Objective
The objective of the study was to determine whether pretreatment of fetal or maternal placental vasculature with 17-hydroxyprogesterone caproate (17-P) attenuates the vasoactive effect of the thromboxane mimetic U46619.
Study Design
Two cotyledons were obtained from each placenta studied. For the first 5 placentas, the fetal artery of 1 cotyledon from each pair was infused with 17-P. After 30 minutes, a bolus dose of U46619 was administered to both cotyledons. An identical procedure was carried out on the next 5 placentas except that 17-P was infused into the intervillous space.
Results
The pressure excursion caused by bolus administration of U46619 was less in the cotyledons infused with 17-P, both in the 5 cases in which the fetal vasculature was infused with 17-P ( P = .0035) and in the 5 cases in which the maternal vasculature was infused with 17-P ( P = .038).
Conclusion
Pretreatment of either the fetal or maternal circuits of the placenta with 17-P attenuates U46619-mediated fetoplacental vasoconstriction.
During normal pregnancy, the placenta produces a large quantity of progesterone essential to establishment and maintenance of the gravid state. Progesterone and the progestogen, 17-hydroxyprogesterone caproate (17-P), have been demonstrated to decrease recurrent preterm delivery. Progesterone’s mechanism of action regarding the prevention of preterm delivery is not clear.
Most theories have focused on progesterone’s activity in the uterine myometrium and on progesterone’s antiinflammatory effects secondary to the differential expression and activation of nuclear progesterone receptors and their control of various transcription factors. Very little research has been devoted to the study of progesterone’s activity within the placenta itself and the role of this activity, if any, on the prevention of preterm labor.
Previous work with the ex vivo placental cotyledon model has demonstrated that thromboxane-induced vasoconstriction of fetoplacental arteries can be rapidly reversed by a single dose of 17-P. A study of nonpregnant women being treated with weekly intramuscular injections of 17-P for endometrial cancer demonstrated that steady-state serum concentrations of 17-P are reached within a few weeks of starting treatment. Therefore, a constant 17-P infusion followed by a pressor challenge in the fetoplacental arteries should more accurately reflect the physiology within the placental circulation of women receiving weekly 17-P injections for the prevention of preterm delivery. In the current study, we set out to determine whether pretreatment with a continuous infusion of 17-P in either the fetoplacental circulation or the intervillous space of an isolated placental cotyledon would attenuate the vasoconstrictive effect of thromboxane mimetic.
Materials and Methods
This protocol was approved by the Investigational Review Board of Madigan Army Medical Center. A total of 10 placentas were obtained at the time of cesarean section from unlabored patients at 37–42 weeks of gestation. Pregnancies complicated by multiple gestation, hypertensive disorders, diabetes, intrauterine growth restriction, or fetal anomalies were excluded.
The placenta perfusion technique was modified from Glance et al, as previously described. Briefly, within 15 minutes of delivery, each placenta was transported to the laboratory in which a peripherally located matched chorionic artery-vein pair perfusing a single cotyledon was identified, cannulated with 22-gauge intravenous catheters, and slowly flushed with perfusate. Once adequate venous return was confirmed, an 8 cm diameter circular section of the placenta containing the selected cotyledon was excised and placed in a holder designed to prevent leakage from the cut edges. Two 21-gauge butterfly needles were inserted through the chorionic surface into the intervillous space and infused with perfusate to simulate maternal uteroplacental circulation.
The perfusate consisted of a sterile preparation of Hanks’ balanced salt solution, gentamicin 5 mg/L, bovine albumin 2.0 gm/L, and sodium heparin 2000 U/L. A 95% oxygen and 5% carbon dioxide mixture was bubbled into the perfusate, which was maintained at 37°C with a pH between 7.35 and 7.45 throughout the experiment. Perfusion to the intervillous space was maintained at a rate of 10 mL/min and perfusion of the fetal artery was maintained at a rate of 4 mL/min. The cotyledon was transferred to a temperature-controlled chamber maintained at 37°C. A second cotyledon from the same placenta was prepared in a similar manner.
