still not known, peripheral vasodilation is responsible for this decrease in blood pressure. I h The present results in spontaneously hypertensive rats confirm these observations. The decrease in blood pressure cannot be solely the result of the number of placentas present because the resistances of virtually all of the vascular beds in the body. not just the uteroplacental circulation, are inversely related to litter size. This ~trongly suggests that the fetal-placental unit plays an important role in regulating maternal systemic vascular tone and blood pressure during pregnancy. The direct relationship between the magnitude of the fall in blood pressure and the naturally occurring fetal number or litter weight was first reported in 1964." It was observed in several models of experimental hypertension in rats that the fall in blood pressure was greatly attenuated in rat~ that delivered few pups compared with those with large litter~. Furthermore, if the
fetuses were destroyed late in pregnancy (postmating day 16) without damage to the placenta, Ihe magnitude of the fall in maternal blood pressure was also greatly reduced. suggesting that it was the fetus that was responsible for the reduction in maternal blood pressure during pregnancy.7 Intraperitoneal transplantation of rat newborn kidney cell suspension into renal hypertensive rats also produced a grad ual decrease in blood pressure, whereas transplantation of placental or newborn liver and spleen cell suspensions had no effect on recipient blood pressure. Intravenous injection of .. crude extract of rat newbOl-n kidney homogenale al~o induced all immediate but tramient fall in blood pressure.' Thus it was proposed that the fetal kidne) s pi a)' an important role in the aI1l ih ypertensive effect 01 pregnancy by secreting some antih yperten~ive substance that is transferred acrm,s the placenta to the maternal circulation. The extensive work by MUIrhead
846
Ahokas and Sibai
et al. 12 demonstrating that adult renomedullary tissue secretes a neutral lipid substance that exerts an antihypertensive action tends to support this hypothesis. Kubota and Yamada 13 also recently reported that plasma from late pregnant spontaneously hypertensive rats injected intravenously into anesthetized male or nonpregnant female spontaneously hypertensive rats produced a marked, transient fall in blood pressure, suggesting the presence of a circulating antihypertensive factor. However, we have not been able to confirm their results. Intravenous bolus injection of termpregnant spontaneously hypertensive rat plasma into conscious, nonpregnant spontaneously hypertensive rats did not acutely induce any change in blood pressure (unpublished results). However, this is not to say that a circulating hypotensive substance does not exist in late pregnancy. The volume of plasma injected relative to the total blood volume of the recipient may have been too small and the dilution of such a factor too great for any effect on blood pressure to be observed. Furthermore, the effect of such a factor may be so short and the substance so labile that continuous infusion is necessary to induce vasodilation. Additional experiments cross-circulating term-pregnant and nonpregnant spontaneously hypertensive rats are in progress to confirm or deny the presence of a circulating hypotensive factor during pregnancy. Alternatively, the fetal-placental unit may regulate maternal vascular tone and blood pressure indirectly rather than directly. Evidence is accumulating indicating' that the function of the corpus luteum is under fetal-placental control in rats. Relaxin, a polypeptide hormone that relaxes uterine smooth muscle and is produced in large amounts by the corpus luteum during the last half of pregnancy in rats, has recently been shown also to have a vasodepressor action when infused chronically into rats. H Furthermore, it has also been shown that corpus luteum production and secretion of relaxin is directly related to fetal-placental number. 15 On the basis of these observations, it is tempting to speculate that endogenous relaxin might playa role in the vasodilating effect of pregnancy. To test this hypothesis, we measured the changes in blood pressure in pregnant spontaneously hypertensive rats and normotensive Wistar-Kyoto rats that were bilaterally ovariectomized on postmating day 13 (pregnancy was maintained by daily administration of 17~-estradiol and progesterone) to eliminate the source of endogenous relaxin. Blood pressure of the ovariectomized rats decreased normally during the last week of gestation, however, and was not significantly different from that of sham-oophorectomized rats. 16 Thus it would seem that endogenous relaxin does not have a vasodilator role in pregnancy. Another possible modulator of maternal vascular
