Implications of prenatal diagnosis of the fetus with both interstitial deletion and a small marker ring originating from chromosome 5.

We describe a patient with 47,XY,del(5)(p11p13), +mar observed in prenatal screening. We performed analyses including G-banding, multi-color fluorescent in situ hybridization (mFISH) for fetal chromosome detection. After birth array-based comparative genomic hybridization (aCGH), bacterial artificial chromosome (BAC)-FISH was carried out to define the chromosomal changes precisely. The mFISH revealed that a ring chromosome that had originated from chromosome 5. The aCGH showed that this fetus had a terminal duplication, an interstitial deletion, and a pericentromeric duplication of the short arm of chromosome 5. This complex alteration resulted in partial trisomy 5p15.33-p15.31, partial monosomy 5p14.3-p13.2, and partial trisomy 5p12-p11. To clarify these alterations, we performed BAC-FISH using BAC clones related to deleted and duplicated regions, and found that a derivative (der) chromosome 5 showed the presence of hybridization signals from the duplicated region at 5p15.33 and the loss of hybridization signals from the deleted region at 5p14.2. In addition, FISH analysis confirmed the origin of the marker chromosome. Hybridization signals from the second intervening sequence at 5p13.1, between the deleted region and the pericentric duplicated region, were present on the marker ring chromosome.

Preoperative serum thrombopoietin levels are higher in patients with ovarian cancer than with benign cysts.

OBJECTIVE: To assess the diagnostic relevance of serum thrombopoietin (TPO) levels, we compared serum TPO levels between the patients with ovarian cancer and benign ovarian cysts. We also correlated serum TPO concentrations in ovarian cancer patients to know prognostic factors such as disease stage, tumor grade, histological subtype, and residual tumor mass. STUDY DESIGN: Preoperative serum TPO levels were measured in women with epithelial ovarian cancer (n=51) and women with benign ovarian cysts (n=25) using an enzyme-linked immunosorbent assay. RESULTS: The serum TPO concentration was significantly elevated in the ovarian cancer patients as compared to the benign patient controls. Platelet counts and TPO in the serum did not correlate in the ovarian cancer patients. However, using a TPO cutoff of 90 pg/ml and a CA125 cutoff of 30 units/ml, when both markers were elevated, the specificity was as high as 92%. CONCLUSION: Serum TPO concentrations may have a role in the diagnostic clinical setting for discerning benign from malignant ovarian tumors.

Elevated serum RANTES levels in patients with ovarian cancer correlate with the extent of the disorder.

OBJECTIVE: To assess the clinical relevance of serum regulated upon activation, normal T-cell expressed and secreted (RANTES) levels in distinguishing patients with ovarian cancers from those with benign ovarian cysts, we measured its concentration with reference to the disease stage, pathological grading, histological subtype, and the residual tumor mass. METHODS: Preoperative serum RANTES levels were measured in women with invasive epithelial ovarian cancer (n = 52), borderline ovarian tumor (n = 6), benign ovarian cysts (n = 28), or normal controls (n = 12) using an enzyme-linked immunosorbent assay. RESULTS: The serum RANTES concentration was significantly elevated in the ovarian cancer patients (median 53 ng/ml, interquartile range 23-104 ng/ml) compared to the benign ovarian cyst patients as controls (38 ng/ml, 5-72 ng/ml) values correlating with the stage of disease and the extent of residual tumor mass. No significant correlation between CA125 and RANTES in the serum was observed in either the controls or the ovarian cancer patients. Using a RANTES cutoff of 45 ng/ml and a CA125 cutoff of 35 units/ml, when either marker was elevated, the specificity improved 94%. CONCLUSION: Our study suggest that preoperative serum RANTES levels may be useful in differentiating benign ovarian tumors from malignancy correlating with the extent of the disorder.

Evaluation levels of cytokines in amniotic fluid of women with intrauterine infection in the early second trimester.

Amniotic fluid was obtained from 180 patients by amniocentesis at 16-22 weeks of gestation and assayed for the levels of interleukin (IL)-6, IL-8, leukocyte elastase (LE), and glucose. Ten of cases had clinical symptoms, such as uterine contraction, genital bleeding, and cervical ripening, and the other 170 were assessed for fetal chromosomal features. Four of the ten cases with uterine contraction developed abortion, while 10 of those screened had findings of fetal chromosomal anomalies, and 7 cases then underwent induced abortion artificially. In the cases of abortion, levels of IL-6, IL-8 and LE were higher than in the samples from the 160 pregnant women without clinical symptoms and a normal karyotype, while glucose in amniotic fluid was lower. Of 6 cases with clinical symptoms, but not developing abortion, 4 developed preterm labor, and in these IL-6 and IL-8 also were significantly elevated, with LE being slight high compared to normal. The results suggest that IL-6, IL-8, LE, and glucose in amniotic fluid at early second trimester can be used as markers of severe infection in the uterus, and with the first two being particularly sensitive.