All perfusions were established within 30 minutes after delivery of the placenta. All perfusion circuits used standard commercial intravenous tubing. Variable flow peristaltic pumps (Fischer Scientific, Fair Lawn, NJ) controlled the intervillous (maternal) circulation, and Corpak 300D Enteral (Corpak, Wheeling, IL) roller-type pumps controlled the fetal circulation. The fetoplacental arterial perfusion pressures were measured with inline transducers upstream from each cotyledon.
Perfusion of the cotyledons, as described in previous text, was unaltered for 30 minutes to establish stable baseline fetoarterial pressures. Next, the test cotyledon’s fetal arterial circuit was switched to perfusate containing 18 ng/mL of 17-P, while infusion of the control cotyledon’s fetal arterial circuit and the maternal-side vasculature of both test and control cotyledons was continued with standard perfusate. After another 30 minutes, a single vasoconstricting dose of thromboxane mimetic U46619 (0.5 μg) was injected into the fetal arterial circuit of each cotyledon, and the resultant change in perfusion pressure was recorded.
The concentration of 17-P in the fetal perfusate was extrapolated from a study of 17-P serum levels in nonpregnant women receiving weekly 17-P injections for endometrial cancer and a longitudinal study of hormone levels in pregnancy. The 17-P, which is delivered as a relatively insoluble powder, was dissolved in a very small amount of dimethylsulfoxide (DMSO; FisherBiotech, Fair Lawn, NJ) as recommended by the company’s technical support department. DMSO was also dissolved in the control circuit to achieve an equal concentration of DMSO in both circuits, 55 μg/mL.
This first part of the experiment consisted of independent runs with cotyledon pairs from 5 different placentas. A random number table was used to select the test and control cotyledons for each run. In 2 of the 5 runs, a blood gas analyzer was used to compare the oxygen content of perfusate upstream from each test and control cotyledon with oxygen content of their downstream effluent to prove continued oxygen utilization throughout the experiment.
At the conclusion of each run, a dose of angiotensin II (10 –10 mol/L) was injected into the fetal circulation of each cotyledon to verify their continued vasoconstrictive potential. In all cases, an elevation in perfusion pressure was observed within 5 minutes of angiotensin II injection, proving viability of our test and control cotyledons for the duration of the experimental runs.
In the second part of the study, we conducted 5 additional independent runs in which the maternal circuit of the test cotyledon was continuously infused with 17-P at a concentration of 1.8 μg/mL, whereas the maternal circulation of the control cotyledon and the fetal circulation of both cotyledons were infused with standard perfusate, without 17-P.
The perfusion pressure in the fetal arterial circuit of each cotyledon was recorded every 5 minutes throughout all experimental runs. Perfusion pressure was recorded for 30 minutes after each U46619 bolus injection: every minute for the first 15 minutes, and every 5 minutes thereafter. The difference between the baseline pressure before U46619 injection and the peak pressure reached after U46619 injection was evaluated for each matched cotyledon pair. Paired Student t tests were used to assess significance of the pressure change between test and control cotyledons. Statistical analysis was performed with STATA 10 statistical software (Stata Corp, College Station, TX).
Results
In the first part of the experiment, continuous infusion of 17-P in the fetal arterial circulation did not change the baseline perfusion pressures of the exposed cotyledons compared with the control cotyledons (average change in baseline perfusion pressure 3.6 ± 1.5 mm Hg vs 2.8 ± 2.3 mm Hg; P = .59). A bolus injection of thromboxane mimetic (U46619) into the control cotyledons caused a significant rise in fetoplacental perfusion pressure compared with starting pressure (97.6 ± 12.5 mm Hg vs 23.8 ± 4.4 mm Hg; P = .0003).
The same dose of U46619 also caused a significant elevation in the fetoplacental perfusion pressure of those cotyledons receiving a continuous infusion of 17-P into the fetoplacental arteries (52.4 ± 10.7 mm Hg vs 27.8 ± 5.3 mm Hg; P = .0018). The pressure excursion (maximum pressure reached after U46619 injection minus starting pressure) was significantly less in the cotyledons infused with 17-P compared with controls (25.6 ± 7.7 mm Hg vs 73.4 ± 13.6 mm Hg; P = .0035). This result is shown on the left side of the Figure . In both the test and control cotyledons, the peak fetal arterial pressure was achieved within 6 minutes of U46619 injection.