March 1990
Am J Obstet Gynecol
tone during pregnancy may be the steroid 17~ estradiol. Infusion of 17~-estradiol into nonpregnant ovariectomized sheep has been shown to increase cardiac output with a significant reduction in systemic vascular resistance, although mean blood pessure remained unchanged. 17 In addition, 17~-estradiol infusion attenuated the pressor responsiveness to angiotensin II, similar to that seen in pregnancy. There is a marked increase in steroid production after day 12 of pregnancy in rats and it has been clearly shown that a direct relationship exists between experimentally determined conceptus number and the production of estrogens and progesterones during the last half of pregnancy.9 Furthermore, naturally occurring differences in litter size have also been shown to be directly related to serum estradiol level. 18 The probable site of 17~ estradiol production is the corpus luteum itself, because the rat placenta has little aromatizing ability and does not produce estradiol, but the question of how the fetalplacental unit regulates corpus luteum 17~-estradiol secretion remains unanswered. 19 Luteotropic activity of rat placental extracts and pregnant rat serum has been well documented, but the identity of the luteotropic substance is uncertain."o In summary, the results of this study suggest that the fetal/placental unit has a prominent role in the regulation of maternal systemic vascular tone and blood pressure during pregnancy, at least in hypertensive animals. Whether this control is direct (i .e., by the production and secretion of a hypotensive hormone into the maternal circulation) or indirect (i.e., by regulating the function of corpus luteum) remains to be clarified. We are grateful to Mr. Ed B. Cannon for his technical assistance and Mr. Kristopher Arheart for his assistance in the statistical analysis of the data. REFERENCES
1. de Swiet M. The physiology of normal pregnancy. In : Rubin PC, ed. Hypertension in pregnancy. vol 10. Handbook of hypertension. Amsterdam : Elsevier, 1988: I. 2. Takeda T. Experimental study on the blood pressure of pregnant hypertensive rats. I. Effect of pregnancy on the course of experimentally and spontaneously hypertensive rats. Jpn Circ J 1964;28:49-54. 3. Aoi W, Gable D, Cleary RE , Young PCM, Weinberger MH . The antihypertensive effect of pregnancy in spontaneously hypertensive rats. Proc Soc Exp Bioi Med 1976;153:13-5. 4. Lundgren Y, Karlsson K, Ljungblod U. Circulatory changes during pregnancy in spontaneously and renal hypertensive rats. Clin Sci 1979;57:337s-9s. 5. Lorenz RP, Picchia LP, Weisz J, Lloyd T. The relationship between reproductive performance and blood pressure in the spontaneously hypertensive rat. AM J OBSTET GvNECOL 1984;150;519-23. 6. Ahokas RA, Reynolds SL, Anderson GD, Lipshitz J. Uteroplacental blood flow in the hypertensive, termpregnant, spontaneously hypertensive rat. AM J OBSTET GVNECOL 1987;156:1010-5. 7. Takeda T. Experimental study on the blood pressure of
Conceptus regulation of maternal blood pressure
Volume 162 Number 3
8.
9.
10.
11.
12.
13.
pregnant hypertensive rats. II. The mechanism of the fall in blood pressure at late pregnancy in experimentally hypertensive rats. Jpn Circ J 1964;28:55-63. Okamoto K, Takeda T, Tabei R, Nosaka S, Matsufuse R. The depressor principle in the kidney of the newborn rat. I. Some biological and physicochemical properites of the kidney extract of the newborn rat. Jpn Circ J 1964;28: 311-9. Kato H, Morishige WK, Rothchild I. A quantitative relation between the experimentally determined number of conceptuses and corpus luteum activity in the pregnant rat. Endocrinology 1979; 105:846-50. Teeuw AH, deJong W. Time course of decrease in blood pressure and in blood pressure response to vasopressor agents during pregnancy in the rat. Pflugers Arch 1973;341: 197-208. Ahokas RA, Reynolds SL, Wang Y-F, Anderson GD, Lipshitz J. The effect of carbohydrate overfeeding on blood pressure in the pregnant spontaneously hypertensive rat. AM J OBSTET GYNECOL 1986; 155: 1113-8. Muirhead EE, Leach BE, Byers LW, Brooks B, Daniels EG, Hinman JW. Antihypertensive neutral renomedullary lipid (ANRL). In: Fisher JW, ed. Kidney hormones. London: Academic Press, 1970:485. Kubota T, Yamada T. Circulating hypotensive factor in pregnant spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 1981 ;8: 125-32.
