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Urinary cGMP levels during pregnancy with and without uterine contractions
European Journal of Obstetrics & Gynecology and Reproductive Biology 63 (1995) 17 19
ELSEVIER
GYNECOLOGY
Urinary cGMP levels during pregnancy with and without uterine contractions J. F r a n c o u a l *'~, J. Taieb a, N. Berkane b, A. L i n d e n b a u m a, R. F r y d m a n b ~'Department o f Bioehemisto,, Antoine Bbelg,re Hospital, 157 rue de la Porte de Trivaux, 92141 Clamart Cedex, France bDepartment o[ Obstetrics and Gynecology, Antoine B&'lbre Hospital, 157 rue de la Porte de Trivaux, 92141 Clamart Cedex, France
Received 13 March 1995; revision received 7 July 1995; accepted 7 July 1995
Abstract Objective: The purpose of this study was to investigate the nitric oxide-cGMP (NO-cGMP) system by urinary cGMP level determinations in pregnant women with and without uterine contractions. Design and subjects: cGMP expressed in nmol/mmol of creatinine was performed by radio immuno-assay (Amersham UK) in urine samples obtained from 94 pregnant women with non-complicated pregnancies. Population A without contractions (n = 62) was divided into three groups according to the gestational age (group I, ~<15 weeks; group II, 16-33 weeks; group III, > 33 weeks). The group III from A population was compared to B population (of the same gestational age) presenting uterine contractions (n = 32). Results: In A population, no significant urinary cGMP level differences were observed whatever the gestational age. Nevertheless, the comparison between patients with or without uterine contractions (A III and B populations) showed a significant difference by a variance analysis (P < 0.05). Lower levels of cGMP were seen when uterine contractions occured. Conclusion: Urinary cGMP levels are significantly decreased in pregnant women with uterine contractions, without any difference from early to late pregnancy. These results, completed by more precise investigations, could suggest that the NO-cGMP system might be implicated in uterine quiescence. Keywords: Nitric oxide; Premature uterine contractility; 3',5'-cyclic guanosine monophoshate
1. Introduction Uterine quiescence during pregnancy is required for successful completion of a normal gestation. The mechanism that modulates changes in uterine activity from a quiescent state to active labor at term remains unknown. Recent reports suggest that the system nitric oxide-cyclic G M P which induces relaxation of the endogenous smooth muscle might be involved [1 3]. The relaxation effects of L-arginine-nitric oxide (NO) and cyclic G M P (cGMP) are substantially decreased during both term and preterm delivery and post partum [4]. H u m a n uterine tissue exhibits nitric oxyde synthase (NOS) activity, with the highest levels during the periods of relative uterine quiescence [5]. Moreover, the observation that nitric oxide donors reduce uterine activity suggests that N O might help to maintain uterine quiescence [2]. The most important target of N O is * Corresponding author, 45374745. 0301-2115/95/$09.50 © 1995 S S D I 0301-211 5(95)02223-T
Tel.:
+ 161 45374527: Fax:
+ 161
the soluble guanylate cyclase in smooth muscle cells. As a consequence of this stimulation, the rate of G M P production is 50-fold greater than those obtained by another way [6]. Therefore, the determination of c G M P in biological samples may be considered as a reflection of the N O production. By urinary c G M P quantification in pregnant women with and without early uterine contractions, we attempt to demonstrate if the N O - c G M P system is involved in the uterine contractility.
