Venu Jain

The twin-twin transfusion syndrome

Centre for Fetal Care, Queen Charlotte’s & Chelsea Hospital & Institute of Reproductive and Developmental Biology, Imperial College of Science, Technology and Medicine, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK The aetiology of twin–twin transfusion syndrome (TTTS), which affects 10–15% of monochorionic (MC) twin pregnancies, remains poorly understood. Although all MC twins have placental vascular anastomoses, unbalanced intertwin transfusion has been shown by ex vivo injection and in vivo Doppler studies of chorionic plate vasculature to be mediated by 1 arterio-venous anastomoses (AVA) in association with absent bi-directional arterio-arterial anastomoses (AAA). TTTS presents in the mid trimester with the oligo-polyhydramnios sequence, the donor may have a small or non-visible bladder and abnormal umbilical artery Doppler, while the recipient has a large bladder and may develop cardiac hypertrophy, triscupid regurgitation, and eventually hydrops. Recently, discordant renal renin angiotensin expression, endothelin and atrial natriuretic peptide have been implicated in the pathogenesis. Survival has increased from <20% to <60–70% with modern treatments, although survivors remain at increased risk of antenatally acquired cerebral white matter injury, and neurodevelopmental sequelae are documented in c. 10% (range 5–23%). The recent introduction of a staging system for TTTS facilitates selection of therapy with less invasive amnioreduction and septostomy preferred for early stage disease, and more aggressive modalities such as laser ablation and cord occlusion with their attendant risk of procedure related fetal loss, reserved for advanced stage disease. 2002 Elsevier Science Ltd. All rights reserved.

The Placenta Contributes to Activation of the Renin Angiotensin System in Twin–Twin Transfusion Syndrome

The renin-angiotensin system (RAS) in twin-twin transfusion syndrome (TTTS) is up-regulated in the donor fetuss kidneys, but down-regulated in the recipients. Ultrasonographic and echocardiographic features suggest that the recipient is also exposed to RAS components. In this study we investigated the role and origin of RAS components in the recipient fetus. Monochorionic diamniotic (MCDA) pregnancies were recruited from a tertiary fetal medicine service. Cord blood was collected from MCDA twins (TTTS and control non-TTTS) at delivery for renin and angiotensin II immunoassays. Placental tissue was flash-frozen for mRNA and protein expression or formalin-fixed for immunohistochemistry. Archival placenta and kidney samples were used for immunohistochemistry and in-situ hybridization. Plasma renin levels were elevated (p<0.05) in recipients (median 201 pg/ml, range 54-315 pg/ml) and donors (125 pg/ml, 25-296) with TTTS compared to controls (2.5 pg/ml, 1.1-1.5 pg/ml). The same was found with angiotensin II with high levels in both recipients (300.5 pg/ml, 86.1-488 pg/ml) and donors (239 pg/ml, 76.6-422) compared to controls (169.5 pg/ml, 89-220 pg/ml, p<0.05). Renin mRNA expression, and protein appeared qualitatively higher in the placental territory of the recipient compared to that of the donor and non-TTTS controls. We conclude that both fetuses in TTTS are exposed to high levels of RAS components; these appear to be produced from different sites, namely the kidney of the donor, and the placenta of the recipient. Given the markedly different phenotypes in the genetically identical fetuses with TTTS, we suggest that the source of RAS components may influence their clinical manifestations.

Effect of chronic treatment with 17β-estradiol and progesterone on endothelium-dependent and endothelium-independent relaxation in isolated aortic rings from ovariectomized rats