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14. St-Louis J, Massicotte G. Chronic decrease of blood pressure by rat relaxin in spontaneously hypertensive rats. Life Sci 1985;37:1351-7. 15. Golos TG, Sherwood OD. Control of corpus luteum function during the second half of pregnancy in the rat: a direct relationship between conceptus number and both serum and ovarian relaxin levels. Endocrinology 1982; 111:872-8. 16. Ahokas RA, Sibai BM, Anderson GD. Lack of evidence of a vasodepressor role for relaxin in spontaneously hypertensive and normotensive pregnant rats. AM J OBSTET GYNECOL 1989;161:618-22. 17. Rosenfeld CR, Jackson GM. Estrogen-induced refractoriness to the pressor effects of infused angiotensin II. AM J OBSTET GYNECOL 1984;148:429-35. 18. Csapo AI, Wiest WG. Plasma steroid levels and ovariectomy-induced placental hypertrophy in rats. Endocrinology 1973;93: 1173-7. 19. Gibori G, Sridaran R. Sites of androgen and estradiol production in the second half of pregnancy in the rat. Bioi Reprod 1981 ;24:249-56. 20. Talamantes F, Ogren L. The placenta as an endocrine organ: polypeptides. In: Knobil E, Neill JD. Ewing LL, et ai, eds. The physiology of reproduction. New York: Raven Press, 1988:2093. (vol 2).
Bound volumes available to subscribers Bound volumes of the AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY are available to subscribers (only) for the 1990 issues from the Publisher, at a cost of $56.00 ($79.00 international) for Vol. 162 (January-June) and Vol. 163 (July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 60 days after publication of the last issue in the volume. The binding is durable buckram with the JOURNAL name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact The C.V. Mosby Company, Circulation Department, 11830 Westline Industrial Drive, St. Louis, Missouri 63146-3318, USA; phone (800) 325-4177, ext. 7351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular JOURNAL subscription.
12. Steer HW. Implantation of the rabbit blastocyst: the invasive phase. J Anat 1971 ; 110:445-62. 13. Enders AC, Schlafke S. Penetration of the uterine epithelium during implantation in the rabbit. Am J Anat 1971; 132:219-40. 14. Davies J, Hoffman LH. Studies of the progestational endometrium of the rabbit. I. Light microscopy, day 0-13 of gonadotropin-induced pseudopregnancy. Am J Anat 1973; 137:423-45. 15. Der ynck R. Transforming growth factor-alpha: structure and biological activities. J Cell Physiol 1986;32:293-304. 16. Brown MJ, Zogg JL, Schult GS, Hilton FK. Increased binding of epidermal growth factor at preimplantation sites in mouse uteri. Endocrinology 1989;124 :2882-8.
Epidermal growth factor receptor during implantation
17. Chakraborty C, Tawfik OW, Dey SK. Epidermal growth factor binding in rat uterus during the peri-implantation period. Biochem Biophys Res Commun 1988; 153:564-9. 18. Jaffe RB. Protein hormones of the placenta, decidua and fetal membranes. In: Yen SSC, RB Jaffe, eds. Reproductive endocrinology: physiology, pathology, and clinical managements. Philadelphia: WB Saunders, 1986:758-69. 19. Hay DL, Lopata A. Chorionic gonadotropin secretion by human embryos in vitro. J Clin Endocrinol Metab 1988;67: 1322-4. 20. Downward J, Yarden Y, Mayes E. Close similarity of the epidermal growth factor receptor and v-erb-B oncogene protein sequence. Nature 1984;307:521-7.
