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Objectives: The purpose is to investigate how umbilical arterial blood flow changes by an intraamniotic distilled water infusion and to determine whether the changes in umbilical circulation have any relationship with fetal cardiovascular status and osmolality in amniotic fluid and fetal plasma. Methods: Eleven chronically catheterizedpregnant sheep were used in this study. After a 1-hour control period, 1.5 L of warmed sterile distilled water was injected over 10 minutes into the amniotic cavity. Fetal heart rate and carotid arterial pressure, blood flow of the umbilical and fetal carotid arteries were continuously measured. Fetal arterial blood sampled twice during the control period and then at 30, 60, 90, 120, 180, 240, 300, and 360 minutes after the start of the infusion, was analyzed for blood gases, pH, plasma electrolytes, and osmolality. Results: Data obtained from seven sheep with normoxemic fetuses were studied statistically. Umbilical arterial blood flow decreased significantly from 229.5 ± 3.83 mL/min in the control to 167 A ± 11.1 mL/min at 30 minutes after water infusion (P <.001). Umbilical arterial vascular resistance increased rapidly and reached its peak at approximately 60 minutes after infusion and then showed a gradual recovery to the control level (P <.001). Amniotic fluid osmolality had a high degree of correlation with umbilical arterial blood flow and vascular resistance, while fetal arterial blood pressure and heart rate had only little correlation with umbilical blood flow. Conclusion: A distilled water infusion into the amniotic cavity in near-term pregnant sheep led to an acute drop in umbilical arterial blood flow. The changes in umbilical flow were closely correlated with those in amniotic fluid osmolality. Hemolysis in the capillary networks in the fetal membranes seems to be one of the main causes of umbilical vasoconstriction. It is speculated that the fetal membranes, including capillary networks, intramembranous pathway, and amnion epithelial cells, sense the changes in amniotic fluid osmolality, which leads to a fetal adaptation to the hypotonic environment.

Objectives:The purpose is to investigate how umbilical arterial blood flow changes by an intraamniotic distilled water infusion and to determine whether the changes in umbilical circulation have any relationship with fetal cardiovascular status and osmolality in amniotic fluid and fetal plasma.Methods:Eleven chronically catheterized pregnant sheep were used in this study. After a 1-hour control period, 1.5 L of warmed sterile distilled water was injected over 10 minutes into the amniotic cavity. Fetal heart ratte and carotid arterial pressure, blood flow of the umbilical and fetal carotid arteries were continuously measured. Fetal arterial blood sampled twice during the control period and then at 30, 60, 90, 120, 180, 240, 300, and 360 minutes after the start of the infusion, was analyzed for blood gases, pH, plasma electrolytes, and osmolality.Results:Data obtained from seven sheep with normoxemic fetuses were studied statistically. Umbilical arterial blood flow decreased significantly from 229.5 ± 3.83 mL/min in the control to 167.4 ± 11.1 mL/min at 30 minutes after water infusion (P <.001). Umbilical arterial vascular resistance increased rapidly and reached its peak at approximately 60 minutes after infusion and then showed a gradual recovery to the control level (P < .001). Amniotic fluid osmolality had a high degree of correlation with umbilical arterial blood flow and vascular resistance, while fetal arterial blood pressure and heart rate had only little correlation with umbilical blood flow.Conclusion:A distilled water infusion into the amniotic cavity in near-term pregnant sheep led to an acute drop in umbilical arterial blood flow. The changes in umbilical flow were closely correlated with those in amniotic fluid osmolality. Hemolysis in the capillary networks in the fetal membranes seems to be one of the main causes of umbilical vascoconstriction. It is speculated that the fetal membranes, including capillary networks, intramembranous pathway, and amnion epithelial cells, sense the changes in amniotic fluid osmolality, which leads to a fetal adaptation to the hypotonic environment.

