The deregulation of regulatory T cells on interleukin-17-producing T helper cells in patients with unexplained early recurrent miscarriage.

BACKGROUND: CD4(+)CD25(+) regulatory T cells (Tregs) are important for the maintenance of immune homeostasis by virtue of their ability to control T-cell proliferation in the peripheral blood (PB). We recently demonstrated that the prevalence of Tregs is decreased, whereas that of Th17 cells is increased, in the PB and decidua samples of patients with unexplained recurrent miscarriage (RM). In this study, we investigated whether the cytokine production of Th17 cells can be suppressed by the Tregs and elucidated the mechanism by which Tregs exert this suppressive effect. METHODS: Flow cytometry was used to analyze the surface phenotype and cytokine production of Th17 cells in the PB of women with unexplained RM (n = 17) and healthy women in early stages of pregnancy who underwent elective abortion (n = 20). The suppressive ability of Tregs on Th17 cells was assessed in in vitro co-cultures and transwell experiments. The amount of secreted interleukin-17 (IL-17) in the supernatants was measured by enzyme-linked immunosorbent assay (ELISA). The inhibitory activity of transforming growth factor-ß (TGF-ß) and IL-10 on IL-17 expression in CD4(+) T cells was assessed using ELISA. RESULTS: The proportions of IL-17-positive CD4(+) T cells, CC chemokine receptor type 6 (CCR6)-positive CD4(+) T cells and CCR6 expression of IL-17-positive CD4(+) T cells were higher in the PB samples of patients with unexplained RM than in PB of healthy control subjects. In vitro, Tregs could inhibit the expression of IL-17; more Th17 cells were inhibited in the control group than in the unexplained RM group. High-dose TGF-ß inhibited the expression of IL-17, whereas IL-10 inhibited IL-17 expression in a dose-dependent manner. CONCLUSIONS: IL-17 expression can be inhibited by Tregs. The suppressive activity of Tregs on Th17 cells was decreased in patients with unexplained RM. The ability of Tregs to suppress cytokine secretion might be effected by a cell-cell contact. TGF-ß and IL-10 could inhibit the expression of IL-17.

[A multi-center study to evaluate the dynamic changes of uterine artery and umbilical artery flow in a normal pregnancy and hypertensive disorders in pregnancy].

OBJECTIVE: To investigate the dynamic changes of uterine artery and umbilical artery in the first, second, and third trimester of normal pregnancy and hypertensive disorders in pregnancy (HDP). METHODS: A multi-center prospective study was conducted on 1098 women with normal singleton pregnancies at the first prenatal visit in the Second West China Hospital of Sichuan University, First Affiliated Hospital with Nanjing Medical University, Beijing Obstetrics and Gynecology Hospital Affiliated to Capital Medical University, Wuhan Union Hospital Affiliated to Medical School of Huazhong University of Science and Technology and Renji Hospital Affiliated to Medical School of Shanghai Jiao Tong University from April 2005 to July 2006, with the average age of (28.3 ± 3.3). The pulsatility indices (PI), resistance indices (RI) and systolic to diastolic velocity ratios (S/D) of uterine artery and umbilical artery were measured for all subjects in the first (10th-14th gestational weeks), second (20th-26th gestational weeks) and third trimester (30th-36th gestational weeks), respectively. In this longitudinal study, women who developed HDP were classified into HDP group, and the rest into normal pregnancy group. RESULTS: (1) Among the 1098 pregnant women, 44 developed HDP during the index pregnancy, including 20 gestational hypertension, 15 mild pre-eclampsia and 9 severe pre-eclampsia, giving an incidence of 4.17%, and the rest 1054 were normal until delivery. (2) In the normal pregnancy group, the RI, PI and S/D of uterine artery were decreased with the progress of pregnancy (RI: 0.64, 0.57, 0.50; PI: 1.24, 0.98, 0.80; S/D: 3.26, 2.58, 2.20; P < 0.01). However, the above indices showed an increasing trend with the increase of gestations in the HDP group (RI: 0.55, 0.67, 0.64; PI: 1.22, 1.36, 1.20; S/D: 3.18, 3.41, 3.05; P < 0.01). In the second and third trimester, the RI, PI and S/D of uterine artery in the HDP group were higher than those in the normal pregnancy group (P < 0.01). (3) In the normal pregnancy group, the RI, PI and S/D of the umbilical artery decreased from the second to the third trimester (RI: 0.71 and 0.58; PI: 1.16 and 0.87; S/D: 3.58 and 2.48; P < 0.01). However, no significant difference was found in the RI, PI and S/D value of umbilical artery in the second and third trimester between the normal and HDP group (RI: 0.71 and 0.63; PI: 1.20 and 0.95; S/D: 3.71 and 2.69; P > 0.05, respectively), despite the decreasing trend in the HDP group. CONCLUSIONS: In uncomplicated pregnancies, the blood flow resistance of uterine artery decreases and the end-diastolic blood flow of uterine artery increases with the progress of pregnancy. However, in pregnant women with HDP, the blood flow resistance of uterine artery increases significantly with the increase of gestations which shows significant difference to that in normal pregnancies. The blood flow resistance of umbilical artery decreases in both normal and HDP pregnant women with the increasing gestational age.