The relationship between experimentally determined litter size and maternal blood pressure in spontaneously hypertensive rats Robert A. Ahokas, PhD, and Baha M. Sihai, MD M emphis, Tennessee Pregnancy lowers blood pressure in hypertensive rats. To evaluate the role of the conceptus in maternal blood pressure regulation, we measured the changes in systolic blood pressure (by tail-cuff plethysmography) throughout gestation and mean arterial pressure, cardiac output, and organ blood flows (with radioactive microspheres) on postmating day 21 for calculation of total peripheral and organ vascular resistances in spontaneously hypertensive rats with litter size surgically adjusted to 0 to 10 conceptuses on postmating day 7. Blood pressure remained elevated in those rats with zero fetuses but decreased during the last week of pregnancy in those rats with three or more fetuses. The magnitude of the decrease was directly related to litter size. At term, cardiac output was positively correlated (r = 0.61; p < 0.001), whereas mean arterial pressure and total peripheral resistance were negatively correlated (r = - 0.74; p < 0 .001 and r = -0.79; P < 0 .001, respectively) with litter size. Resistances of all the vascular beds in the body, except the kidneys, spleen, and hepatic artery were also negatively correlated with fetal number. Thus pregnancy is characterized by a generalized maternal vasodilation, and the fetal/placental unit may playa significant role in modulating maternal vascular tone. (AM J OBSTET GVNECOL 1990;162:841-7.)
Key words: Blood pressure, vascular resistance, pregnancy, spontaneously hypertensive rat
During the course of normal mammalian pregnancy, maternal blood volume and cardiac output increase to meet the demands of the expanding uteroplacental circulation, whereas systemic vascular resistance decreases to maintain perfusion of other maternal organs.' The From the DivlSlon of Maternal-Fetal Medlcme, Department of Obstetncs and G.~necology, and the Department of Ph.~slOlogy and BlOph}sics, UnIVersIty of Tennessee, MemphIS. Supported by the Tennessee Affihate of the Amencan Heart AssociatIOn, Grant No. TN-87-G-9 . R eceived for publuatlOn July 17. 1989; accepted October 17. 1989. R epnllt requests: Robert A. Ahokas. PhD. Department of Obstetrzes and G}necology, UniversIty of T ennessee. M emphlS. MemphlS , TN
38163. 6/1 /17474
net effect of these opposing cardiovascular changes is a moderate decrease or maintenance of blood pressure constant. Although evidence suggests that a blunting of vascular responsiveness to angiotensin II, and perhaps other vasoconstrictors, may play a role in these adaptations, the precise mechanisms are still not completely understood. In hypertensive animals, such as the spontaneously hypertensive rat, the vasodilator effect of pregnancy has a profound blood pressurelowering effect. During the last week of gestation, blood pressure progressively falls and usually reaches normotensive levels by term.'" Thus the spontaneously hypertensive rat is a unique model in which to study the cardiovascular adaptations to pregnancy.
841
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It has been reported that the number of live-born pups is directly related to the magnitude of the reduction in blood pressure and the perinatal mortality rate is greater in spontaneously hypertensive rats than in normotensive control rats, suggesting that abnormal blood pressure regulation during pregnancy adversely affects reproductive performance. 5 Recently we also reported that litter size was negatively correlated with mean arterial pressure at term in spontaneously hypertensive rats fed a high-carbohydrate, low-protein diet. 6 Furthermore we observed that maternal placental blood flow was also negatively correlated with mean arterial pressure, suggesting that maintenance of hypertension during pregnan~y could adversely affect the fetus. An alternative explanation for the inverse relationship of maternal blood pressure with litter size could be that the conceptus regulates maternal vascular tone. More than two decades ago it was reported that the magnitude of the antihypertensive effect of pregnancy was reduced in renal hypertensive rats by fetal ablation at mid pregnancy with maintenance of viable placentas until term. 7 In addition, extracts of newborn rat kidney homogenate induced a rapid, transient fall in blood pressure when administered intravenously into hypertensive rats." Thus there is evidence to support a role by the conceptus in the regulation of maternal vascular tone during pregnancy. To further test the hypothesis that the fetal! placental unit plays a role in the regulation of maternal blood pressure, this study was undertaken to determine the effect of experimental reduction of litter size shortly after implantation on the changes in maternal blood pressure during the last 2 weeks of pregnancy. In ad-