2. Patients and methods 2. 1. Patients Ninety four normal pregnant women were included in two populations (A and B) defined as follows: Sixtytwo (A population) without uterine contractions (less than five a day and non-painful) were separated into three groups according to their gestational age. G r o u p I included pregnancies below 15 weeks (n = 16), group II (n = 23) pregnancies from 16 to 33 weeks and group
Elsevier Science [reland Ltd. All rights reserved
18
J. Fram'oual el ell.' European Journal o1 Obstetric.s & Gynecology and Reproductite Biology 63 (1995) 17 19
III (n = 23) from 33 weeks until delivery. G r o u p III was compared to 32 pregnant women at the same gestational age (B population) presenting more than five non-painful uterine contractions a day. An oral treatment using progesterone (Utrogestran*, Lab. Besins Iscovesco) had been administrated to 35% of them. We excluded pregnancies presenting preeclampsia or premature labor, all patients had a bishop score ~<4. During regular mensual consultations, uterine contractions were noted by physicians and urine samples were collected and stored at - 2 0 ° C until assayed. G r o u p Ill of A population was compared to B population. These two populations have the same gestational age (37 _+ 1.7 weeks). It is mainly the parity one (50%) that is represented in A population and parity zero (45%) in B population. 2.2 Methods" c G M P determination was performed by an immunoreactive method and measured by R I A ( c G M P assay RPA 525; Amersham, Bucks, UK) [7]. Urine samples were previously diluted 10- and 100-fold in buffer-assay. Creatinine concentrations in urine were determined enzymatically and the c G M P concentration was expressed as nmol per mmol of creatinine. The intraassay and interassay variations were each < 10%. All results were expressed as mean _+ S.D. D a t a analysis of variance and the differences were considered significant when the P value was less than 0.05. 3. Results
Table 1 summarizes the urine c G M P values for the three groups of pregnant women without uterine contractions. No significant differences were found at any gestional age. The S.D.s are high and show that important differences are observed for each pregnant patient. Table 2 compares normal pregnant women at the last trimester with (B population) and without (A population, group III) uterine contractions. A significant difference (P < 0.05) is observed between the two Table 1 Urinary cGMP levels (nmol,'mmol of creatinine) in 62 pregnant women without uterine contractions: population A Gestational age (weeks)
Mean
S.D.
Max
Min
Group I _<15 (n = 16) Group II 16 33 (/i = 23) Group 1II >33 (n 23)
121
56
292
52
I 18
59
328
48
147
66
328
68
Table 2 Comparison by an analysis of variance between cGMP levels (nmol mmol of creatinine) obtained from group Ill pregnant women (gestational age > 33 weeks) with or without uterine contractions
Uterine contraction n = 32 No uterine contraction n - 23
Mean
S.D.
Max
Mill
106"
60
263
9
147
66
317
6(1
* significant difference P < 0.05.
populations; lower levels of urinary c G M P are obtained in patients with uterine contractility, whatever the parity. Moreover, progesterone treatment does not influence the c G M P rate, respectively 98 _+ 72 and 112 + 51 for treated and not treated women. 4. Discussion
The N O - c G M P system plays a prominent role during pregnancy, particularly in maintaining uterine quiescence and attenuating the actions of vasoconstrictor agents [8]. By the determination of urinary cGMP, the second messenger of the NO system, we try to investigate its implication during pregnancy. In a previous work (unpublished data) we found, by a variance analysis, a significant increase (P < 0.01) of urinary c G M P in normal pregnant women when compared to nonpregnant ones (respectively 127 _+ 63 and 74 + 30 nmol/mmol of creatinine). In the present study, we first show that no significant differences in urinary c G M P levels are observed in pregnant women without uterine contractions from the first to the last trimester of pregnancy. However, when uterine contractility occurs the c G M P rate is significantly decreased. These results are observed albeit the contractions are non-painful, not exactly quantified, whatever the parity and even when the women are under progesterone treatment. This fact suggests, in accordance with others [3], that the c G M P - N O system is possibly implicated in the uterine quiescence. NO, synthesized by nitric oxide synthase (NOS) from L-arginine, acts through the stimulation of soluble guanylate cyclase increasing c G M P levels in smooth muscle and producing relaxation. NOS has been found in at least two isoforms in the genital tract. One of these is an endothelial constitutive form (ecNOS) found mainly in the human placenta [9]. The other inductible form is present in the human uterine artery [10] and in mouse uterine myocytes [11]. The uterine tissue does not seem to be homogeneous [2]; particularly the myometrium in pregnant rats is less sensitive to c G M P relaxation at term [12].