[Regulative effects of regulatory T cells on dendric cells in peripheral blood and deciduas from unexplained recurrent spontaneous abortion patients].

OBJECTIVE: To study the effect of CD(4)(+)CD(25)(+) regulatory T(Tr) cells on dendric cells (DC) in peripheral blood and deciduas from unexplained recurrent spontaneous abortion (URSA) patients. METHODS: Four URSA patients (abortion group) and 4 normal early pregnant women (control group) were enrolled in this study. Tr cells and DC in the peripheral blood and deciduas were isolated using Ficoll density gradient centrifugation and magnetic cell sorting (MACS). DC were cultured alone (DC alone) or in combination with Tr cells (DC + Tr) for 6 days, during which the release of interferon (IFN)-gamma and interleukin (IL)-10 in the medium was subsequently measured by enzyme linked immunoadsorbent assay (ELISA). RESULTS: (1) Peripheral blood: there was no significant difference in IFN-gamma level between DC alone (23.2 +/- 0.7) ng/L and DC + Tr (22.5 +/- 3.0) ng/L in abortion group (P > 0.05). The similar level of IL-10 was observed between DC alone (37 +/- 7) ng/L and DC + Tr (35 +/- 4) ng/L in abortion group (P > 0.05). IL-10 level, but not IFN-gamma, was significantly higher in DC alone (54 +/- 20) ng/L than that in DC + Tr (36 +/- 9) ng/L in control group (P < 0.01). (2) Deciduas: there was no significant difference in IFN-gamma level between DC alone (23.4 +/- 2.6) ng/L and DC + Tr (24.4 +/- 2.5) ng/L in abortion group (P > 0.05). Moreover, Similar IL-10 level was found between DC alone (28 +/- 7) ng/L and DC + Tr (25 +/- 5) ng/L in abortion group (P > 0.05). IFN-gamma level in DC alone (30.7 +/- 4.6) ng/L was significantly higher than that in DC + Tr (22.6 +/- 3.8) ng/L in control group (P < 0.01); whereas IL-10 level was much lower in DC alone (27 +/- 6) ng/L than that in DC + Tr (31 +/- 9) ng/L in control group (P < 0.05). CONCLUSION: The decreasing of immunosuppressive function of Tr cell of URSA patients affect its regulation on DC, resulting in imbalance of Th1/Th2 and abnormality of maternal-fetal immuno-tolerence in URSA.

[Multi-center study of motherwort injection to prevent postpartum hemorrhage after caesarian section].