PROBLEM: The aim of this study was to investigate the gene frequencies and shared alleles of human leukocyte antigen (HLA)‐E gene in Japanese couples with or without recurrent abortion.
 METHOD OF STUDY: Polymerase chain reaction (PCR)‐single strand conformation polymorphism (SSCP) analysis was carried out to detect polymorphism in exon 3 of the HLA‐E gene in 30 Japanese couples with recurrent abortion and 38 normal Japanese couples with proven fertility.
 RESULTS: No point mutation was detected in exon 3 of HLA‐E in both recurrent aborters and normal controls. HLA‐EG and HLA‐ER alleles were detected with frequencies of 66.7% and 33.3% in couples with recurrent abortion and 69.2% and 30.8% in normal couples, respectively. The gene frequency of HLA‐EG was higher than that of HLA‐ER, which is contrary to that found in Caucasian, African–American and Hispanic people but similar to Chinese people. The frequency of each allele was not significantly different between recurrent aborters and normal controls. The number of shared alleles between each couple with recurrent abortion is not significantly different from that with normal controls.
 CONCLUSION: Allele frequencies of HLA‐E were suggested to be different in Asian people from those in other ethnic people. In light of no specific distribution pattern in recurrent aborters, HLA‐E polymorphism does not seem to play a role in the pathogenesis of recurrent abortion.

PROBLEM: To better understand the role of human leukocyte antigen (HLA)‐G in regulating the T helper (Th)1/Th2 cytokine balance, one of key conditions in determining the fate of pregnancy, we asked whether the presence of HLA‐G protein altered the release of cytokines from both decidual mononuclear cells and peripheral blood mononuclear cells (PBMCs).
 METHOD OF STUDY: The amounts of cytokines released from decidual mononuclear cells and PBMCs were compared in the presence or absence of HLA‐G‐expressing cells.
 RESULTS: When cocultured with HLA‐G‐expressing cells, the amounts of tumor necrosis factor‐α and interferon‐γ released from decidual mononuclear cells and PBMCs were decreased, while the amounts of interleukin (IL)‐4 from PBMCs was increased, with IL‐4 release from decidual mononuclear cells being unchanged.
 CONCLUSIONS: Upon contact with HLA‐G, decidual mononuclear cells, and PBMCs as well, modulate their ability to release cytokines in a way that may shift the Th1/Th2 balance towards relative Th2 dominance, suggesting a role for HLA‐G in maintaining pregnancy.

PROBLEM AND METHOD OF STUDY: To get insight into the basis for the empirical usage of herbal medicines in the treatment of recurrent abortion, we examined whether Tokishakuyaku‐san (Toki) and Sairei‐to (Sai) modulate T helper‐1 (Th1) and T helper‐2 (Th2) cytokine release from peripheral blood mononuclear cells (PBMCs). The effects of these medicines were investigated as related to human leukocyte antigen (HLA)‐G, a non‐classical HLA class I antigen expressed on trophoblasts and a putative crucial player involved in fetomaternal immune interplay.
 RESULTS: Toki and Sai increased the release of Th1 group cytokines, tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ while preserving the inhibitory effect of HLA‐G on the release of these cytokines. As for Th2 group cytokine release, Toki was without effect in modulating interleukin (IL)‐4 release, regardless of the presence of HLA‐G, whereas Sai nullified the effect of the presence of HLA‐G to stimulate the release of IL‐4 without affecting its release in the absence of HLA‐G.
 CONCLUSION: Toki and Sai may have therapeutic potential, particularly in autoimmunity‐related recurrent abortion where Th2 response is pathologically enhanced, but not in recurrent abortion involving alloimmune fetomaternal derangement, a condition of, rather, an enhanced Th1 response.

AbstractClinical features of Prader-Willi syndrome in neonates are marked hypotonia with the absence of crying and feeding difficulty so that prenatal diagnosis of Prader-Willi syndrome is strongly hoped in order to provide appropriate medical and psychological care for neonates and their families. However, the clinical picture of Prader-Willi syndrome in utero has not been well described. We report a pregnancy associated with Prader-Willi syndrome manifesting polyhydramnios, large biparietal diameter of the fetus and characteristic fetal heart rate pattern: prolonged inactive periods and diurnal variation of the incidence of heart rate accelerations. These findings may offer a clue to the prenatal diagnosis of Prader-Willi syndrome, although molecular cytogenetics is mandatory for the definite diagnosis.Copyright © 2000 S. Karger AG, Basel