J. Francoual et al. European Journal qf Obstetrics & Qvnecoh~gy amt Reprmh~ctive Biology 63 (1995) 17 19
The regulation of the N O - c G M P system is not yet completely understood. In vitro, it seems to be regulated during the pregnancy by changes in steroid hormone levels [13 14]. A rise in estrogen and a decrease in progesterone at term could inhibit this system and thus initiate labor [15]. How the NO donors really participate in uterine relaxation [16] remains unclear. In conclusion, we show that no significant differences of urinary c G M P levels are observed during pregnancy without uterine contractions. On the contrary, when contractions occurs, urinary c G M P levels are decreased. This decrease could be due to modifications in the N O S - N O - c G M P system. To determine their implications, others studies concerning (i) the quantification of uterine contractility, (ii) the evaluation of the steroid hormone status and (iii) the NOS isoforms induction in the genital tract, are necessary. Moreover, regarding the high range observed in the results complementary studies using each pregnant woman as her own reference should be done.
Acknowledgments We are grateful to Mrs. Jarry and the nurses of obstetrics and gynecology department for collecting urinary samples. This work was supported by a 'contrat de recherche clinique AP-HP'.
References [l] Kerwin Jr JF, Heller M. The arginine-nitric oxide pathway: a target for new drugs. Med Res Rev 1994; 14:23 74. [2] Izumi H, Yallampalli C, Garfield RE. Gestational changes in L-arginine-induced relaxation of pregnant rat and human myometrial smooth muscle. Am J Obstet Gynecol 1993: 169:1327 1337. [3] Izumi H, Garfield RE, Makino Y, Shirakawa K, ltoh T. Gestational changes in endothelium-dependent vasorelaxation in human umbilical artery. Am J Obstet Gynecol 1994; 170:236 245.
19
[4] Yallampalli C, Byam-Smith M, Nelson S, Garfield RE. Synthesis of nitric oxide and cGMP in the rat uterus are increased during pregnancy and decreased during labor. In: Society for gynecology investigation, Chicago, Illinois, March 1994. Abstract 4. [5] Sladek SM, Regenstein AC, Lykins D, Roberts JM. Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. Am J Obstet Gynecol 1993: 169:1285 1291. [6] Knowles RG, Moncada S. Nitric oxide as a signal in blood vessels. TIBS 1992; 17:399 402. [7] Jakob G, Mair J, Vorderwinkler KP et al. Clinical significance of urinary cyclic guanosine monophosphate in diagnosis of heart failure. Clin Chem 1994; 40:96 100. [8] Myatt L, Brewer AS, Langdon G, Brockman DE. Attenuation of the vasoconstrictor effects of thromboxane and endothelin by nitric oxide in the human fetal-placental circulation. Am J Obstet Gynecol 1994; 166:224 230. [9] Buttery LDK, McCarthy A, Springall DR, Sullivan M H F Elder MG, Michel T, Polak JM. Endothelial nitric oxide synthase in the human placenta: regional distribution and proposed regulatory role at the feto-maternal interface. Placenta 1994; 15:257 265. [10] Jovanovie A, Grbovi6 L, Tuli6 I. L-arginine induces relaxation of human uterine artery with both intact and denuded endothelium. Eur J Pharmacol 1994; 256:103 107. [11] Natuzzi ES, Kurtzman JT, Riemer RK. TGF-~I interaction with CSF-I: induction of nitric oxide synthase activity in primary mouse uterine myocytes. In: Society for gynecology investigation, Chicago, Illinois, March 1994. Abstract 129. [12] Word RA, Cornwell TL. Roles of cyclic G M P and cGMP-dependent protein kinase in myometrial relaxation during pregnancy. In: Society for gynecology investigation, Chicago Illinois, March 1994. Abstract 153. [13] Weiner CP, Baylis S, Lizasoain I, Knowles RG, Charles I, Moncada S. Regulation of NO-synthase by sex hormones. In: Society for gynecology investigation, Chicago, Illinois, March 1994. Abstract 266. [14] Yallampalli C, Byam-Smith M, Nelson SO, Garfield RE. Steroid hormones modulate the production of nitric oxide and cGMP in the rat uterus. Endocrinology 1994; 134:1971 1974. [15] Yallampalli C, lzumi H, Byam-Smith M, Garfield RE. An L-arginine-nitric oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy. Am J Obstet Gynecol 1993; 170:175 185. [16] Lees C, Campbell S, Jauniaux E et al. Arrest of preterm labour and prolongation of gestation with glyceryl trinitrate, a nitric oxide donor. Lancet 1994; 343:1325 1326.