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Material and methods This project confOl'ms with the "Guiding PrinCiples in the Care and Use of Laboratory Animals" approved hy the Council of the American Physiological SOCiety and with federal laws and regulations. The protocol used was approved by the Unive rsity of Tennessee. Memphis. Animal Care and Use Committee. Virgin female spontaneously hypertensive rats (10 to 12 weeks old) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, Ind .). The animals were housed four per cage in a room with temperature controlled at 22° ± 2° C with the lights on from 7 AM to 7 PM and were fed standard laborato"y rodent chow (Ralston Purina. St. Louis) with tap water to drink as desired. FOI' breeding. females were caged 1: I with mature spontaneously hypertens ive rat male breeders and vaginal smears were checked each morning for the presence of spermatozoa. The day vaginal smears were sperm po~itive was designated as day 0 of gestation and the pregnant rats were sub,equently housed singly in stainless steel wire-bottom cages thnmghout gestation. Systolic blood pressure was measured on days O. 7, and 14 and semidaily during the last week of pregnancy by tail-cuff plethys mograph y after pl'ewarming the rats at 300 C for 15 minutes. On day 7 the rats (n = 40) were light!} anesthetized with methoxyflurane and a midline laparotomy was perf'lrmed. Litter size wa~ adjusted variably to O. 2, 4. G, 8. or 10 conceptuses by aspirating embryos throug-h small inciSIOns in the antime some trial uterine wall.O The uterine wall and abdominal incisions were sutured closed and the rats were injected with penicillin G (30,000 I UI rat, administered intramuscu larl y) a nd returned to their cages. On day 2 I, the day before delivery. 'he ,'ats were ag-ain anesthetized with methox yHut'ane and polyethylene catheters were inserted into the left ventricle of the hearl , through the right carotid artery. a nd into the left fem oral artery. The catheters. filled with heparinized 0.9 % saline solution. were tunneled subcutaneow.ly to the
back of the neck and led out through a small incision , The rats were placed in a Plexiglas restrainer to recover from anesthesia. and the arterial catheter was connected to a Statham P23Gb pressure transducer (Gould Inc., Cleveland, Ohio) for continuous recording of blood pressure on a Gould 22005 physiologic recorder (Gould Inc.. Cleveland). After a recovery period of 2 to 3 hours, cardiac output and organ blood flows were measured according to the radioactive-labeled microsphere technique." Briefly, approximately 1 x 10' microspheres ( 15 ± 3 f.Lm in diameter suspended in 0.9% saline solution With 0.01 % Tween HO added and labeled with tin 11 301' gadolinium 153) were infused into the left ventricle of the heart during a 30-second period while simultaneously withdrawing an arterial blood reference sample at 0.5 ml /min . The arterial blood reference withdrawal was continued for 1 minute after microsphere infusion to ensure that all microspheres in transit were collected. The a rterial blood reference sam pie was transferred to a -v-counting vial. The rat \,'as killed with an overdose of anesthetic and the organs were removed by dissection, weighed, and placed in individual -v-counting vials. The remaining carcass was skinned. weighed, and cut into small pieces. The skin and carcass sections were distributed among several -v-counting vials. The arterial blood reference sample and all organs and tissues were counted in a -v-well counter (Minaxi Gamma Counter A5530; Packard Instrument Co., Inc.. Downers Grove, III.) set at the photopeak of the isotope used. The counts per minute were transferred directly to a DEC PDP 1170 computer (Digital Equipment Corp ., Marlboro, Mass .) for background correction and calculation of cardiac output and organ blood flows by the following formulas: Cardiac output (mil min) = (cpm in the whole rat x 0.5 ml / min)/cpm in the arterial reference ; organ blood How (mIl min) = (cpm in organ x 0.5 ml /min)/cp m in arterial reference. Total peripheral resistance and organ vascular resistances were calculated by dividing mean arterial
844
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Mdrch 1990 Am.J Obstet Gynecol
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pressure by cardiac output and organ blood flows, respectively. The data are presented as mean ± SEM. The systolic blood pressure data were analyzed by two-way analysis of variance, where factor 1 was group (litter size) and factor 2 was gestational age. Differences between means were determined by the least-squares means method. The relationships between litter size and mean arterial pressure, cardiac output, total peripheral resistance, organ blood flows, and organ vascular resistances were analyzed by linear regression analysis. The 95% confidence interval was accepted as statistical significance in all analyses.