Reduced nitric oxide in amniotic fluid of patients with chorioamnionitis.

OBJECTIVE: Levels of nitric oxide (NO) and cytokines were assessed in amniotic fluid obtained from patients with severe chorioamnionitis (CAM) and appropriate controls. METHODS: Amniotic fluid was obtained from 12 patients with CAM (17-24 weeks of gestation) and 89 patients undergoing diagnostic amniocentesis (16-18 weeks of gestation). The concentrations of NO, interleukin-6 (IL-6), and leukocyte elastase (LE) in amniotic fluid were then measured and compared. RESULTS: The concentrations of NO, IL-6, and LE were all higher in CAM cases than in normal pregnant women. Furthermore, an inverse correlation between NO and LE was suggested in the CAM group. CONCLUSIONS: These results indicate that in severe CAM, the action of NO might be reduced, not only due to blockage of action but also by degradation, despite increased production.

Elevated aldosterone in amniotic fluid and maternal blood has diagnostic potential in pregnancies complicated with a fetus of Bartter syndrome.

Pregnancies with fetuses affected with the Bartter syndrome, an autosomal recessive disorder of hyperreninism and hyperaldosteronism, are complicated by early onset of polyhydramnios which results in preterm deliveries. We have assessed biochemical changes in amniotic fluid and the mother's blood with a view to early diagnosis. Aldosterone levels of both amniotic fluid and the mother's blood were found to be increased at 27 weeks of gestation, while electrolyte levels did not differ significantly from those reported earlier for controls. After birth the baby suffered from polyuria with hyponatremia, hypomagnesemia and hypercalciuria which could be controlled by treatment with sodium chloride and magnesium. Elevated aldosterone thus might be a useful marker for early diagnostic purposes.

Increased granulocyte chemotactic protein-2 concentrations in peritoneal fluid of women with endometriosis.

BACKGROUND: To evaluate the release of granulocyte chemotactic protein-2 (GCP-2) into peritoneal fluid in women with endometriosis, we measured its concentration with reference to the disease stage and the phase of the menstrual cycle. METHODS: Surgery was scheduled in the proliferative or secretory phase of the menstrual cycle for 64 women with endometriosis (n = 38) or cystadenomas (n = 26). GCP-2 concentrations in the peritoneal fluid were measured using an enzyme-linked immunosorbent assay. RESULT: Our findings indicated elevated concentrations of GCP-2 in peritoneal fluid from women with endometriosis during the proliferative phase, which were positively correlated with the stage of endometriosis. CONCLUSION: Inflammation associated with endometriosis may be involved in the pathogenesis of the disease through increasing levels of peritoneal fluid GCP-2.

Chronic nitroglycerine administration reduces endothelial nitric oxide production in rabbit mesenteric resistance artery.

We investigated whether 10 days' in vivo treatment with nitroglycerine (NTG) would inhibit nitric oxide production by the endothelial cells of resistance arteries ex vivo and, if so, what the underlying mechanism might be. ACh increased the intracellular nitric oxide concentration ([NO]i; estimated using the nitric oxide-sensitive fluorescent dye diaminofluorescein-2) within the endothelial cells of rabbit mesenteric resistance arteries. This effect was significantly smaller in arteries isolated from NTG-treated rabbits than in those from control rabbits. The reduction in endothelial [NO]i in NTG-treated rabbits was prevented when olmesartan (blocker of type 1 angiotensin II receptors (AT1Rs)) was coadministered in vivo with NTG and also when the superoxide scavenger manganese (III) tetrakis-(4-benzoic acid) porphyrin (Mn-TBAP), the protein kinase C (PKC) inhibitor GF109203X or L-arginine (with or without the active form of folate (5-methyltetrahydrofolate)) was incubated with the arteries in vitro. Endothelial cell superoxide production (estimated by ethidium fluorescence) was greatly increased in arteries from NTG-treated rabbits. This was normalized by in vivo coadministration of olmesartan with NTG and also by in vitro application of Mn-TBAP or GF109203X (but not of 5-methyltetrahydrofolate+L-arginine). ACh increased the intracellular Ca2+ concentration (estimated using the Ca2+-sensitive dye Fura 2) within endothelial cells, the increase being not significantly different between NTG-treated rabbits and control rabbits. We conclude that in NTG-treated rabbits, endothelial nitric oxide production in mesenteric resistance arteries is reduced, possibly through a reduction in the bioavailability of L-arginine via an action mediated by superoxide. Activation of the AT1R-PKC pathway may be involved in increasing superoxide production.