PROBLEM: We assessed the expression of natural killer (NK) receptors in recurrent aborters before and after immunotherapy using their husbands’ peripheral blood mononuclear cells (PBMCs). METHOD OF STUDY: Using stored PBMCs from recurrent aborters before and after the immunotherapy, the expression of NK receptors, CD158a, CD158b, CD159 and CD94, were analyzed using monoclonal antibodies for respective receptors. The diversity of killer activatory receptors (KARs) and killer inhibitory receptors (KIRs) was also examined using reverse transcriptase‐polymerase chain reaction (RT‐PCR)‐single strand conformation polymorphism (SSCP) method. RESULTS: In recurrent aborters, no apparent changes in NK receptor expression and the balance between KARs and KIRs were found before and after the immunotherapy. CONCLUSION: The allo‐human leukocyte antigen (HLA)–stimulation caused by the immunotherapy for recurrent aborters did not affect the expression of NK receptors and the ratio of KARs to KIRs regardless of the outcome of subsequent pregnancies, suggesting that recurrent aborters may benefit from the immunotherapy through mechanisms unrelated to alteration in NK receptor status.

Background and Objectives: Red cell type RhD^Va lacks epD1 and 5 and is encoded by hybrid RHD-CE(5)-D alleles. We analyzed RhD^Va and RhD^Va-like samples in Japanese blood donors. Materials and Methods: Ten RhD^Va samples lacked epD1 and 5 and 3 RhD^Va-like variants also lacked, epD2 and a part of 6/7. We identified the full-length nucleotide sequences of the complementary DNA (cDNA) synthesized from 4 samples: 3 of type D^Va and the 4th a D^Va-like variant. Results: Although their sequences differed from each other, all the substitutions were exclusively in exon 5. Three D^Va samples had hybrid RHD-CE(5)-D alleles, but the D^Va-like variant had a unique nucleotide substitution with a single amino acid change, E233K. Exon 5 of the genomic DNA from all 13 samples was analyzed by sequencing. No other sequences were identified. Conclusion: All RhD^Va and RhD^Va-like variants had the substitution for E233. E233 seems to be a determinant of epD1 and 5. A new category of RhD variant, DYO, was identified.

PROBLEM: We have shown the attenuated human leukocyte antigen (HLA)‐G expression on trophoblasts and an aberrant expression of interleukin (IL)‐2, a cytotoxic cytokine, in decidual tissue in preeclampsia, where deteriorated trophoblastic invasion into decidual layers may constitute a crucial pathogenesis. We hypothesized that the absence of HLA‐G might make trophoblasts susceptible to compromise by IL‐2.

PROBLEM: The purpose of this study was to clarify whether there is a difference between the allele frequency of human leukocyte antigen (HLA)‐G in healthy Japanese people and that of Japanese couples with habitual abortion.

Thirty-five women with normal menstrual cycles underwent transvaginal and high-frequency (15- or 20-MHz) intrauterine sonographic examinations for the assessment of the endometrium. Five sonographic patterns of endometrium were recognized by both sonographic techniques, but the pattern visualized differed between techniques in 9 women (26%). The higher resolution of intrauterine sonography provided more detail of the endometrium. Our preliminary experience indicates that high-frequency intrauterine sonography enables more detailed visualization of changes in the endometrium during the menstrual cycle than does transvaginal sonography. © 1999 John Wiley & Sons, Inc. J Clin Ultrasound 27:195–198, 1999.

PROBLEM: In search for pathogenesis of recurrent abortion, we examined whether lymphocytes/macrophages from women with recurrent abortion exhibited an aberrant ability to release cytokines upon the direct contact of human leukocyte antigen (HLA)‐G.