ELSEVIER
GYNECOLOGY
Urinary cGMP levels during pregnancy with and without uterine contractions J. F r a n c o u a l *'~, J. Taieb a, N. Berkane b, A. L i n d e n b a u m a, R. F r y d m a n b ~'Department o f Bioehemisto,, Antoine Bbelg,re Hospital, 157 rue de la Porte de Trivaux, 92141 Clamart Cedex, France bDepartment o[ Obstetrics and Gynecology, Antoine B&'lbre Hospital, 157 rue de la Porte de Trivaux, 92141 Clamart Cedex, France
Received 13 March 1995; revision received 7 July 1995; accepted 7 July 1995
Abstract Objective: The purpose of this study was to investigate the nitric oxide-cGMP (NO-cGMP) system by urinary cGMP level determinations in pregnant women with and without uterine contractions. Design and subjects: cGMP expressed in nmol/mmol of creatinine was performed by radio immuno-assay (Amersham UK) in urine samples obtained from 94 pregnant women with non-complicated pregnancies. Population A without contractions (n = 62) was divided into three groups according to the gestational age (group I, ~<15 weeks; group II, 16-33 weeks; group III, > 33 weeks). The group III from A population was compared to B population (of the same gestational age) presenting uterine contractions (n = 32). Results: In A population, no significant urinary cGMP level differences were observed whatever the gestational age. Nevertheless, the comparison between patients with or without uterine contractions (A III and B populations) showed a significant difference by a variance analysis (P < 0.05). Lower levels of cGMP were seen when uterine contractions occured. Conclusion: Urinary cGMP levels are significantly decreased in pregnant women with uterine contractions, without any difference from early to late pregnancy. These results, completed by more precise investigations, could suggest that the NO-cGMP system might be implicated in uterine quiescence. Keywords: Nitric oxide; Premature uterine contractility; 3',5'-cyclic guanosine monophoshate
1. Introduction Uterine quiescence during pregnancy is required for successful completion of a normal gestation. The mechanism that modulates changes in uterine activity from a quiescent state to active labor at term remains unknown. Recent reports suggest that the system nitric oxide-cyclic G M P which induces relaxation of the endogenous smooth muscle might be involved [1 3]. The relaxation effects of L-arginine-nitric oxide (NO) and cyclic G M P (cGMP) are substantially decreased during both term and preterm delivery and post partum [4]. H u m a n uterine tissue exhibits nitric oxyde synthase (NOS) activity, with the highest levels during the periods of relative uterine quiescence [5]. Moreover, the observation that nitric oxide donors reduce uterine activity suggests that N O might help to maintain uterine quiescence [2]. The most important target of N O is * Corresponding author, 45374745. 0301-2115/95/$09.50 © 1995 S S D I 0301-211 5(95)02223-T
Tel.:
+ 161 45374527: Fax:
+ 161
the soluble guanylate cyclase in smooth muscle cells. As a consequence of this stimulation, the rate of G M P production is 50-fold greater than those obtained by another way [6]. Therefore, the determination of c G M P in biological samples may be considered as a reflection of the N O production. By urinary c G M P quantification in pregnant women with and without early uterine contractions, we attempt to demonstrate if the N O - c G M P system is involved in the uterine contractility.