Can preimplantation genetic diagnosis improve success rates in recurrent aborters with translocations?

Preimplantation genetic diagnosis (PGD) for people suffering recurrent miscarriages is increasingly being performed worldwide. However, there is limited information on whether PGD can improve success rates in translocation carriers. We therefore compared pregnancy outcomes between PGD and natural pregnancy cases, reviewing the clinical research database. No improvement in the success rate at the first oocyte retrieval was evident in reciprocal translocation carriers. In the natural course of events, patients with translocations can hope for a baby in the long term. However, with PGD, rates can reach 68% after IVF failure and the duration to eventual birth may be shorter than with natural pregnancies. In the particular case of Robertsonian translocations, PGD may not be necessary because natural success rates are relatively good.

Mutational analysis of the homeobox region of the human NOBOX gene in Japanese women who exhibit premature ovarian failure.

To determine whether mutations of the homeobox region of the human NOBOX gene are present in Japanese women with premature ovarian failure (POF), genomic DNA of blood samples from 30 women with POF and 20 normal controls were extracted for polymerase chain reaction (PCR) and analyzed by direct sequencing. No mutations in exons 2-6 of the NOBOX gene, including the homeobox region, could be identified in Japanese women with POF. Our data suggest that mutations of the homeobox region of the NOBOX gene are uncommon in Japanese patients with POF.

Exposure to bisphenol A is associated with recurrent miscarriage.

BACKGROUND: Little is known about the influence of high exposure to bisphenol A on recurrent miscarriage and immunoendocrine abnormalities. METHODS: Serum bisphenol A, antiphospholipid antibodies (aPLs), antinuclear antibodies (ANAs), natural killer cell (NK) activity, prolactin, progesterone, thyroid-stimulating hormone (TSH) and free T4 were examined in 45 patients with a history of three or more (3-11) consecutive first-trimester miscarriages and 32 healthy women with no history of live birth and infertility. Subsequent pregnancy outcome and embryonic karyotype of abortuses were examined prospectively. RESULTS: The mean+/-SD values for bisphenol A in patients were 2.59+/-5.23 ng/ml, significantly higher than the 0.77+/-0.38 ng/ml found for control women (P=0.024). High exposure to bisphenol A was associated with the presence of ANAs but not hypothyroidism, hyperprolactinaemia, luteal phase defects, NK cell activity or aPLs. A high level of bisphenol A in itself did not predict subsequent miscarriage. CONCLUSION: Exposure to bisphenol A is associated with recurrent miscarriage.

Role of cathepsins and cystatins in patients with recurrent miscarriage.

In the implantation, trophoblasts penetrate maternal decidua by secreting proteases. It has been reported that cathepsins are highly expressed in the mouse villi, and play an important role in normal embryonal growth and decidualization. In this study, we evaluated cathepsins and their endogenous inhibitors, cystatins, in tissue and serum of patients with recurrent miscarriage. Decidua and villi were surgically collected from 22 patients and 12 healthy women. Immunohistochemistry was performed with antibodies against cathepsins, stefin A (cystatin A), stefin B (cystatin B) and cystatin C. The concentrations of cathepsins, stefins and cystatin C were measured by Enzyme-linked immunosorbent assay. In addition, we measured the serum level of cystatin C in 85 Japanese women with recurrent miscarriage. Staining of cathepsin B, D, H, L, stefin B and cystatin C was observed in the cytoplasm of epithelial cells in decidua. Stefin A was expressed on the surface of the trophoblast. The concentration of cathepsin B and H in patients' decidua was significantly higher than in control individuals. The serum level of cystatin C was significantly lower in patients than in control individuals. Our findings suggest that the regulation of the cathepsin-cystatin system may play an important role in patients with recurrent miscarriage.

Paternal uniparental disomy of chromosome 14 and unique exchange of chromosome 7 in cases of spontaneous abortion.

To investigate the involvement of uniparental disomies (UPDs) in spontaneous abortion, the polymorphic patterns of microsatellites on each chromosome were analyzed in 164 cases of abortion. Eighty-three of the 164 cases had chromosomal abnormalities. In 79 of the remaining 81 cases with normal karyotypes, the microsatellite analysis revealed that biparental patterns were present in the informative microsatellites in all chromosomes. In one of the remaining two cases, however, the polymorphic patterns of chromosome 14 appeared to be both of paternal origin. The patterns of the distal of the long arm were homozygous, and those of the remaining region were heterozygous. That is, this fetus had paternal UPD 14, originating from meiosis I nondisjunction. In the other case, the polymorphic patterns of the distal one third of the long arm of chromosome 7 were uniparental (maternal) in origin whereas those of the remaining region of this chromosome were biparental. These findings thus suggested that this chromosome might have originated from chromatid exchange between the long arms of paternal and maternal chromosome 7 at the first mitotic division. Microsatellite analysis, however, produced no evidence of duplication or deletion of any segments. The findings also suggest the possibility that some UPDs may cause spontaneous abortion.

