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.

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.

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.

Peritoneal fluid concentrations of epithelial neutrophil-activating peptide-78 correlate with the severity of endometriosis.

OBJECTIVE: To assess the release of epithelial neutrophil-activating peptide-78 (ENA-78) into peritoneal fluid in women with and without endometriosis. DESIGN: Retrospective study. SETTING: Nagoya City University Hospital. PATIENT(S): Surgery was scheduled in the proliferative or secretory phase of the menstrual cycle for 59 women with (n = 35) and without (n = 24) endometriosis. INTERVENTION(S): Peritoneal fluid samples were obtained at laparotomy or laparoscopy. MAIN OUTCOME MEASURE(S): The ENA-78 concentrations in the peritoneal fluid were measured using enzyme-linked immunosorbent assay (ELISA). RESULT(S): The concentrations of ENA-78 in the peritoneal fluid were markedly elevated in the endometriosis patients as compared with the controls, especially in women with severe stage disease. CONCLUSION(S): We conclude that ENA-78 is an important factor that may contribute to the pathogenesis of endometriosis, possibly promoting neovascularization.

Elevated angiogenin levels in the peritoneal fluid of women with endometriosis correlate with the extent of the disorder.

OBJECTIVE: To assess the release of angiogenin into peritoneal fluid in women with and without endometriosis by measuring its concentration with reference to disease stage, presence of red lesions, and phase of the menstrual cycle. DESIGN: Retrospective study. SETTING: Nagoya City University Hospital. PATIENT(S): Sixty-four women with endometriosis (n = 38) and cystadenomas (n = 26) for whom surgery was scheduled in the proliferative or secretory phase of the menstrual cycle. INTERVENTION(S): Peritoneal fluid samples were obtained at laparotomy or laparoscopy. MAIN OUTCOME MEASURE(S): Angiogenin concentrations in the peritoneal fluid, as measured by ELISA. RESULT(S): Angiogenin concentration in the peritoneal fluid was markedly elevated in the endometriosis patients (median 515 ng/mL, interquartile range 151-1763 ng/mL) compared with the cystadenoma (control) patients (195 ng/mL, 98-324 ng/mL), with values correlating with the extent of the disease. No significant differences between the proliferative phase and the secretory phase were observed in either the controls or the endometriosis patients. CONCLUSION(S): The inflammation associated with endometriosis, through increasing levels of peritoneal fluid angiogenin, might promote angiogenesis for progression of the disease and correlate with the extent of the disorder.

Phosphatidylserine-dependent antiprothrombin antibodies are not useful markers for high-risk women with recurrent miscarriages.

To determine the possible association between antiprothrombin antibodies and conventional antiphospholipid antibody (aPL) 1 (1.0%), 2 (2.0%) and 17 (17.0%) were found to be positive for phosphatidylserine-dependent antiprothrombin antibody (aPS/PT) IgG, beta2glycoprotein I-dependent anticardiolipin antibody, and lupus anticoagulant by activated partial thromboplastin time (LA), respectively, in 100 recurrent aborters. Because patients with aPS/PT were included in the 17 with LA, we should not add aPS/PT measurement to routine testing in addition to conventional aPL for patients with recurrent miscarriage.

Poor prognosis of recurrent aborters with either maternal or paternal reciprocal translocations.

OBJECTIVE: To determine whether the miscarriage rate in recurrent miscarriage patients with an abnormal karyotype, especially reciprocal translocations, in either partner is worse than without an abnormal karyotype. DESIGN: Retrospectively analyzed prospectively obtained database. SETTING: Nagoya City University Hospital. PATIENT(S): One thousand and two hundred eighty-four couples with a history of 2 or more (2 to 12) consecutive first-trimester miscarriages. INTERVENTION(S): Patients with antiphospholipid antibodies were treated with low-dose aspirin and combined therapy. MAIN OUTCOME MEASURE(S): Subsequent miscarriages were compared for cases with and without an abnormal karyotype in either partner. A karyotype analysis was also conducted for each aborted conceptus and offspring of 95 pregnancies of 47 patients with reciprocal translocations. RESULT(S): Of the total of 1,284 couples, 58 (4.5%) had translocations, 11 being Robertsonian translocations. Eleven of the 18 cases (61.1%) where the husband had a reciprocal translocation suffered further miscarriage; this also was the case for 21 of the 29 cases (72.4%) where the wives had a reciprocal translocation. Those with reciprocal translocations in either partner miscarried significantly more frequently than those without an abnormal karyotype. Only one infant with an unbalanced translocation was found in 34 cases of successful pregnancy following habitual abortion. CONCLUSION(S): The pregnancy prognosis with either maternal or paternal reciprocal translocations is poorer than without them. The presence of a reciprocal translocation is thus a risk factor in couples who have recurrent miscarriages.

