Carlo Romanini

Relation between decreased anandamide hydrolase concentrations in human lymphocytes and miscarriage

BACKGROUND Endocannabinoids such as anandamide are thought to have adverse effects on pregnancy and embryonic development. The activity of the degradative enzyme anandamide hydrolase may therefore be crucial for prevention of excessive concentrations of anandamide in the uterus, and thus prevention of pregnancy failure or female infertility. We tested this hypothesis in a preliminary study, and then used the results to find out whether anandamide hydrolase activity could predict miscarriage in a group of pregnant women. METHODS We assessed anandamide hydrolase activity in peripheral lymphocytes from 50 healthy, pregnant women at weeks 6-11 of gestation by a specific radiochromatographic method. The expression of the enzyme at the protein level was measured by ELISA with specific polyclonal antibodies. In a further study, we measured anandamide hydrolase concentration in 120 women who were 7-8 weeks pregnant and compared these findings with subsequent pregnancy outcome. FINDINGS In the first study, seven of the 50 women had a miscarriage. Anandamide hydrolase activity was lower in the seven women who miscarried than in the 43 who did not (60.43 pmol/min per mg protein [SD 29.34] vs 169.60 pmol/min per mg protein [30.20]; difference 109.17 pmol/min per mg protein [95% CI 26.64-191.70]; p<0.0001 by the Mann-Whitney test). Enzyme activity correlated with enzyme concentration, and a threshold concentration represented by an optical density (after ELISA) of 0.15 absorbance units at 450 nm separated the women who had miscarriages from those who did not. In the second study, 15 women had anandamide hydrolase concentrations below the threshold, and 105 had concentrations at or above the threshold. All 15 women in the low anandamide hydrolase group had miscarriages, compared with one of the 105 women with high concentrations (p<0.0001 by Fishers exact test). INTERPRETATION Decreased anandamide hydrolase activity and expression in peripheral lymphocytes is an early (<8 weeks of gestation) marker of spontaneous abortion, and may prove useful as a diagnostic tool for large-scale, routine monitoring of gestation. Our results also suggest that endocannabinoids might be critical in regulating the lymphocyte-dependent cytokine network associated with human fertility and successful pregnancy.

Progesterone Up-Regulates Anandamide Hydrolase in Human Lymphocytes: Role of Cytokines and Implications for Fertility

Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human lymphocytes. At the same concentrations, the membrane-impermeant conjugate of progesterone with BSA was ineffective, suggesting that binding to an intracellular receptor was needed for progesterone activity. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level, and was partly mediated by the Th2 cytokines. In fact, lymphocyte treatment with IL-4 or with IL-10 had a stimulating effect on FAAH, whereas the Th1 cytokines IL-12 and IFN-γ reduced the activity and the protein expression of FAAH. Human chorionic gonadotropin or cortisol had no effect on FAAH activity. At variance with FAAH, the lymphocyte anandamide transporter and cannabinoid receptors were not affected by treatment with progesterone or cytokines. Good FAAH substrates such as anandamide and 2-arachidonoylglycerol inhibited the release of leukemia-inhibitory factor from human lymphocytes, but N-palmitoylethanolamine, a poor substrate, did not. A clinical study performed on 100 healthy women showed that a low FAAH activity in lymphocytes correlates with spontaneous abortion, whereas anandamide transporter and cannabinoid receptors in these cells remain unchanged. These results add the endocannabinoids to the hormone-cytokine array involved in the control of human pregnancy.

The value of fetal fibronectin in cervical and vaginal secretions and of ultrasonographic examination of the uterine cervix in predicting premature delivery for patients with preterm labor and intact membranes.

OBJECTIVE We compared the diagnostic performances of fetal fibronectin assay of cervical and vaginal secretions and of transvaginal ultrasonographic evaluation of the uterine cervix in the prediction of preterm delivery of patients presenting with preterm labor and intact membranes. STUDY DESIGN One hundred eight patients admitted to the hospital for preterm labor and with intact membranes underwent assay of fibronectin in the cervical and vaginal secretions and transvaginal ultrasonographic evaluation of the uterine cervix. The ultrasonographic parameters evaluated were cervical length, presence of funneling, and cervical index ([Funnel length + 1]/Cervical length). Outcome measures were occurrence of preterm delivery, defined as birth before the thirty-seventh week of gestation, and the admission-to-delivery interval. RESULTS Forty-seven patients (43.5%) were delivered preterm. Receiver characteristic curve analysis showed that a level of fetal fibronectin in cervical secretions > or = 60 ng/ml had the highest diagnostic performance in predicting preterm delivery (sensitivity 80.9%, specificity 83.6%). Multiple stepwise logistic regression analysis indicated that the cervical index significantly improved the prediction of preterm delivery achieved by the fetal fibronectin assay. In patients with cervical secretion fibronectin levels > or = 60 ng/ml, survival analysis showed a shorter admission-to-delivery interval in the presence of an abnormal cervical index (p < or = 0.001). CONCLUSIONS The assay of fetal fibronectin in cervical secretions is more accurate than ultrasonographic evaluation of the uterine cervix in the prediction of preterm delivery. Combined use of the fetal fibronectin assay and the cervical index improves the diagnostic efficiency and allows prediction of the admission-to-delivery interval, identifying a subgroup of patients who may require aggressive treatment.