Hypothesis:We hypothesized that there might be differences in the outcomes of post-term pregnancy according to fetal size. Methods:A retrospective analysis of 143 women with uncomplicated pregnancy that persisted beyond 42 weeks was conducted. Ultrasound dating was performed between 8 and 14 weeks in all cases. The obstetrical outcomes were compared between the cases in which neonatal birth weight was <3,600 g (non-large group) and ≥ 3,600 g (large group). Results:In primiparous women, there was no significant difference between the non-large and large groups in terms of the rate of cesarean section, but the rate of cesarean section for fetal distress in the non-large group was significantly higher than that found in the large group. In addition, the non-large group showed a significantly higher incidence of low pH (<7.20) compared with the large group (14/58 vs. 0/22, p < 0.01). In contrast, the rate of prolonged labor (>24 h) and induction failure in the non-large group were significantly lower than that in the large group. In multiparous women, there was no significant difference in obstetrical outcomes between the non-large and large groups. Conclusions:Primiparous women who were delivered of fetuses weighing not less than 3,600 g at post-term periods were at lower risk of fetal distress on the one hand and at higher risk of induction failure associated with infavorable cervix on the other. This study suggests that post-term pregnancy should be dealt with differently based on fetal weight.

PROBLEM: Human leukocyte antigen‐G (HLA‐G) is thought to be at play in maternal‐fetal immune interplay during pregnancy. Whether the expression of HLA‐G protein on the target cells altered the release of cytokines from effector mononuclear cells was questioned. METHOD OF STUDY: The amounts of cytokines released from peripheral blood mononuclear cells (PBMC) cocultured with or without HLA‐G‐expressing target cells were compared. RESULTS: When cocultured with HLA‐G‐expressing target cell lines, the amounts of interleukin‐3 (IL‐3) and interleukin‐lβ (IL‐1β) released from PBMC were increased, whereas the amounts of tumor necrosis factor‐α (TNF‐α) were decreased.

PROBLEM: The purpose of this study is to clarify whether IL‐2 expressed in the decidua in preeclampsia affects the angiogenesis of the placenta.

PROBLEM: The purpose of this study is to clarify whether the disruption of immune regulation occurs in early pregnancy before the clinical manifestations of preeclampsia.

PROBLEM: The appropriate modality of immunotherapy with the husband's mononuclear cells in women with a history of recurrent abortion who aborted despite the immunotherapy performed before pregnancy was explored.

Abstract: We compared various methods to control blood flow for extrauterine incubation of goat fetuses without lung respiration using arterio‐venous extracorporeal membrane oxygenation via umbilical vessels to develop a suitable system for a long‐term life support system for premature neonates. In twenty‐seven consecutive preparations of goat fetuses, 4 designs (System 1, manual flow control without a reservoir; System 2, manual flow control with an arterial open‐top reservoir; System 3, semiautomatic flow control with an arterial open top reservoir; and System 4, semiautomatic flow control with a tube occluder and an arterial open top reservoir) were tested in terms of the duration of incubation periods during which fetuses were kept under stable conditions. The incubation periods be‐came prolonged with the advancement of the system (System 1, 5.4 h; System 2, 11.6 h; System 3, 64.8 h; and System 4, 159.8 h medians of the incubation periods). The incubation periods with System 4 were significantly longer compared to those with System 1 and System 2. These results suggest that, for extrauterine incubation of goat fetuses using arterio‐venous extracorporeal membrane oxygenation via umbilical vessels, the method to control blood flow is of utmost importance for long‐term maintenance, and thus a control system with the fixed vascular resistance of the extracorporeal circuit is applicable to long‐term maintenance of premature goat fetuses.

To investigate developmental aspects of metabolic and endocrine responses to cold exposure in fetuses, we conducted experiments on six goat fetuses, three aged 95-116 d of gestation (dGA; group I), and three aged 122-134 dGA(group II), using an extrauterine fetal incubation system that provided arteriovenous extracorporeal membrane oxygenation (A-V ECMO). The fetuses were cannulated via the umbilical vessels, and their blood gas exchange was totally supported by A-V ECMO, while they were maintained in an isothermal incubator containing artificial amniotic fluid. After confirming that fetuses were in metabolically stable condition in the extrauterine incubation system, fetal core temperature was lowered by 2°C over 2 h by decreasing the temperature of incubating fluid from 39.5°C. During and after cold exposure, fetal heart rate and arterial blood pressure remained unchanged. We observed significant increases in oxygen consumption and plasma concentrations of norepinephrine, epinephrine, adrenocorticotropic hormone, and cortisol in group II but not in group I fetuses. In addition, based on regression analysis, maximal changes of these parameters during cold exposure were linearly correlated with gestational age significantly, and the regression lines were found to intersect the x (gestational age) axis at around 98-106 dGA. These results suggest that metabolic and endocrine responses to cold exposure develop with gestational age in the goat fetus, the responses being manifested around 100 dGA.