Coagulation factor XII activity, but not an associated common genetic polymorphism (46C/T),is linked to recurrent miscarriage.

OBJECTIVE: To investigate whether a factor XII genetic polymorphism is associated with first-trimester embryonal loss. DESIGN: Prospective case-control study. SETTING; Nagoya City University Hospital. PATIENT(S): Eighty-three patients with a history of two or more unexplained first-trimester recurrent miscarriages and 67 controls with no obstetric complications or history of miscarriage. MAIN OUTCOME MEASURE(S): Plasma factor XII activity, a genetic polymorphism (46 C-->T) of factor XII, lupus anticoagulant, and beta(2)glycoprotein I dependent anticardiolipin antibodies. RESULT(S): Ten of the 83 patients and 1 of the 67 controls had decreased factor XII activity; the difference in frequency was statistically significant. Wild-type (CC), heterozygote (CT), and homozygote (TT) allele patterns were observed in 8, 36, and 39 patients, respectively, compared with 11, 20, and 36 of the patients and controls, respectively. The mean (+/- SD) corresponding factor XII activity was 154.8 +/- 44.8%, 112.7 +/- 30.2%, and 66.2 +/- 29.2% in patients and 164.6 +/- 26.7%, 114.3 +/- 28.1%, and 70.4 +/- 18.1% in controls. The two groups did not differ in the frequency of the T allele or categories of factor XII activity. CONCLUSION(S): Factor XII activity overall, but not the 46C/T common genetic polymorphism, is associated with recurrent miscarriage.

Conservative treatment by angiographic artery embolization of an 11-week cervical pregnancy after a period of heavy bleeding.

OBJECTIVE: To describe a rare case of conservative treatment of an 11-week cervical pregnancy after a period of heavy bleeding. DESIGN: Case report. SETTING: A university hospital. PATIENT(S): A 33-year-old woman was admitted to our hospital for treatment of a cervical pregnancy. Two-and-a-half years thereafter, she gave birth to a healthy baby by vaginal delivery at 38 weeks of gestation. INTERVENTION(S): Systemic methotrexate treatment, ligation of descending branches of uterine arteries, cervical cerclage, and unilateral internal iliac artery embolization. MAIN OUTCOME MEASURE(S): Transvaginal ultrasound, magnetic resonance imaging, and arteriography findings. RESULT(S): The patient was successfully treated with unilateral internal iliac artery embolization on the same side as the pregnancy in the 11th gestational week. CONCLUSION(S): After failed methotrexate and vessel ligation in cervical pregnancy, unilateral internal iliac artery embolization is an effective and conservative treatment that allows preservation of reproduction potential.

DNA-based prenatal exclusion of bullous congenital ichthyosiform erythroderma at the early stage, 10 to 11 weeks' of pregnancy, in two consequent siblings.

The proband was a Japanese woman with bullous congenital ichthyosiform erythroderma harboring a keratin 10 gene mutation M150T. DNA-based prenatal exclusion of bullous congenital ichthyosiform erythroderma was successfully performed in her two consequent pregnancies using chorionic villus samples at 10 to 11 weeks' gestation, several weeks earlier than the previously reported cases.

Enrichment of fetal cells from maternal blood by magnetic activated cell sorting (MACS) with fetal cell specific antibodies: one-step versus two-step MACS.