OBJECTIVE Our purpose was to study the influence of chronic treatment with sex hormones on endothelium-dependent and endothelium-independent relaxation of rat aortic rings. STUDY DESIGN Rings of aortas, with and without endothelium, from rats treated with sex hormones or vehicle for 10 days were mounted in organ baths for isometric tension recording. Indomethacin (10(-5) mol/L) and N(omega)-nitro-L-arginine methyl ester (10(-4) mol/L), alone or in combination, were used to block cyclooxygenase and nitric oxide synthase, respectively. Mean data of contraction induced by potassium chloride (60 mmol/L), the relaxation by acetylcholine (10(-6) mol/L) in potassium chloride-contracted rings, tension induced by phenylephrine, and the negative logarithm of the concentration of acetylcholine or 3-morpholinosydnonimine producing a 50% relaxation, and area under the curve were calculated. RESULTS Treatment with 17beta-estradiol (10 microg/rat/day) decreased the tension induced by 60 mmol/L potassium chloride and increased the relaxation by acetylcholine in the rings with endothelium precontracted with potassium chloride. Contraction induced by potassium chloride and relaxation induced by acetylcholine were not influenced by the treatment with progesterone (2 mg/rat/day) or estrogen-progesterone combination. Treatment with estradiol, progesterone, or both hormones had no effect on tension developed in intact rings in response to phenylephrine and did not influence endothelium-dependent relaxation to acetylcholine or endothelium-independent relaxation to 3-morpholinosydnonimine in rings contracted with phenylephrine. The inhibition by N(omega)-nitro-L-arginine methyl ester of endothelium-dependent relaxation by acetylcholine was attenuated after the treatment with the sex hormones. CONCLUSIONS Chronic treatment with sex hormones did not increase production or release of endothelium-derived relaxing factor and did not change the sensitivity of rat aortic smooth muscle to nitric oxide. The treatment slightly counteracted the inhibition of endothelium-dependent relaxation produced by nitric oxide synthase blocker.

Expression of receptors for corticotropin-releasing factor in the vasculature of pregnant rats.

OBJECTIVE To identify and localize the receptor(s) responsible for modulating vascular effects of corticotropin-releasing factor (CRF) during pregnancy. METHODS Reverse transcriptase-polymerase chain reaction (RT-PCR), competitive RT-PCR, and Western blot analyses were used to study the expression of CRF receptors (CRFR(1), CRFR(2alpha), and CRFR(2beta)) in the aorta and uterine vascular bed of nonpregnant and late (day 18) and term pregnant (day 22) Sprague-Dawley rats. Immunohistochemistry was done to localize the CRF receptor in the aortic wall. There were six rats in each study group. RESULTS Only CRFR(2beta) was identified in the aorta and uterine vascular bed by RT-PCR and Western blot analyses. The PCR product was sequenced to confirm its identity. Competitive RT-PCR and Western blot analyses showed that expression of CRFR(2beta) is not different in late pregnancy compared with the nonpregnant but is decreased at term. Immunohistochemistry showed high expression of CRFR(2beta) on the aortic endothelial surface but low expression in the smooth-muscle layer. CONCLUSION Only CRFR(2beta) is expressed in vasculature of nonpregnant and pregnant rats and may mediate the vasorelaxant effect of CRF. This receptor is present predominantly in the vascular endothelium and to a lesser extent in the smooth muscle. The expression of CRF receptor in pregnant rat vasculature is down-regulated at term of gestation.

Effect of nitric oxide and carbon monoxide on uterine contractility during human and rat pregnancy

OBJECTIVE We sought to study the effects of authentic nitric oxide and carbon monoxide on the contractile activity of pregnant human and rat myometrium. STUDY DESIGN Strips were prepared from uterine biopsy specimens of 10 pregnant, nonlaboring women at term gestation undergoing cesarean delivery. In addition, rings were prepared from the uteri of pregnant rats at midterm (day 14) and at term (day 22) gestation (n = 10-12). The tissues were mounted in organ chambers filled with Krebs-Henseleit solution continuously aerated with 5% carbon dioxide in air (37 degrees C, pH approximately 7.4) for isometric tension recording. The effects of nitric oxide and carbon monoxide gases on spontaneous contractile activity were studied. Responses to hemin (hemoxygenase substrate), which produces endogenous carbon monoxide, were also examined. Responses to nitric oxide and carbon monoxide were also studied in aortic and tail artery rings from pregnant rats after contraction with phenylephrine. RESULTS Nitric oxide significantly inhibited contractility of human myometrium at term (area under the concentration-response curve, 145.36 +/- 30.02 vs 40.56 +/- 22.81 in controls; P <.05) and rat myometrium at midterm gestation (264.23 +/- 47.86 vs 121.82 +/- 23.50; P <.05) but not at term. No statistically significant inhibition was induced in human or rat myometrium by carbon monoxide, whereas hemin significantly attenuated contractility in human myometrium at term and in rat myometrium at midterm gestation (P <. 05). Nitric oxide, carbon monoxide, and hemin relaxed aortic and tail artery rings. CONCLUSIONS Authentic nitric oxide inhibits rat uterine contractile activity at midterm gestation but not at term. However, nitric oxide inhibits human myometrium activity at term. Authentic carbon monoxide does not appear to modulate uterine contractility, whereas hemin may have some inhibitory properties.