Results Fig. 1 illustrates the changes in systolic blood pressure throughout gestation in rats with experimentally adjusted litter size. There was a significant interaction between group and gestational age (F = 7.42; p ~ 0.001). There were no significant differences between groups in systolic blood pressure on the day of conception (day 0). Blood pressure remained high through pbstmating day 21 in those rats from which all the embryos were removed on postmating day 7. Similarly, blood pressure also remained high throughout pregnancy in those rats in which one or two embryos remained and blood pressure was not significantly lower than that of the rats with zero fetuses on postmating day 21. In the rats with litters of three or more fetuses, however, blood pressure fell progressively during the last 7 days of pregnancy. The magnitude of this decrease was directly related to litter size and was greatest in those rats carrying 8 to 10 fetuses. Fig. 2 illustrates the relationship between litter size and maternal hemodynamics on postmating day 21 for the 36 rats from which we were able to obtain cardiac output and organ blood flow data. Mean arterial blood pressure was inversely and significantly (r = -0.74; p ~ 0.001) correlated with litter size, whereas cardiac output was significantly positively
(r = 0.61; p ~ 0.001) correlated with litter size. Therefore total peripheral vascular resistance was negatively (r = -0.79; P ~ 0.001) correlated to conceptus number. Blood flows and resistances of the gastrointestinal tract, the major fraction of the splanchnic circulation, are illustrated in Fig. 3. Blood flow was not correlated with conceptus number in the stomach and large intestine, but blood flow to the small intestine was positively correlated with litter size. The calculated vascular resistances of the gastrointestinal organs were significantly negatively correlated with litter size. Hepatic artery and spleen blood flows and resistances were not significantly related to litter size (data not shown). Cardiac, but not brain, perfusion was borderline significantly positively correlated to litter size, whereas both cardiac and brain vascular resistances were significantly negatively correlated with litter size (Fig. 4). Neither renal blood flow nor resistance was significantly correlated with litter size. Blood flows to the skin and carcass (skeletal muscle and bone) were both significantly positively correlated to conceptus number and their vascular resistances were negatively correlated to conceptus number (Fig. 5). The relationships between reproductive organ hemodynamics and litter size are illustrated in Fig. 6. Ovarian and uterine wall blood flows were not significantly related to conceptus number, but there was a significant positive correlation between placental perfusion and litter size. There was a significant negative correlation between the vascular resistances of all three fractions of reproductive organ blood flow and litter size.
Comment The antihypertensive effect of pregnancy in various models of experimental hypertension, including genetic hypertension, in rats has been well documented."" 10 " The reduction in blood pressure is manifest in the second half of pregnancy and reaches a maximum 1 to 2 days before delivery. Although the
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factors involved are still not known, peripheral vasodilation is responsible for this decrease in blood pressure. I h The present results in spontaneously hypertensive rats confirm these observations. The decrease in blood pressure cannot be solely the result of the number of placentas present because the resistances of virtually all of the vascular beds in the body. not just the uteroplacental circulation, are inversely related to litter size. This ~trongly suggests that the fetal-placental unit plays an important role in regulating maternal systemic vascular tone and blood pressure during pregnancy. The direct relationship between the magnitude of the fall in blood pressure and the naturally occurring fetal number or litter weight was first reported in 1964." It was observed in several models of experimental hypertension in rats that the fall in blood pressure was greatly attenuated in rat~ that delivered few pups compared with those with large litter~. Furthermore, if the