OBJECTIVE: To evaluate the efficacy and safety of motherwort (herba leonuri/leonurus heterophyllus sweet) injection for preventing postpartum hemorrhage after caesarian section. METHODS: The prospective study was designed as a randomized and single blind multi-center research matched with positive agent as controls from Apr 2007 to Aug 2007. 440 women underwent caesarian section (CS) indicated by obstetric factors were enrolled from 15 teaching hospitals in China and assigned into three groups: group of motherwort: 147 cases were administered by motherwort 40 mg uterine injection during CS and 20 mg intramuscular injection per 12 hours 3 times after CS; group of motherwort+oxytocin: 144 cases were administered by motherwort 40 mg and oxytocin 10 U uterine injection during CS and motherwort 20 mg intramuscular injection per 12 hours 3 times after CS and group of oxytocin: 149 cases were administered by oxytocin 10 U uterine injection and oxytocin 10 U+5% glucose 500 ml intravenously injection during operation and oxytocin 10 U intramuscular injection per 12 hours 3 times after CS. The following clinical parameter were collected and analyzed: (1) The amount of blood loss during operation, at 2, 6, 12, 24, 48 hours after operation. (2) The total amount of blood loss in 24 hours after CS and the incidence of postpartum hemorrhage. (3) The change of level of hemoglobin (Hb) and counting of red blood cell (RBC) from prepartum to postpartum. (4) Adverse reaction. RESULTS: (1) The mean amount of blood loss during operation were (368+/-258) ml in group of motherwort, (255+/-114) ml in group of motherwort+oxytocin and (269+/-141) ml in group of oxytocin, which exhibited significant difference among three groups (P<0.01). Meanwhile, no statistical different amount of blood loss among three groups were observed at 2, 6, 12, 24, 48 hours after CS. (2) The amount of blood loss of postpartum at 24 hours were (480+/-276) ml in group of motherwort, (361+/-179) ml in group of motherwort+oxytocin, (381+/-179) ml in group of oxytocin, which showed significant difference among 3 groups (P<0.01). (3) The incidence of postpartum hemorrhage were 32.0% (47/147) in group of motherwort, 11.1% (16/144) in group of motherwort+oxytocin, and 18.8% in (28/149) in group of oxytocin. When comparing the lowest rate of postpartum blood loss in group of motherwort+oxytocin and the highest rate in group of motherwort, it displayed statistical difference (P<0.01). (4) The decreased level of RBC and Hb were shown that RBC (0.3+/-0.5)x10(12)/L and Hb (9+/-13) g/L in group of motherwort, RBC (0.2+/-0.4)x10(12)/L and Hb (6+/-10) g/L in group of motherwort+oxytocin and RBC (0.2+/-0.4)x10(12)/L and Hb (7+/-30) g/L in group of oxytocin respectively. However, the comparison of different value of RBC and Hb in group of oxytocin and motherwort+oxytocin showed significant difference (P<0.05). (5) Two cases with allery reaction was observed. CONCLUSION: It is safe and efficacious that combined use of motherwort injection and oxytocin was to prevent postpartum hemorrhage during or after caesarian section.

[Changes in proportion of decidua and peripheral blood CD(4)(+)CD(25)(+) regulatory T cells in unexplained recurrent spontaneous abortion patients].

OBJECTIVE: To study the changes in the percentage of CD(4)(+)CD(25)(+) regulatory T (Tr) cells in peripheral blood and deciduas in unexplained recurrent spontaneous abortion (URSA) patients, normal non-pregnant and pregnant women respectively. METHODS: The percentage of CD(4)(+)CD(25)(+) Tr cells in deciduas and peripheral blood from 25 URSA patients, 22 normal non-pregnant (NNP) women, and 34 normal early pregnant (NP) women were measured by double-staining followed by flow cytometric analysis. RESULTS: (1) The percentage of CD(4)(+)CD(25)(bright) T cells in peripheral blood in both URSA and NP [(1.55 +/- 0.77)%, (2.65 +/- 1.10)%, respectively] women were increased significantly than that in NNP women [(0.39 +/- 0.14)%, P < 0.05]. The percentage of CD(4)(+)CD(25)(bright) T cells in peripheral blood in URSA women was significantly lower than that in NP women (P < 0.05). (2) The percentage of CD(4)(+)CD(25)(bright) T cells in decidua in URSA women was significantly lower than that in NP women [(0.59 +/- 0.23)%, (1.24 +/- 0.55)%, respectively, P < 0.01]. There was no significant difference in the percentage of CD(4)(+)CD(25)(dim) T cells in decidua between URSA women and NP women [(4.23 +/- 1.52)%, (3.75 +/- 1.88)%, respectively, P > 0.05]. (3) The proportion of CD(4)(+)CD(25)(bright)/CD(4)(+) cells in deciduas was significantly higher than that in peripheral blood in NP women [(13.10 +/- 10.25)%, (5.59 +/- 2.62)%, respectively, P < 0.05]. However, a significant difference in the proportion of CD(4)(+)CD(25)(bright)/CD(4)(+) between decidua and peripheral blood was not found in URSA patients [(5.16 +/- 2.83)%, (4.64 +/- 2.07)%, respectively, P > 0.05)]. CONCLUSIONS: The number of CD(4)(+)CD(25)(+) Tr cells is increased in normal pregnancy and decreased in URSA. Therefore, CD(4)(+)CD(25)(+) Tr cells may play an important role in maintaining maternal-fetal tolerance and may be involved in the pathogenesis of URSA.