The relaxation responses to corticotropin-releasing factor in rat aorta are endothelium dependent and gestationally regulated

OBJECTIVE Our purpose was to examine the hypothesis that the relaxant response to corticotropin-releasing factor is endothelium dependent and changes during pregnancy. STUDY DESIGN Relaxant responses to cumulative concentrations of corticotropin-releasing hormone were measured in rings of thoracic aorta, precontracted with phenylephrine, from pregnant and nonpregnant female rats. Each group consisted of 5 to 10 rats. RESULTS The relaxation induced by corticotropin-releasing factor in aortic rings from nonpregnant rats decreased after deendothelization and treatment with N omega-nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor), LY-83,583 (a guanylate cyclase inhibitor), or alpha-helical corticotropin-releasing factor 9-41 (a corticotropin-releasing factor antagonist). The percent decrease in relaxation at 1 nmol/L of corticotropin-releasing factor was about 88% with deendothelization, 100% with N omega-nitro-L-arginine methyl ester, 76% with LY-83,583, and 100% with alpha-helical corticotropin-releasing factor 9-41. The responses to corticotropin-releasing factor in intact rings from rats in early pregnancy were not significantly different from those in the nonpregnant female rats, but the responses were decreased during the later stages of gestation (percent decrease in relaxation at 1 nmol/L of corticotropin-releasing factor about 71% at day 20 and 98% at term). The relaxation induced by corticotropin-releasing factor during pregnancy was also inhibited by deendothelization, N omega-nitro-L-arginine methyl ester, or LY-83,583. CONCLUSIONS The relaxant response to corticotropin-releasing factor in the rat aorta is endothelium dependent and is mediated by the nitric oxide-cyclic guanosine monophosphate pathway. The receptor involved in this effect is alpha-helical corticotropin-releasing factor 9-41 sensitive. This inhibitory response is unchanged during early pregnancy but reduced toward the end of gestation.

In vivo effects of corticotropin-releasing factor in pregnant rats

OBJECTIVES Our purpose was to study the effects of corticotropin-releasing factor on (1) maternal blood pressure, (2) uterine vasculature, and (3) parturition in pregnant rats. STUDY DESIGN Infusion minipumps containing vehicle, corticotropin-releasing factor, or alpha-helical corticotropin-releasing factor 9-41 (corticotropin-releasing factor receptor antagonist) were inserted subcutaneously in timed pregnant rats on day 16 of gestation. Systolic blood pressure was measured daily by the tail-cuff method. The time of onset of labor was determined and the newborn pups were weighed. Circulating levels of corticotropin-releasing factor were measured in untreated controls by radioimmunoassay. Relaxant responses to corticotropin-releasing factor were studied in isolated segments of uterine artery from late (day 18) and term (day 22) pregnant rats mounted in a wire myograph. RESULTS The blood pressure was decreased by corticotropin-releasing factor and increased by alpha-helical corticotropin-releasing factor 9-41 (p < 0.05). The time of onset of labor was not affected by either treatment. Pup weight was decreased by corticotropin-releasing factor (p < 0.05). Circulating levels of corticotropin-releasing factor (immunoreactive) were not changed in pregnancy. In vitro, corticotropin-releasing factor caused relaxation of the uterine artery in a concentration-dependent manner and the relaxation was decreased at term compared with late pregnancy (p < 0.05). CONCLUSIONS Endogenously produced corticotropin-releasing factor lowers blood pressure during pregnancy in rats. It is a relaxant of uterine vasculature and this effect is decreased at term. It does not play an essential role in the initiation of labor in rats.