fetuses were destroyed late in pregnancy (postmating day 16) without damage to the placenta, Ihe magnitude of the fall in maternal blood pressure was also greatly reduced. suggesting that it was the fetus that was responsible for the reduction in maternal blood pressure during pregnancy.7 Intraperitoneal transplantation of rat newborn kidney cell suspension into renal hypertensive rats also produced a grad ual decrease in blood pressure, whereas transplantation of placental or newborn liver and spleen cell suspensions had no effect on recipient blood pressure. Intravenous injection of .. crude extract of rat newbOl-n kidney homogenale al~o induced all immediate but tramient fall in blood pressure.' Thus it was proposed that the fetal kidne) s pi a)' an important role in the aI1l ih ypertensive effect 01 pregnancy by secreting some antih yperten~ive substance that is transferred acrm,s the placenta to the maternal circulation. The extensive work by MUIrhead
846
Ahokas and Sibai
et al. 12 demonstrating that adult renomedullary tissue secretes a neutral lipid substance that exerts an antihypertensive action tends to support this hypothesis. Kubota and Yamada 13 also recently reported that plasma from late pregnant spontaneously hypertensive rats injected intravenously into anesthetized male or nonpregnant female spontaneously hypertensive rats produced a marked, transient fall in blood pressure, suggesting the presence of a circulating antihypertensive factor. However, we have not been able to confirm their results. Intravenous bolus injection of termpregnant spontaneously hypertensive rat plasma into conscious, nonpregnant spontaneously hypertensive rats did not acutely induce any change in blood pressure (unpublished results). However, this is not to say that a circulating hypotensive substance does not exist in late pregnancy. The volume of plasma injected relative to the total blood volume of the recipient may have been too small and the dilution of such a factor too great for any effect on blood pressure to be observed. Furthermore, the effect of such a factor may be so short and the substance so labile that continuous infusion is necessary to induce vasodilation. Additional experiments cross-circulating term-pregnant and nonpregnant spontaneously hypertensive rats are in progress to confirm or deny the presence of a circulating hypotensive factor during pregnancy. Alternatively, the fetal-placental unit may regulate maternal vascular tone and blood pressure indirectly rather than directly. Evidence is accumulating indicating' that the function of the corpus luteum is under fetal-placental control in rats. Relaxin, a polypeptide hormone that relaxes uterine smooth muscle and is produced in large amounts by the corpus luteum during the last half of pregnancy in rats, has recently been shown also to have a vasodepressor action when infused chronically into rats. H Furthermore, it has also been shown that corpus luteum production and secretion of relaxin is directly related to fetal-placental number. 15 On the basis of these observations, it is tempting to speculate that endogenous relaxin might playa role in the vasodilating effect of pregnancy. To test this hypothesis, we measured the changes in blood pressure in pregnant spontaneously hypertensive rats and normotensive Wistar-Kyoto rats that were bilaterally ovariectomized on postmating day 13 (pregnancy was maintained by daily administration of 17~-estradiol and progesterone) to eliminate the source of endogenous relaxin. Blood pressure of the ovariectomized rats decreased normally during the last week of gestation, however, and was not significantly different from that of sham-oophorectomized rats. 16 Thus it would seem that endogenous relaxin does not have a vasodilator role in pregnancy. Another possible modulator of maternal vascular
March 1990
Am J Obstet Gynecol
tone during pregnancy may be the steroid 17~ estradiol. Infusion of 17~-estradiol into nonpregnant ovariectomized sheep has been shown to increase cardiac output with a significant reduction in systemic vascular resistance, although mean blood pessure remained unchanged. 17 In addition, 17~-estradiol infusion attenuated the pressor responsiveness to angiotensin II, similar to that seen in pregnancy. There is a marked increase in steroid production after day 12 of pregnancy in rats and it has been clearly shown that a direct relationship exists between experimentally determined conceptus number and the production of estrogens and progesterones during the last half of pregnancy.9 Furthermore, naturally occurring differences in litter size have also been shown to be directly related to serum estradiol level. 18 The probable site of 17~ estradiol production is the corpus luteum itself, because the rat placenta has little aromatizing ability and does not produce estradiol, but the question of how the fetalplacental unit regulates corpus luteum 17~-estradiol secretion remains unanswered. 