[The skewed usage of T cell receptor beta variable chain at the maternal-fetal interface of women with unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate T cell receptor (TCR) variable beta (BV) chain usage at the maternal-fetal interface and explore the relationship between the skewed TCR BV usage and unexplained recurrent spontaneous abortion (RSA). METHODS: Eighteen cases with unexplained RSA, together with matched 41 women with normal pregnancies in first trimester from Renji Hospital, Shanghai Jiao Tong University were studied. A high-resolution spectrum typing analysis of complementarity-determining region 3 (CDR3) was used to detect and compare the degree and frequency of TCR BV family expression in deciduas between RSA patients and normal controls. RESULTS: (1) The expression degree of BV19 (0.029 +/- 0.031 vs. 0.013 +/- 0.010, P = 0.038) in RSA group showed a higher usage, while BV5.2 (0.040 +/- 0.035 vs. 0.067 +/- 0.052, P = 0.046) showed a significantly lower usage when compared with normal controls. No significant difference in the expression of the other TCR BV families between RSA and controls were observed (P > 0.05). (2) TCR BV2, 3, 6, and 7 were the four most common BV families in deciduas of patients with RSA and normal controls, whose frequencies were all more than 50%. In RSA group, higher frequencies of BV15 (33.3% vs. 7.3%, P = 0.018), BV19 (38.9% vs. 14.6%, P = 0.049) and BV20 (33.3% vs. 7.3%, P = 0.018) were observed; meanuhile lower frequencies of BV4 (33.3% vs. 65.9%, P = 0.026) and BV7 (66.7% vs.92.7%, P = 0.018) distributions were observed. The other TCR BV families did not display significantly different freqencies of distribution (P > 0.05). CONCLUSIONS: It is suggested that a significant skewed TCR BV family occurs at the maternal-fetal interface in patients who undergo abortion. The specific skewed usages of TCR BV might be associated with the susceptibility to unexplained pregnancy loss.

[Study on the expression of transcription factor GATA-3 and T-bet mRNA in decidua of women with unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate the roles of transcription factor GATA-3 and T-bet at the fetal-maternal interface in the pathogenesis of unexplained recurrent spontaneous abortion (URSA). METHODS: The expression of GATA-3 and T-bet mRNA was examined by in situ hybridization. Decidua was obtained from 20 women with URSA and 20 normal pregnant (NP) women. RESULTS: (1) The number of GATA-3 positive cells per high power field in women with URSA (25 +/- 16) was significantly lower than those in NP women (38 +/- 16) (P < 0.05). (2) The number of T-bet positive cells per high power field in women with URSA (59 +/- 17) was significantly higher than those in NP women (46 +/- 18) (P < 0.05). (3) Numbers of cells expressing GATA-3 mRNA correlated significantly with T-bet mRNA positive cell numbers in decidua (r = -0.55, P < 0.01). CONCLUSIONS: The results demonstrate that upregulated expression of T-bet and downregulated expression of GATA-3 are found in the decidua of women with URSA. Imbalance of GATA-3/T-bet transcription factors might interfere with type 1 helper T cell (Th1)/type 2 helper T cell (Th2) differentiation at the fetal-maternal interface and cause URSA.

[Expression of trophoblast invasion related genes mRNA and protein in human placenta in preeclampsia].

OBJECTIVE: To study the mRNA and protein expression of matrix metalloproteinase (MMP) 9, 2, metastasis suppressor gene KiSS-1, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in placenta of preeclampsia patients and their relation to the pathogenesis of preeclampsia. METHODS: Expression of MMP-9, MMP-2, KiSS-1, TIMP-1, TIMP-2 mRNAs and proteins in placenta from 27 cases of preeclampsia, 10 cases of gestational hypertension and 30 cases of normal term pregnant women was detected by semi-quantitative RT-PCR, western blotting, and enzyme activity of MMP-9 and MMP-2 was measured by gelatin zymography. RESULTS: (1) Expression of MMP-9 and MMP-2 mRNA in placenta of preeclampsia (0.39 +/- 0.05 and 0.71 +/- 0.16) was significantly lower than that in normal term pregnancy (0.78 +/- 0.11 and 1.63 +/- 0.31, P < 0.05). Expression of KiSS-1 and TIMP-1 mRNAs in placenta of preeclampsia (1.97 +/- 0.21 and 1.11 +/- 0.18) was significantly higher than that in normal term pregnancy (0.69 +/- 0.27 and 0.65 +/- 0.19) (P < 0.05.). There was no significant difference in TIMP-2 mRNA level between preeclampsia and normal pregnancy (P > 0.05). (2) Expression of MMP-9 and MMP-2 proteins in preeclampsia (1.07 +/- 0.35 and 0.74 +/- 0.23) was significantly lower than that in normal term pregnancy (2.43 +/- 0.92 and 1.48 +/- 0.78) (P < 0.05). Expression of KiSS-1 and TIMP-1 proteins in placenta of preeclampsia (2.46 +/- 0.39 and 1.51 +/- 0.40) was significantly higher than that in normal pregnancy (0.91 +/- 0.35 and 0.93 +/- 0.56) (P < 0.05). There was no significant difference in TIMP-2 protein level between preeclampsia and normal pregnancy (P > 0.05). (3) Enzyme activity of MMP-9 and MMP-2 in placenta of preeclampsia [(2.67 +/- 0.53) gray level.g(-1).L(-1) and (1.13 +/- 0.28) gray level.g(-1).L(-1))] was significantly lower than that in placenta of normal pregnancy [(8.44 +/- 3.70) gray level.g(-1).L(-1) and (3.87 +/- 1.43) gray level.g(-1).L(-1)] (P < 0.05). CONCLUSION: Abnormal expression of MMP-9, MMP-2, KiSS-1 and TIMP-1 can cause insufficiency invasion of trophoblast in preeclampsia and superficial placentation, which may play an important role in the pathogenesis and development of preeclampsia.