2. Patients and methods 2. 1. Patients Ninety four normal pregnant women were included in two populations (A and B) defined as follows: Sixtytwo (A population) without uterine contractions (less than five a day and non-painful) were separated into three groups according to their gestational age. G r o u p I included pregnancies below 15 weeks (n = 16), group II (n = 23) pregnancies from 16 to 33 weeks and group
Elsevier Science [reland Ltd. All rights reserved
18
J. Fram'oual el ell.' European Journal o1 Obstetric.s & Gynecology and Reproductite Biology 63 (1995) 17 19
III (n = 23) from 33 weeks until delivery. G r o u p III was compared to 32 pregnant women at the same gestational age (B population) presenting more than five non-painful uterine contractions a day. An oral treatment using progesterone (Utrogestran*, Lab. Besins Iscovesco) had been administrated to 35% of them. We excluded pregnancies presenting preeclampsia or premature labor, all patients had a bishop score ~<4. During regular mensual consultations, uterine contractions were noted by physicians and urine samples were collected and stored at - 2 0 ° C until assayed. G r o u p Ill of A population was compared to B population. These two populations have the same gestational age (37 _+ 1.7 weeks). It is mainly the parity one (50%) that is represented in A population and parity zero (45%) in B population. 2.2 Methods" c G M P determination was performed by an immunoreactive method and measured by R I A ( c G M P assay RPA 525; Amersham, Bucks, UK) [7]. Urine samples were previously diluted 10- and 100-fold in buffer-assay. Creatinine concentrations in urine were determined enzymatically and the c G M P concentration was expressed as nmol per mmol of creatinine. The intraassay and interassay variations were each < 10%. All results were expressed as mean _+ S.D. D a t a analysis of variance and the differences were considered significant when the P value was less than 0.05. 3. Results
Table 1 summarizes the urine c G M P values for the three groups of pregnant women without uterine contractions. No significant differences were found at any gestional age. The S.D.s are high and show that important differences are observed for each pregnant patient. Table 2 compares normal pregnant women at the last trimester with (B population) and without (A population, group III) uterine contractions. A significant difference (P < 0.05) is observed between the two Table 1 Urinary cGMP levels (nmol,'mmol of creatinine) in 62 pregnant women without uterine contractions: population A Gestational age (weeks)
Mean
S.D.
Max
Min
Group I _<15 (n = 16) Group II 16 33 (/i = 23) Group 1II >33 (n 23)
121
56
292
52
I 18
59
328
48
147
66
328
68
Table 2 Comparison by an analysis of variance between cGMP levels (nmol mmol of creatinine) obtained from group Ill pregnant women (gestational age > 33 weeks) with or without uterine contractions
Uterine contraction n = 32 No uterine contraction n - 23
Mean
S.D.
Max
Mill
106"
60
263
9
147
66
317
6(1
* significant difference P < 0.05.
populations; lower levels of urinary c G M P are obtained in patients with uterine contractility, whatever the parity. Moreover, progesterone treatment does not influence the c G M P rate, respectively 98 _+ 72 and 112 + 51 for treated and not treated women. 4. Discussion
The N O - c G M P system plays a prominent role during pregnancy, particularly in maintaining uterine quiescence and attenuating the actions of vasoconstrictor agents [8]. By the determination of urinary cGMP, the second messenger of the NO system, we try to investigate its implication during pregnancy. In a previous work (unpublished data) we found, by a variance analysis, a significant increase (P < 0.01) of urinary c G M P in normal pregnant women when compared to nonpregnant ones (respectively 127 _+ 63 and 74 + 30 nmol/mmol of creatinine). In the present study, we first show that no significant differences in urinary c G M P levels are observed in pregnant women without uterine contractions from the first to the last trimester of pregnancy. However, when uterine contractility occurs the c G M P rate is significantly decreased. These results are observed albeit the contractions are non-painful, not exactly quantified, whatever the parity and even when the women are under progesterone treatment. This fact suggests, in accordance with others [3], that the c G M P - N O system is possibly implicated in the uterine quiescence. NO, synthesized by nitric oxide synthase (NOS) from L-arginine, acts through the stimulation of soluble guanylate cyclase increasing c G M P levels in smooth muscle and producing relaxation. NOS has been found in at least two isoforms in the genital tract. One of these is an endothelial constitutive form (ecNOS) found mainly in the human placenta [9]. The other inductible form is present in the human uterine artery [10] and in mouse uterine myocytes [11]. The uterine tissue does not seem to be homogeneous [2]; particularly the myometrium in pregnant rats is less sensitive to c G M P relaxation at term [12].