We report here the results of fetal cell enrichment from maternal blood in 58 pregnant women by the use of magnetic activated cell sorting (MACS) with erythroblast-specific and/or maternal cell specific antibodies. Two approaches were compared; one-step MACS to enrich CD71+ (a membrane-bound marker) or GPA+ (another marker, glycophorin A) fetal cells versus two-step MACS to deplete CD14+ maternal cells and subsequently to enrich fetal (CD71+ or GPA+) cells. The existence of fetal cells was ensured by both FISH with Y-specific probes and karyotyping of respective amniotic and/or chorionic vullus cells, the results being applied for comparison of detection rate for XY fetuses between the two MACS procedures. In 24 (38.8%) of the 58 blood samples examined, Y-positive cells were observed by FISH, whereas there were 38 true XY fetuses later confirmed by karyotyping, including two cases of 47,XY,+21. On the other hand, in Y-negative cells by FISH, there were two cases of 47,XX,+18. The average number of cells sorted did not differ among one-step MACS procedures with anti-CD14, anti-CD71 and anti-GPA antibodies. With the latter, 12 (75%) of 16 Y-positive fetuses were detected, while only one (20%) of 5 Y-positive fetuses was detected by two-step MACS with anti-CD14/anti-GPA antibodies. The detection rate significantly varied (p = 0.0024) between the two procedures, although the numbers of cases examined were small. There was no statistical difference (p > 0.05) between one-step and two-step MACS with other combinations of antibodies. These findings indicate that one-step MACS using the anti-GPA antibody is more effective than two step MACS for enrichment of fetal cells from maternal blood.

An examination of different fetal specific antibodies and magnetic activated cell sorting for the enrichment of fetal erythroblasts from maternal blood.

The aim of the present study was to compare the rates of fetal cells obtained after separation from maternal blood by magnetic activated cell sorting (MACS) using different fetal specific antibodies, and to evaluate the potential role of this method in the prenatal diagnosis of fetal trisomies. Peripheral blood samples were obtained from 42 women carrying chromosomally normal fetuses and from 4 women with aneuploid fetuses (2 cases of 47,XX,+18 and 2 of 47,XY,+21) at 9-20 weeks of gestation. After fetal cells were enriched by MACS with three different monoclonal antibodies (GPA, CD71, CD14), fluorescence in situ hybridization (FISH) with chromosome X, and Y-specific probes was performed to detect the rates of fetal cells in the samples sorted. FISH with chromosome 13-, 18-, and 21-specific probes was carried out to compare proportions of cells with three-signal nuclei in chromosomally normal and abnormal groups. In male infants, X- and Y-positive cells were detected in 80%, 73.3%, and 66.6% of samples after the separation by antibodies CD14, GPA, and CD71, respectively. The percentage of nuclei with three signals was increased in pregnancies with trisomy, ranging between 2% and 5.18%. Pregnancies with normal fetuses showed 0 to 3.7% of nuclei with three signals. The data demonstrate that fetal cell detection varies depending on the antibodies used for cell sorting. This study provides further evidence on the feasibility of screening for fetal chromosomal abnormalities by enriching maternal blood for fetal cells and using FISH.

Low dose gonadotropin-releasing hormone agonist treatments with early discontinuation for controlled ovarian hyperstimulation in an in vitro fertilization program.

Aim: The purpose of the present study was to investigate the applicability of a protocol for controlled ovarian hyperstimulation (COH) featuring early discontinuation of low dose gonadotropin-releasing hormone agonist (GnRHa) for ovulation induction for in vitro fertilization (IVF). Methods: Four hundred and eighty-seven women undergoing 555 IVF cycles were recruited into the study. Controlled ovarian hyperstimulation was achieved by using either a short protocol of low dose GnRHa (for 5 days only; groups 1 and 2) or a modified long protocol with early discontinuation of GnRHa (groups 3 and 4). Groups 1 and 3 received urinary follicle-stimulating hormone (FSH) and groups 2 and 4 received recombinant FSH. Oocyte retrieval was performed 34 to 36 h after human chorionic gonadotropin (hCG) injection, followed by embryo transfer 3 days later. Results: Luteinizing hormone (LH) levels on the hCG injection day were lower with the modified long protocol (groups 3 and 4) than with the short 5-day treatment (groups 1 and 2). There were higher LH levels in group 1 than in groups 2, 3 and 4, resulting in a worse fertilization rate and clinical pregnancy rate. There were no statistically significant differences between groups 2, 3 and 4 in the rates of fertilization, clinical pregnancy and delivery. A higher estradiol (E2) level in group 3 than in groups 1, 2 and 4 resulted in a worse implantation rate. Conclusion: Early cessation of GnRHa may not induce a premature LH surge in controlled ovarian hyperstimulation, while a low dose also offers a useful alternative to a long protocol of IVF. Ovarian stimulation with recombinant follicle-stimulating hormone (rFSH) is considered to be favorable in this low dose GnRHa treatment. (Reprod Med Biol 2003; 2: 25-30).

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.

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.