19 Luteotropic activity of rat placental extracts and pregnant rat serum has been well documented, but the identity of the luteotropic substance is uncertain."o In summary, the results of this study suggest that the fetal/placental unit has a prominent role in the regulation of maternal systemic vascular tone and blood pressure during pregnancy, at least in hypertensive animals. Whether this control is direct (i .e., by the production and secretion of a hypotensive hormone into the maternal circulation) or indirect (i.e., by regulating the function of corpus luteum) remains to be clarified. We are grateful to Mr. Ed B. Cannon for his technical assistance and Mr. Kristopher Arheart for his assistance in the statistical analysis of the data. REFERENCES
1. de Swiet M. The physiology of normal pregnancy. In : Rubin PC, ed. Hypertension in pregnancy. vol 10. Handbook of hypertension. Amsterdam : Elsevier, 1988: I. 2. Takeda T. Experimental study on the blood pressure of pregnant hypertensive rats. I. Effect of pregnancy on the course of experimentally and spontaneously hypertensive rats. Jpn Circ J 1964;28:49-54. 3. Aoi W, Gable D, Cleary RE , Young PCM, Weinberger MH . The antihypertensive effect of pregnancy in spontaneously hypertensive rats. Proc Soc Exp Bioi Med 1976;153:13-5. 4. Lundgren Y, Karlsson K, Ljungblod U. Circulatory changes during pregnancy in spontaneously and renal hypertensive rats. Clin Sci 1979;57:337s-9s. 5. Lorenz RP, Picchia LP, Weisz J, Lloyd T. The relationship between reproductive performance and blood pressure in the spontaneously hypertensive rat. AM J OBSTET GvNECOL 1984;150;519-23. 6. Ahokas RA, Reynolds SL, Anderson GD, Lipshitz J. Uteroplacental blood flow in the hypertensive, termpregnant, spontaneously hypertensive rat. AM J OBSTET GVNECOL 1987;156:1010-5. 7. Takeda T. Experimental study on the blood pressure of
Conceptus regulation of maternal blood pressure
Volume 162 Number 3
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pregnant hypertensive rats. II. The mechanism of the fall in blood pressure at late pregnancy in experimentally hypertensive rats. Jpn Circ J 1964;28:55-63. Okamoto K, Takeda T, Tabei R, Nosaka S, Matsufuse R. The depressor principle in the kidney of the newborn rat. I. Some biological and physicochemical properites of the kidney extract of the newborn rat. Jpn Circ J 1964;28: 311-9. Kato H, Morishige WK, Rothchild I. A quantitative relation between the experimentally determined number of conceptuses and corpus luteum activity in the pregnant rat. Endocrinology 1979; 105:846-50. Teeuw AH, deJong W. Time course of decrease in blood pressure and in blood pressure response to vasopressor agents during pregnancy in the rat. Pflugers Arch 1973;341: 197-208. Ahokas RA, Reynolds SL, Wang Y-F, Anderson GD, Lipshitz J. The effect of carbohydrate overfeeding on blood pressure in the pregnant spontaneously hypertensive rat. AM J OBSTET GYNECOL 1986; 155: 1113-8. Muirhead EE, Leach BE, Byers LW, Brooks B, Daniels EG, Hinman JW. Antihypertensive neutral renomedullary lipid (ANRL). In: Fisher JW, ed. Kidney hormones. London: Academic Press, 1970:485. Kubota T, Yamada T. Circulating hypotensive factor in pregnant spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 1981 ;8: 125-32.
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14. St-Louis J, Massicotte G. Chronic decrease of blood pressure by rat relaxin in spontaneously hypertensive rats. Life Sci 1985;37:1351-7. 15. Golos TG, Sherwood OD. Control of corpus luteum function during the second half of pregnancy in the rat: a direct relationship between conceptus number and both serum and ovarian relaxin levels. Endocrinology 1982; 111:872-8. 16. Ahokas RA, Sibai BM, Anderson GD. Lack of evidence of a vasodepressor role for relaxin in spontaneously hypertensive and normotensive pregnant rats. AM J OBSTET GYNECOL 1989;161:618-22. 17. Rosenfeld CR, Jackson GM. Estrogen-induced refractoriness to the pressor effects of infused angiotensin II. AM J OBSTET GYNECOL 1984;148:429-35. 18. Csapo AI, Wiest WG. Plasma steroid levels and ovariectomy-induced placental hypertrophy in rats. Endocrinology 1973;93: 1173-7. 19. Gibori G, Sridaran R. Sites of androgen and estradiol production in the second half of pregnancy in the rat. Bioi Reprod 1981 ;24:249-56. 20. Talamantes F, Ogren L. The placenta as an endocrine organ: polypeptides. In: Knobil E, Neill JD. Ewing LL, et ai, eds. The physiology of reproduction. New York: Raven Press, 1988:2093. (vol 2).
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