Interleukin-6 concentrations in cervical secretions identify microbial invasion of the amniotic cavity in patients with preterm labor and intact membranes

OBJECTIVE The objectives of this study were to determine whether cytokine levels in cervical secretions were increased in the presence of microbial invasion of the amniotic cavity in patients with preterm labor and intact membranes and to relate concentrations to cytokine levels in amniotic fluid, cervicovaginal microflora, and the presence of chorioamnionitis. STUDY DESIGN Cervical secretions were sampled immediately before amniocentesis in 92 patients admitted for preterm labor with singleton pregnancies and intact membranes. Amniotic fluid was cultured and the following cytokines were measured in amniotic fluid and cervical secretions: interleukin-1 beta, interleukin-1 receptor antagonist, tumor necrosis factor-alpha, and interleukin-6. The cervicovaginal microflora and placentas (n = 42) were also analyzed. RESULTS A total of 19.56% (18/92) of the amniotic fluid cultures had positive results. All the cytokines tested showed significantly higher levels in cervical secretions in the presence of intraamniotic infection. There were significant relationships between the concentrations of interleukin-6 and interleukin-1 receptor antagonist in amniotic fluid and cervical secretions. A concentration of interleukin-6 in cervical secretions > 410 pg/ml had a sensitivity of 66.8% and a specificity of 90.5% and a relative risk of 7.7 for intraamniotic infection, higher than the other cytokines tested. There were no relationships between the presence of bacterial vaginosis and cervicovaginal pathogens and cervical cytokine levels. In the presence of chorioamnionitis, cervical concentrations of interleukin-6 and interleukin-1 receptor antagonist were significantly increased in spite of negative amniotic fluid culture results. CONCLUSION The measurement of interleukin-6 in cervical secretions may help to noninvasively identify intraamniotic infection among pregnancies with preterm labor and intact membranes.

Mild thyroid abnormalities and recurrent spontaneous abortion: diagnostic and therapeutical approach.

PROBLEM: The aim of this study is to evaluate the role of mild thyroid abnormalities in recurrent spontaneous abortion, and to assess the effects of two different therapeutical protocols.
 METHOD: A prospective study in the population of recurrent aborters with mild thyroid abnormalities, evaluating the obstetric outcome in 42 patients. Sixteen thyroid autoantibodies positive patients were treated with thyroid replacement therapy, while 11 patients received intravenous immunoglobulins (IVIG). Fifteen patients, characterized by negative antithyroid antibodies, and having underlying thyroid pathology, were treated with thyroid replacement therapy.
 RESULTS: Among patients with thyroid antibodies, 6 out of the 11 pregnancies (54.5%) treated with IVIG ended in live birth. In the thyroid supplementation group, 13 out of 16 pregnancies (81.2%) ended in live birth. Only one pregnancy loss occurred among patients with a mild underlying thyroid pathology treated with thyroid replacement therapy.
 CONCLUSIONS: Mild thyroid abnormalities are associated with an increased rate of miscarriage. This poor obstetrical prognosis seems to be related to an impaired thyroid adaptation to pregnancy. Thyroid replacement therapy appears to be more effective than IVIG in preventing a new miscarriage.

The development of abnormal heart rate patterns after absent end-diastolic velocity in umbilical artery : analysis of risk factors

OBJECTIVES Our objectives were to evaluate the time interval elapsing between the occurrence of absent end-diastolic velocity in the umbilical artery and either the development abnormal fetal heart rate patterns or delivery and to establish the maternal and fetal factors that may affect this interval. STUDY DESIGN Thirty-seven fetuses free of structural and chromosomal abnormalities in which the development of absent end-diastolic velocity in umbilical artery was evidenced by serial Doppler recordings were studied. At the first occurrence the following factors were considered: gestational age, presence of hypertension or preeclampsia, amniotic fluid index, severity of growth retardation, and 10 different Doppler indices calculated from extracardiac and intracardiac vascular districts. Actuarial statistical methods were applied, with the occurrence of antepartum late heart rate deceleration as the censoring variable. RESULTS The interval between the first occurrence of absent end-diastolic velocity in umbilical artery and delivery ranged from 1 to 26 days. Indications for delivery were the development of antepartum late heart rate decelerations in 23 fetuses (62.1%) and different maternal or fetal complications in the remaining 14 fetuses. Multivariate analysis revealed that gestational age and the presence of hypertension and pulsations in umbilical vein were the dominant factors in determining the length of this time interval. CONCLUSION The duration of the time interval between the occurrence of absent end-diastolic velocity in umbilical artery and abnormal heart rate pattern differs considerably among fetuses, and it is mainly determined by gestational age and presence of maternal hypertension and pulsations in umbilical vein.