[A/G polymorphism at position 49 in exon 1 of CTLA-4 gene in Chinese women with unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate whether A/G polymorphism at position 49 in exon 1 of cytotoxic T lymphocyte antigen-4 (CTLA-4) gene confers the susceptibility to unexplained recurrent spontaneous abortion in Chinese population. METHODS: One hundred and sixty-eight restrictive Chinese women with unexplained recurrent spontaneous abortion (URSA) and 117 women with normal pregnancy as control were included in this study. Polymerase chain reaction restrictive fragment length polymorphism (PCR-RFLP) was used to detect the polymorphism at position 49 in exon 1 of CTLA-4 gene. The frequency of alleles G/A, genotypes AA/AG/GG and phenotypes A+ (AA + AG)/G+ (GG + AG) of CTLA-4 were compared between URSA patients and controls. RESULTS: The different distributions of alleles G/A, genotype AA/AG/GG and phenotypes A+/G+ of CTLA-4 were observed between URSA patients and controls. The frequencies of both G allele [68.4% (230/336) vs 59.4% (139/234), P < 0.05] and GG genotype [48.8% (82/168) vs 33.3% (39/117), P < 0.05] were significantly higher in URSA group than that in control group, while the frequencies of AG genotype [39.3% (66/168) vs 52.1% (61/117), P < 0.05] and A+ (AA + AG) phenotype [51.2% (86/168) vs 66.7% (78/117), P < 0.05] were significantly lower in URSA group than that in control group. CONCLUSIONS: The results suggest that A/G polymorphism in exon-1 of CTLA-4 might confer the susceptibility to RSA in Chinese women.

[The role of KiSS-1 and matrix metalloproteinase-9 in regulation of invasion of trophoblasts].

OBJECTIVE: To explore the role of metastasis-related gene KiSS-1 and matrix metalloproteinase (MMP)-9 in regulation of invasion of trophoblasts. METHODS: RT-PCR and Western blotting were used to detect the MMP-9 and KiSS-1 expression levels in the placental tissues obtained from 90 cases of normal pregnant women undergoing artificial abortion, induction of labor with water bag or selective cesarean section among which 30 cases were in the first trimester, 30 in second-trimester and 30 cases of term pregnancy, and in the placental tissues of 40 cases of preeclampsia (15 cases of mild and 25 cases of severe preeclampsia) undergoing cesarean section, and tissues of 90 cases of hydatidiform mole, 9 cases of invasive mole and 8 cases of choriocarcinoma, all undergoing surgery. RESULTS: The expression levels of MMP-9 mRNA and protein were higher in first-trimester [A value 0.391 +/- 0.215, (36 +/- 7) microg/100 microg total protein] and then decreased gradually with the progress of gestation. The expression levels of MMP-9mRNA and protein in the term placental samples were significantly lower than those of first-trimester (both P < 0.01). The expression levels of KiSS-1mRNA and metastin in normal placenta increased along with the progress of gestation (both P < 0.01). The KISS-1 mRNA expression level and MMP-9 protein expression level in the placental tissue of preeclampsia were 0.09 +/- 0.06 (A value) and (9.6 +/- 4.3) microg/100 microg total protein respectively, both significantly lower than those of the term placenta (both P < 0.01). The expression levels of MMP-9 mRNA and protein in the tissues of gestational trophoblastic disease were significantly higher than those in the first-trimester placenta (both P < 0.01). The expression level of KISS-1 mRNA and metastin in the tissues of hyddatidiform mole and invasive mole were both significantly lower than those in the first trimester placenta (P < 0.05, P < 0.01). The expression of KiSS-1mRNA and metastin in the tissues of choriocarcinoma could not be detected. CONCLUSION: The expression of the invasion-related gene, MMP-9, is positively related with, while the invasion suppressor gene, KiSS-1, is negatively related with the invasive ability of trophoblasts. The interaction of these two genes plays an important role in regulation of the invasion of trophoblasts.