Structure and function of the myometrium

Abstract Uterine smooth muscle is very similar to the smooth muscle of other visceral organs, but with a significant difference i.e. its contractile activity. During pregnancy, it is maintained in a quiescent state. Inefficient cell-to-cell coupling due to low level of expression of gap junctions and the inhibitory influence of nitric oxide are important factors responsible for minimal contractile activity in the uterine smooth muscle as gestation progresses. Labor is a state in which the uterus contracts frequently and forcefully to expel the products of conception. The instability of membrane potential of the uterine smooth muscle forms the basis for the spontaneity of contractions in the uterine smooth muscle. There occur changes in the calcium regulatory mechanisms that favor more forceful contractility of the muscle. A prerequisite for the effectiveness of these contractions is the ability of the myometrium to propagate its electrical activity, wherein lies the importance of gap junctions. Term of pregnancy is marked by a favorable estrogen: progesterone ratio and an upregulation of gap junctions, rendering the myometrium a functional syncitium. Superimposed on this enhanced activity and reactivity of the uterus is an array of uterine stimulants like prostaglandins and oxytocin, which, in the absence of uterine relaxants, result in forceful, well-sustained, rhythmic, and regular uterine contractions, and initiation of labor.

Relaxation Kinetics of the Aorta in Nω-nitro-L-arginine Methyl Ester-Treated Pregnant Rats

Objective: To test the hypothesis that vascular relaxation kinetics are prolonged in pregnant rats treated chronically with Nω-nitro-L-arginine methyl ester (L-NAME). Methods: Timed pregnant rats (on day 13 of a 22-day gestation) were implanted with infusion pumps containing vehicle (controls) or L-NAME (50 mg/d). L-NAME pumps were retained until day 22 (group 1), or removed on day 20 (group 2). All rats were killed at term. Aortic rings were mounted in organ chambers containing physiologic salt solution (PSS) for isometric tension recording, contracted with high-K+ PSS (60 mM), and allowed to relax in normal-K+ PSS. Relaxation kinetics were quantified as time for 50% and 80% relaxation. After contraction with phenylephrime, responses to cumulative concentrations of methacholine were studied in the absence and presence of L-arginine (L-Arg) (10-3 M). Results: Responses to methacholine were inhibited completely in group 1 and partially in group 2 P < .05). The inhibition in both groups was reversed by L-Art. The rate of relaxation was significantly slower in groups 1 and 2 (P < .05) as compared with controls. Mechanical removal of the endothelium caused prolongation of relaxation in controls and group 2 (P < .05), but not in group 1. Preincubation of aortic rings from untreated control with L-NAME (in vitro, 10-4 M) did not affect relaxation. Conclusion: The endothelium medulates the are of vascular relaxation by a factor other than nitric oxide. Nω-nitro-L-arginine methyl ester (L-NAME) prolongs vasorelaxation by endothelium-dependent and -independent mechanisms. Prolongation of vascular relaxation kinetics may be a mechanism to elevate blood pressure and peripheral vascular resistance in preeclampsia.

Relaxation Kinetics of Rat Aorta During Pregnancy

Objective: To examine the hypothesis that kinetics of vasorelaxation are altered during pregnancy. Methods: Rings of aorta from rats at different stages of pregnancy (early, late, and term) and from nonpregnant female rats were precontracted with high-K+ and then allowed to relax in normal-K+ physiologic saline solution. The time to reach 50% and 80% relaxation were determined in the absence or presence of a cycloxygenase inhibitor (indomethacin) and a nitric oxide synthase inhibitor (Nω-nitro-L-arginine methyl ester) or after removal of the endothelium. Results: The aortic relaxation was progressively faster in later stages of gestation. Nω-nitro-L-arginine methyl ester and indomethacin had no significant effect whereas removal of hte endothelium caused a slowing of relaxation in all the groups. Even in the presence of Nω-nitro-L-arginine methyl ester and indomethacin or after de-endothelization, the relaxation remained faster at term as compared with the other groups. Conclusion: Aortic relaxation is faster in the presence of endothelium. The effect of endothelium on relaxation is independent of nitric oxide synthase or cycloxygenase systems. Progression of gestation is associated with acceleration of aortic relaxation, which cannot be totally ascribed to an endothelial factor and may involve a change intrinsic to the vascular smooth muscle. Faster relaxation kinetics of the vasculature during pregnancy may be a mechanism to decrease peripheral vascular resistance.