Doppler echocardiographic assessment of atrioventricular velocity waveforms in normal and small‐for‐gestational‐age fetuses

Summary. A prospective longitudinal study determined the physiological patterns of blood flow velocity waveforms in normal and in small‐for‐gestational‐age (SGA) fetuses. Using a Pulsed Doppler Duplex system, 125 normally grown fetuses and 35 SGA fetuses were studied longitudinally at between 27 and 42 weeks gestation. In normal fetuses the ratio between the E velocity (early passive ventricular filling) and the A velocity (active ventricular filling during atrial contraction) increased progressively during pregnancy in both transmitral and transtricuspid waveforms, approaching 1 at term. In SGA fetuses, the E/A ratios did not increase during pregnancy and the values obtained were significantly lower than in normal fetuses. The ratio between the transtricuspid and transmitral mean temporal velocities remained almost constant throughout pregnancy in the normal fetuses with transtricuspid velocity slightly exceeding the transmitral velocity. On the other hand, in SGA fetuses the ratio between the mean temporal velocities was inversed with the transmitral velocity progressively greater than the transtricuspid velocity suggesting the existence of intracardiac hacmodynamic changes in these fetuses.

Inferior vena cava flow velocity waveforms in appropriate- and small-for-gestational-age fetuses.

Reference ranges of inferior vena cava flow velocities were constructed from a cross-sectional study of 118 appropriate-for-gestational-age fetuses of 18 to 40 weeks of gestation. Blood flow velocity waveforms were recorded with color and pulsed Doppler equipment. Peak velocities and time velocity integrals were measured from inferior vena cava during systole, early diastole, and atrial contraction. The systolic-to-diastolic ratios between the peak velocities and time velocity integrals were calculated, and the reverse flow with atrial contraction was quantified as the percentage of forward flow (percentage of reverse flow). Recordings were also obtained from 79 small-for-gestational-age fetuses free of structural and chromosomal abnormalities, divided into three groups according to umbilical artery velocity waveforms: normal pulsatility index values (group 1, n = 26), pulsatility index greater than 95th percentile of our reference limits but presence of end-diastolic velocities (group 2, n = 33), and absence of end-diastolic velocities (group 3, n = 20). Fourteen fetuses of groups 2 and 3 were also studied at weekly intervals until the onset of antepartum late heart rate decelerations. In appropriate-for-gestational-age fetuses no changes were evident in peak velocities and time velocity integrals ratios, whereas the percentage of reverse flow significantly decreased with gestation. No significant differences were found between these values and those obtained in small-for-gestational-age fetuses of group 1. A significant increase of peak velocities and time velocity integrals ratios and of percentage of reverse flow was evidenced in fetuses of both group 2 and 3. The fetuses of groups 2 and 3 with a percentage of reverse flow above the 95% confidence interval showed a poorer perinatal outcome when compared with the fetuses of the same groups but with values inside the normal range. In the 14 fetuses longitudinally followed up until the onset of late heart rate decelerations a significant and progressive increase of peak velocities and time velocity integrals ratios and percentage of reverse flow was evidenced in spite of minimal changes in the pulsatility index from both umbilical artery and different peripheral fetal vessels. This study presents evidence that in small-for-gestational-age fetuses with abnormal Doppler-measured placental resistance the modified flow velocity patterns in the inferior vena cava seem to deteriorate progressively with advancing gestation.

Utero-placental blood flow velocity waveforms as predictors of pregnancy-induced hypertension

Sixty high-risk pregnancies were studied in order to define the validity of the analysis of utero-placental blood flow velocity waveforms in early screening for developing hypertensive diseases. Recordings were obtained at 18-20 weeks gestation, in normotensive patients, using a pulsed duplex Doppler system at the level of uterine vessels. The patients (n = 22) who developed hypertension showed a higher resistance index value (p less than 0.001) than normotensive patients (n = 38). The validity of uteroplacental waveform analysis was as follows: specificity = 84.2%; sensitivity = 63.6%; positive predictive value = 70%; negative predictive value = 80%; accuracy = 76.6%. The high specificity attained suggests that this test can adequately identify, among a high-risk population, patients destined to remain normotensive during pregnancy.

The value of fetal arterial, cardiac and venous flows in predicting pH and blood gases measured in umbilical blood at cordocentesis in growth retarded fetuses

Objective To assess the value of Doppler indices, calculated from fetal arterial peripheral vessels, cardiac outflow tracts and venous vessels, in the identification of acidaemia, hypercapnia and hypoxaemia as determined by pH and gas analysis of fetal blood obtained by cordocentesis in growth retarded fetuses.