[Clinical significance of KiSS-1 and matrix metalloproteinase-9 expression in trophoblasts of women with preeclampsia and their relation to perinatal outcome of neonates].

OBJECTIVE: To investigate the role of KiSS-1 and matrix metalloproteinase (MMP) 9 in trophoblasts in the pathogenesis of preeclampsia and their relation to perinatal outcome of neonates. METHODS: RT-PCR and western blot analyses were used to detect the MMP-9 and KiSS-1 expression levels in trophoblast of 40 patients with preeclampsia (15 cases of mild and 25 cases of severe preeclampsia) (preeclampsia group) and 20 cases of term pregnancy (normal pregnancy group) and their correlations with symptoms and perinatal outcome of neonates were analyzed. RESULTS: (1) The KiSS-1mRNA and metastin expression levels in trophoblasts of preeclampsia group were 1.73 +/- 0.24 (A value) and (78.4 +/- 8.0) microg/100 microg total protein separately, those of mild preeclampsia were (1.50 +/- 0.15) and (72.4 +/- 6.9) microg/100 microg total protein, and severe preeclampsia were (1.87 +/- 0.20) and (83.52 +/- 3.57) microg/100 microg total protein, which were all significantly higher than those of normal pregnancy group [1.24 +/- 0.25, P < 0.01, and (63.4 +/- 2.7) microg/100 microg total protein, P < 0.01], especially those in severe preeclampsia. (2) The MMP-9 mRNA and MMP-9 expression levels in trophoblasts of preeclampsia group were 0.09 +/- 0.06 and (9.6 +/- 4.2) microg/100 microg total protein, those of mild preeclampsia were 0.11 +/- 0.08 and (10.0 +/- 3.2) microg/100 microg total protein, and severe preeclampsia were 0.07 +/- 0.05 and (7.8 +/- 2.0) microg/100 microg total protein, which were all significantly lower than those of normal pregnancy group [0.17 +/- 0.10, P < 0.01, and (17.9 +/- 7.3) microg/100 microg total protein, P < 0.01]. (3) The expression level of KiSS-1mRNA and metastin in preeclampsia group were positively correlated with MAP, urinary protein per 24 hours, the correlation coefficients between KiSS-1mRNA and MAP, urinary protein per 24 hours were 0.610 (P = 0.023), 0.397 (P = 0.003), and between metastin and MAP, urinary protein per 24 hours were 0.713 (P = 0.011), 0.638 (P = 0.002), separately. The expression level of KiSS-1mRNA and metastin in preeclampsia group was significantly positively correlated with the occurrence of fundus oculi artery spasm, correlation coefficients were 0.499 (P = 0.000) and 0.511 (P = 0.000). The MMP-9mRNA and MMP-9 expression levels were negatively correlated with MAP, urinary protein per 24 hours and the occurrence of fundus oculi artery spasm, correlation coefficients between MMP-9mRNA and MAP, urinary protein per 24 hours, the occurrence of fundus oculi artery spasm were 0.561 (P = 0.042), 0.275 (P = 0.039), 0.346 (P = 0.001), between MMP-9 and MAP, urinary protein per 24 hours, the occurrence of fundus oculi artery spasm were 0.571 (P = 0.022), 0.375 (P = 0.048), 0.543 (P = 0.000), separately. (4) The expression level of MMP-9 mRNA and MMP-9 of preeclampsia was positively correlated with neonatal birth weight significantly, Pearson correlation coefficient was 0.651 (P = 0.000) and 0.544 (P = 0.004) separately, while the expression levels of KiSS-1mRNA and KiSS-1 of preeclampsia were negatively correlated with neonatal birth weight significantly, Pearson correlation coefficients were 0.759 (P = 0.000) and 0.865 (P = 0.000) separately. (5) The expression level of MMP-9 showed decreased tendency with the severity of newborn asphyxia, while the expression level of KiSS-1 showed increased tendency (P < 0.05). CONCLUSION: The expression imbalance of MMP-9 and KiSS-1 in trophoblasts may play an important role in the pathogenesis of preeclampsia, and correlate to perinatal outcome of neonates.

Association of HLA-DQB1 coding region with unexplained recurrent spontaneous abortion.