Parental origin and cell stage of non-disjunction of double trisomy in spontaneous abortion.

Using polymorphic analysis of microsatellites, we investigated the parental origin and mechanism of double trisomies seen in cases of spontaneous abortion. We obtained chorionic villi from spontaneous abortions, and peripheral blood from females who experienced abortion and their spouses. Chromosomal analysis of 170 cases revealed four cases with double trisomy. The karyotypes of these cases are 48,XX,+16,+22, 48,XXY,+18, 48,XX,+15,+21 and 48,XX,+2,+5. In the present study, the incidence of double trisomy was 2.4% of spontaneous abortions. Polymorphic analysis of microsatellites indicated that extra chromosomes were all of maternal origin in the four cases of double trisomy. The predominance of maternal origin in cases of double trisomy is similar to cases of single trisomy. The result also indicated that both extra chromosomes in two cases occurred by non-disjunction at the first meiotic division, and extra chromosomes in the other two cases occurred by non-disjunction at the first mitotic division. The mean maternal age in cases of double trisomy was significantly higher than that in cases of single trisomy. These findings suggest the possibility that abnormal separation of two or more chromosomes may occur simultaneously in oogonia, and that this phenomenon may increase in relation to the increase in age of women.

Reduced flow-mediated vasodilation is not due to a decrease in production of nitric oxide in preeclampsia.

OBJECTIVE: Our aim was to determine the reduced function of endothelial nitric oxide in preeclampsia by use of noninvasive techniques in vivo. STUDY DESIGN: With the use of a high-resolution ultrasound transducer, diameters of brachial artery were measured after reactive hyperemia in 20 nonpregnant women, 20 normotensive pregnant women, and 15 women with preeclampsia. The concentrations of cyclic guanosine monophosphate were measured in samples of platelets from all groups. RESULTS: Flow-mediated vasodilation at 1 minute after deflation was higher in the normotensive pregnant women (115.1% +/- 6.5%) than in the nonpregnant women (108.7% +/- 3.9%); flow-mediated vasodilation was lower in women with preeclampsia (106.8% +/- 2.7%) than in the normotensive pregnant women. The concentration of platelet cyclic guanosine monophosphate was higher in the normotensive pregnant women than in the nonpregnant women (2.21 +/- 1.10 pmol/mL/10(8) cells vs 0.746 +/- 0.381 pmol/mL/10(8) cells). There was no difference between the normotensive pregnant and the preeclamptic group (2.81 +/- 1.82 pmol/mL/10(8) cells). Furthermore, the increase in cyclic guanosine monophosphate by sodium nitroprusside in platelet samples that were obtained from the normotensive pregnant women was larger than the samples from the nonpregnant women (6.20 +/- 4.2 pmol/mL/10(8) cells vs 1.62 +/- 0.81 pmol/mL/10(8) cells). The increase in cyclic guanosine monophosphate from the women with preeclampsia did not differ from that in the normotensive pregnant women (5.84 +/- 3.73 pmol/ml/10(8) cells). CONCLUSION: These results indicate that reduced endothelial nitric oxide activity might be due to a reduction of nitric oxide-cyclic guanosine monophosphate activity rather than its production in preeclampsia.

Role of apoptosis controlled by cytochrome c released from mitochondria for luteal function in human granulosa cells.

PROBLEM: The aim of this study was to investigate the relationship between apoptosis by the mitochondrial pathway and luteal function in human granulosa cells. METHOD OF STUDY: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization and embryo transfer. After the addition of RU486, cells were stained with a mitochondria-specific fluorescent dye, MitoTracker Red CM x Ros. Using flow cytometry and National Institute of Health image, the mitochondrial fluorescent area was measured. After staining with Hoechst 33258 dye, the number of apoptotic bodies per 1000 cells were counted at random on photomicrographs. Homogenates were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against cytochrome c or caspase-3. RESULTS: The incidence of apoptotic bodies increased and the mitochondrial membrane potential decreased time dependently. The opposite effect was observed dose dependently with RU486 treatment. Western blot analysis showed increased cytochrome c expression, after treatment with 1-2 microg/mL of RU486 which then decreased with 5-10 microg/mL of RU486. Caspase-3 expression increased dose dependently with RU486. CONCLUSIONS: These results suggest that the activation of caspase-3 caused by cytochrome c released from mitochondria plays an important role in apoptosis-related luteal function in human granulosa cells.