PROBLEM: The objective of this study was to establish an enzyme‐linked immunosorbent assay (ELISA) system, in an attempt to quantify the amount of human leukocyte antigen (HLA)‐G protein in amniotic fluid.

A 31-year-old woman, gravida 2 para 1, visited our clinic for routine follow-up at 20 weeks' gestation. Although she had no abdominal pain or pressure, digital vaginal examination revealed dilatation of the internal cervical os of 1.5 cm, and transvaginal sonography demonstrated dynamic changes in the shape of the cervical canal. The patient underwent Shirodkar's operation. Routine postoperative assessment of the cervix with transvaginal sonography showed dynamic dilatation of the upper cervix (above the cerclage), which was accompanied by a sensation of pelvic pressure but no apparent uterine contractions. A healthy male infant weighing 2,980 g was delivered at 38 weeks' gestation. © 1998 John Wiley & Sons, Inc. J Clin Ultrasound 26:371–373, 1998.

PROBLEM: The objective of this study was to evaluate the activated status of helper T cells in women with recurrent abortion.

[Background and purposes] Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. The use of Notch ligands or soluble IL-6 receptor combined with IL-6 has been a major technique that revealed several fold expansion of human cord blood SCID repopulating cells (SRCs). These studies, however, have been conducted in an independent manner, which hampered direct comparison and evaluation of the effect of combination of these methods. Our purpose of this study is to clarify these issues in a chemically defined serum-free medium that allows us to develop clinical usage of the culture condition. We also compared the efficiencies of SRC isolation with magnetic beads targeting CD34 and CD133. [Methods] Human cord blood CD133-sorted cells were cultured on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, flt-3 ligand, IL-3 and IL-6/soluble IL-6 receptor chimeric protein (FP6) for three weeks, and cultured cells were transplanted into NOD/SCID mice after limiting dilution to calculate the number of SRCs. To confirm whether full multipotency and self-renewal capacity of SRCs were maintained during the culture, cells were transplanted serially into NOD/SCID/γcnull (NOG) mice, and hematopoietic reconstitution was examined. To compare the efficiencies of CD34- and CD133-sorting, we divided each sample into two aliquots and separated CD34+ and CD133+ cells, and calculated the SRC numbers recovered by both separation methods. [Results and discussion] The frequencies of SRCs in the culture-initiating CD133-sorted cells and cultured progeny were calculated as one out of 1,020 and one out of 175 (adjusted to culture-initiating cells), respectively, indicating 6-fold expansion of SRCs that was statistically significant. Delta1 significantly enhanced the expansion rate of SRCs, and addition of IL-3 to this condition further promoted the expansion. In the serial transplantation assays, we found human myeloid and lymphoid reconstitution both in the primary and secondary NOG recipients, verifying the SRC capacity in the cultured cells. Notably, the CD133-sorting was approximately 4.5 times more efficient in collecting SRCs than the CD34-sorting from the same number of mononuclear cells (MNCs) (308 and 254 SRCs by CD133-sorting vs. 67 and 59 SRCs by CD34-sorting from 108 MNCs). Our study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.

[Background and purposes] Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. The use of Notch ligands or soluble IL-6 receptor combined with IL-6 has been a major technique that revealed several fold expansion of human cord blood SCID repopulating cells (SRCs). These studies, however, have been conducted in an independent manner, which hampered direct comparison and evaluation of the effect of combination of these methods. Our purpose of this study is to clarify these issues in a chemically defined serum-free medium that allows us to develop clinical usage of the culture condition. We also compared the efficiencies of SRC isolation with magnetic beads targeting CD34 and CD133.