BACKGROUND: DNA analysis has shown a lack of significant compatibility between couples affected by unexplained recurrent spontaneous abortion (URSA) compared with normal fertile couples, [8] although one study that made use of a PCR-sequence-specific oligonucleotide (SSO) method did observe evidence of significant compatibility in the HLA-DQA1 and DQB1 alleles between patients and aborted fetuses. [9] This study was designed to investigate whether URSA were associated with particular DQ alleles or promoter alleles. METHODS: Thirty-two patients with URSA and 54 women who had had at least one successful pregnancy were included in this study. HLA-DQ genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The HLA-DQB1 promoter was detected by the SSO and sequence-specific primer (SSP) methods. The DQA1, DQB1, and DQB1 promoter (QBP) gene frequencies in the patients were compared with the gene frequencies in normal controls. The data were analyzed statistically with the chi(2) and Fisher's exact tests. RESULTS: The results showed that the frequency of DQB1 * 0604/0605 was significantly higher and the frequency of DQB1 * 0501/0502 was significantly lower in the patient group as compared with the normal controls. In addition, the frequencies of the DQA1 * 01-DQB1 * 0604/0605 and QBP6.2-DQB1 * 0604/0605 haplotypes were overrepresented in the patients relative to the controls. Our results did not show any differences between URSA patients and the controls with regard to DQA1 and QBP allele frequencies. CONCLUSIONS: Our data suggest that URSA is associated with the HLA-DQB1 coding region, and is not associated with its upstream regulatory region. The DQB1 * 0604/0605, DQA1 * 01-DQB1 * 0604/0605, and QBP6.2-DQB1 * 0604/0605 haplotypes may confer susceptibility to URSA, while the DQB1 * 0501/0502 allele may protect women from URSA.

[Relationship between left ventricular diastolic function and plasma brain natriuretic peptide concentration during severe pregnancy-induced hypertension syndrome].

OBJECTIVE: To evaluate the relationship between the left ventricular diastolic function and plasma level of plasma brain natriuretic peptide (BNP) among patients with pregnancy-induced hypertension (PIH) syndrome. METHODS: The left ventricular diastolic function was evaluated by Doppler echocardiography, and plasma BNP level was tested by radioimmunoassay among 36 pregnant women with severe PIH, 32 normal pregnant women, and 21 normal non-pregnant women of childbearing age. RESULTS: The parameters such as peak mitral flow velocity in early diastole (E), E-wave velocity-time integral (EVTI), the ratio of peak mitral flow velocity in early diastole and peak A-wave velocity in late diastole (E/A), peak pulmonary venous diastolic forward flow velocity (D) and D-wave velocity-time integral (DVTI) of the 36 severe PIH patients were significantly lower than those of the 32 normal pregnant women and those of the 21 normal non-pregnant women of child-bearing age. But the parameters such as A, A-wave velocity integral (AVTI), and peak pulmonary venous diastolic forward flow velocity (AR) were significantly higher than those of the normal pregnant women and those of the normal non-pregnant women of child-bearing age. E/A ratio, D and DVTI of the normal pregnant women were significantly lower than those of the normal non-pregnant women of child-bearing age, however, A, AVTI, S/D, and AR were significantly higher than those of the normal non-pregnant women of child-bearing age. The BNP concentration of the normal pregnant women was significantly higher than that of the normal non-pregnant women of childbearing age, but significantly lower than that of the severe PIH patients. There were significant correlations between left ventricular diastolic function variables (E/A ratio and AR) and BNP concentration in normal pregnant women and in PIH patients. CONCLUSION: The left ventricular diastolic function is slightly damaged in normal pregnant women and significantly damaged in patients with severe PIH. The plasma BNP level of pregnant women can become an excellent index to predict their left ventricular diastolic function.

[Study on the relationship between regulatory T cells and unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate the relationship between CD(4)(+)CD(25)(+) regulatory T cells (CD(4)(+)CD(25)(+)Tr) and unexplained recurrent spontaneous abortion (URSA). METHODS: The expression rate of CD(4)(+)CD(25)(+)Tr in peripheral blood was determined by double-label flow cytometric analysis. The samples were taken from 20 normal non-pregnancy women, 17 URSA women and 20 normal pregnancy women. RESULTS: The expression frequency of CD(4)(+)CD(25)(+)Tr cells was decreased significantly (P < 0.01) in URSA women [(11.9 +/- 2.7)%] than that in normal non-pregnancy women [(15.2 +/- 3.0)%], however, there was no significant difference between normal pregnancy women [(14.4 +/- 4.8)%] and normal non-pregnancy women (P > 0.05). CONCLUSION: The results suggest that downregulation of CD(4)(+)CD(25)(+)Tr may be associated with URSA.