J. Francoual et al. European Journal qf Obstetrics & Qvnecoh~gy amt Reprmh~ctive Biology 63 (1995) 17 19
The regulation of the N O - c G M P system is not yet completely understood. In vitro, it seems to be regulated during the pregnancy by changes in steroid hormone levels [13 14]. A rise in estrogen and a decrease in progesterone at term could inhibit this system and thus initiate labor [15]. How the NO donors really participate in uterine relaxation [16] remains unclear. In conclusion, we show that no significant differences of urinary c G M P levels are observed during pregnancy without uterine contractions. On the contrary, when contractions occurs, urinary c G M P levels are decreased. This decrease could be due to modifications in the N O S - N O - c G M P system. To determine their implications, others studies concerning (i) the quantification of uterine contractility, (ii) the evaluation of the steroid hormone status and (iii) the NOS isoforms induction in the genital tract, are necessary. Moreover, regarding the high range observed in the results complementary studies using each pregnant woman as her own reference should be done.
Acknowledgments We are grateful to Mrs. Jarry and the nurses of obstetrics and gynecology department for collecting urinary samples. This work was supported by a 'contrat de recherche clinique AP-HP'.
References [l] Kerwin Jr JF, Heller M. The arginine-nitric oxide pathway: a target for new drugs. Med Res Rev 1994; 14:23 74. [2] Izumi H, Yallampalli C, Garfield RE. Gestational changes in L-arginine-induced relaxation of pregnant rat and human myometrial smooth muscle. Am J Obstet Gynecol 1993: 169:1327 1337. [3] Izumi H, Garfield RE, Makino Y, Shirakawa K, ltoh T. Gestational changes in endothelium-dependent vasorelaxation in human umbilical artery. Am J Obstet Gynecol 1994; 170:236 245.
19
[4] Yallampalli C, Byam-Smith M, Nelson S, Garfield RE. Synthesis of nitric oxide and cGMP in the rat uterus are increased during pregnancy and decreased during labor. In: Society for gynecology investigation, Chicago, Illinois, March 1994. Abstract 4. [5] Sladek SM, Regenstein AC, Lykins D, Roberts JM. Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. Am J Obstet Gynecol 1993: 169:1285 1291. [6] Knowles RG, Moncada S. Nitric oxide as a signal in blood vessels. TIBS 1992; 17:399 402. [7] Jakob G, Mair J, Vorderwinkler KP et al. Clinical significance of urinary cyclic guanosine monophosphate in diagnosis of heart failure. Clin Chem 1994; 40:96 100. [8] Myatt L, Brewer AS, Langdon G, Brockman DE. Attenuation of the vasoconstrictor effects of thromboxane and endothelin by nitric oxide in the human fetal-placental circulation. Am J Obstet Gynecol 1994; 166:224 230. [9] Buttery LDK, McCarthy A, Springall DR, Sullivan M H F Elder MG, Michel T, Polak JM. Endothelial nitric oxide synthase in the human placenta: regional distribution and proposed regulatory role at the feto-maternal interface. Placenta 1994; 15:257 265. [10] Jovanovie A, Grbovi6 L, Tuli6 I. L-arginine induces relaxation of human uterine artery with both intact and denuded endothelium. Eur J Pharmacol 1994; 256:103 107. [11] Natuzzi ES, Kurtzman JT, Riemer RK. TGF-~I interaction with CSF-I: induction of nitric oxide synthase activity in primary mouse uterine myocytes. In: Society for gynecology investigation, Chicago, Illinois, March 1994. Abstract 129. [12] Word RA, Cornwell TL. Roles of cyclic G M P and cGMP-dependent protein kinase in myometrial relaxation during pregnancy. In: Society for gynecology investigation, Chicago Illinois, March 1994. Abstract 153. [13] Weiner CP, Baylis S, Lizasoain I, Knowles RG, Charles I, Moncada S. Regulation of NO-synthase by sex hormones. In: Society for gynecology investigation, Chicago, Illinois, March 1994. Abstract 266. [14] Yallampalli C, Byam-Smith M, Nelson SO, Garfield RE. Steroid hormones modulate the production of nitric oxide and cGMP in the rat uterus. Endocrinology 1994; 134:1971 1974. [15] Yallampalli C, lzumi H, Byam-Smith M, Garfield RE. An L-arginine-nitric oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy. Am J Obstet Gynecol 1993; 170:175 185. [16] Lees C, Campbell S, Jauniaux E et al. Arrest of preterm labour and prolongation of gestation with glyceryl trinitrate, a nitric oxide donor. Lancet 1994; 343:1325 1326.