[C677T and A1298C mutation of the methylenetetrahydrofolate reductase gene in unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate the association between the C677T and A1298C mutation of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene and unexplained recurrent spontaneous abortion (URSA) in Chinese population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect the mutation of C677T and A1298C of MTHFR in 148 cases with URSA and 82 normal controls. RESULTS: (1) The distribution frequencies of C667T associated 3 genotypes between the URSA and control group showed statistically significant difference (P = 0.012). The frequencies of C677T genotypes were: CC (33.3%), CT (53.1%), TT (13.6%) in URSA group and CC (52.4%), CT (51.5%), TT (6.1%) in control group, respectively. And the frequency of CC genotype in URSA group was decreased significantly (P = 0.005), while the frequency of T allele in URSA was increased (P < 0.005). (2) The prevalence of the MTHFR A1298C associated 3 genotypes and A/C alleles in URSA group did not differ significantly from the control. (3) According to the linkage analysis of C677T and A1298C, 8 linkage genotypes were found, and the frequency of 677CC/1298AA in URSA was significantly lower compared with the control, the linkage of 677 (CT + TT)/1298CC was only observed in URSA group. CONCLUSIONS: The mutations of MTHFR C677T and A1298C play a role in the mechanism of unexplained recurrent spontaneous abortion.

Study on the expressions of PHD and HIF in placentas from normal pregnant women and patients with preeclampsia.

OBJECTIVE: To investigate the relationship between oxygen sensitivity of trophoblast and hypoxia in preeclamptic placenta by the study on the expressions of hypoxia-inducible factor prolyl 4-hydroxylase (PHD) and hypoxia-inducible factor (HIF) in placentas from normal pregnant women and patients with pre-eclampsia. METHODS: Subjects were chosen from the in-patients or the out-patients from May 2003 to May 2004. They were divided into 5 groups: early pregnancy group (EP), 13 cases; middle pregnancy group (MP), 9 cases; late pregnancy group (LP, or control group), 12 cases; preeclampsia (PE) group, 20 cases; gestational hypertension group (GH), 10 cases. The mRNA expressions of PHD-1 and -2 and -3 in placentas from all the subjects were assessed by in situ hybridization and Real-time PCR. The expressions of HIF-1α and -2α in placentas from different groups were assessed by immunohistochemistry and western blot. RESULTS: PHD-1,-2 and -3 mRNA were mainly expressed in cytoplasm of trophoblast, especially strongly expressed in extravillous trophoblast. During the progress of pregnancy, the expression of PHD-1 increased significantly (R=0.616, P<0.001). The PHD-1mRNA expression in placentas from PE group decreased significantly compared with that from control group, P<0.05. A significant direct correlation between the PHD-1 mRNA expression in placentas from PE group and their placenta weight was found (R=0.457, P<0.05). The HIF-2α, not the HIF-1α expression, from PE group was significantly higher than that from control group, P<0.01; The HIF-2α expression in trophoblast from PE was inversely correlated to the date of the onset of the disease (R=-0.730, P<0.01). CONCLUSIONS: PHD-1 played an important role in hypoxic response pathway of trophoblast through modulating the level of HIF-2α. The overly activated hypoxic response pathway of trophoblast in preeclamptic placenta, which is manifested as the result of HIF-2α over-expression, is the key point to hypoxic dysfunction of trophoblast.

Dysregulation of macrophage activation by decidual regulatory T cells in unexplained recurrent miscarriage patients.

CD4(+)CD25(+) T cells (Treg cells) and macrophages play roles in the maintenance of maternal-fetal immunological tolerance. Treg cells suppress the function of macrophages via mechanisms mediated by cell-cell contact and production of soluble factors. The purpose of this study was to investigate regulation of macrophages by Treg cells within decidua from patients with unexplained recurrent miscarriage (RM) and normal control women during early pregnancy. Treg cells and macrophages were isolated from deciduas of unexplained RM (n=15) and control women (n=15) by magnetic cell separation and co-cultured for six days. Regulation of macrophages by Treg cells was assessed in the presence and absence of neutralizing anti-TGFß antibodies and in transwell experiments. Expression of CD80, CD86, IL10, and IFNγ by macrophages was measured by flow cytometry or ELISA. Macrophage expression of CD80 and CD86 was higher in deciduas of unexplained RM patients compared with controls whereas the expression of IL10 was lower. There was no difference in the expression of IFNγ by macrophages between the two groups. Treg cells inhibited macrophage expression of CD80, CD86 and IFNγ and increased the expression of IL10. The regulatory effects of Treg cells were abrogated in the presence of neutralizing anti-TGFß antibodies or by transwell culture. The phenotype of macrophages therefore differed in unexplained RM patients compared with normal early pregnant subjects. Macrophage regulation by Treg cells was shown to be mediated by cell-cell contact and TGFß and this capacity was decreased in unexplained RM patients.