Review: Exploration of placentation from human beings to ocean-living species.

This review covers four topics. 1) Placental pathology in Himalayan mountain people. To determine morphological changes of the placenta at high altitude, pathological examination was made of 1000 Himalayan placentas obtained in Nepal and Tibet and the results compared with Japanese placentas delivered at sea level. Characteristic findings in the placental villi of the Himalayan group included high incidences of villous chorangiosis and chorangioma. These processes were clarified by ultrastructural observation. 2) Placentation in Sirenians. The giant Takikawa sea cow, which lived 5 million years ago, was discovered on Hokkaido, Japan. It was an ancestor of the dugong as well as the manatees. Sirenia, the sea cow group, shares a common ancestor with Proboscidea, the elephants, even though they now inhabit quite different environments. A comparison was made of their zonary endothelial type of placentation. 3) Placentation in sharks and rays. The remarkable placentation of hammerhead sharks and manta rays is described. 4) Placentation in the Antarctic minke whale. Placental tissue samples of this whale were obtained from the Japan Institute of Cetacean Research. In an ultrastructural study of the utero-placental junction, microfilamental processes of the allantochorionic zone and crypt formation were visualized.

IFPA Meeting 2012 Workshop Report I: comparative placentation and animal models, advanced techniques in placental histopathology, human pluripotent stem cells as a model for trophoblast differentiation.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of models and technical issues involved in placenta research: 1) comparative placentation and animal models; 2) advanced techniques in placental histopathology; 3) human pluripotent stem cells as a model for trophoblast differentiation.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Identification of Gaucher cells in the chorionic villi associated with recurrent hydrops fetalis.

Recurrent non-immune hydrops fetalis has rarely been reported. In order to detect the risk of recurrence in a subsequent pregnancy, one should carefully consider the possibility of an inborn error of metabolism. In such cases, placental examination may be useful in detecting such metabolic storage disorders in the fetus, which usually present as vacuolization of placental cells. We describe a rare case of recurrent hydrops that was detected by placental examination. Through light microscopy, electron microscope (EM) studies and beta-glucocerebrosidase activity the disease was identified as Gaucher's disease.

A clinicopathological aspect of chorionic villous hemorrhage leading to formation of intervillous thrombosis.

Recently it was reported that chorionic villous hemorrhage (CVH) had a significant association with retroplacental hemorrhage (RPH). In this study, to determine whether CVH has a correlation with RPH or intervillous thrombosis (IVT), placentas associated with CVH were clinico-histopathologically investigated and compared to those with IVT. Obstetric complications in 32 cases with CVH were compared with 258 cases with IVT. Placental abruption was significantly higher (18.7%) in the CVH group than in the IVT group (5.03%) and the incidences of intrauterine fetal death (IUFD) as well as intrauterine growth retardation (IUGR) were also significantly higher (18.7% and 9.37%) in the CVH group than in the IVT group (3.10% and 2.32%). Of the pathological characteristics of the placentas with CVH, IVT was found in 46.8%, but marginal or retroplacental hemorrhages were seen in 34.3%, compared to 14.2% of the IVT group. Premature infants with low birth weight (under 2000 g) and before 36 weeks of gestation were born in 53.1% and 34.3% of cases with CVH placentas. CVH was not only located near the basal plate of the placentas and decidual hemorrhages, but was also highly associated with chorangiosis in 75% of cases. Using histological and ultrastructural studies in CVH cases, villous stroma was filled with fetal erythrocytes and leakage of blood cells was observed through a disruption of the trophoblast layer of hemorrhagic villi. In conclusion, it is suggested that CVH may rather be related to formation of the intervillous thrombosis than to RPH.

Diagnostic relevance of abortion-associated human embryonic antigen expressed on the cell surface of tumour promoter-treated Bloom syndrome cells.

We detected stable expression of human embryonic antigen associated with spontaneous abortion (HEAA) on the cell surface of a tumour promoter-treated B lymphoblastoid cell line (BS-SHY) originating from Bloom syndrome. We used indirect immunofluorescence and diluted serum from 44 patients who had recurrent spontaneous abortions. With the use of the panning procedure, we separated characteristic cells expressing strong HEAA. The BS-SHY-HEAA cells separated here would be useful for measuring serum antibody (against HEAA) produced by patients with recurrent abortions. It was also noted that aborters who received husbands' leukocyte immunization have lost this antibody, and have delivered successful pregnancies at term. Using HEAA proteins, we conducted Western blotting analysis for the amino acid sequencing (mol. wt 77 kDa). Amino acid sequencing data indicated that HEAA had 87.5% homology to the immunoglobulin (Ig) VHIII region in the framework. Recently, the protective value of high dose i.v. administration of immunoglobulin in the treatment of recurrent spontaneous abortions has been reported to be similar to that of leukocyte immunization. Therefore, the BS-SHY-HEAA cells appear to provide a valuable tool for rapid serological diagnosis and for evaluating the efficacy of immunotherapy with husbands' leukocytes in patients with recurrent spontaneous abortions.

Chorangiosis and chorangioma in three cohorts of placentas from Nepal, Tibet, and Japan.

It has been reported that both chorangiosis and chorangioma are increased in placentas at high altitudes. In this study, 1.14% of 2448 Japanese placentas obtained at 30-300 m had chorangiosis or chorangioma, compared with 3.24% of the Nepalese placentas (1300-3000 m) and 9.09% of the Tibetan samples (3800-4200 m). The incidence of both pathologies was significantly higher in the Himalayan groups than those of the Japanese group (P < 0.05). Obstetric complications of the 28 Japanese placentas with chorangiosis and chorangioma included Caesarean section 16 (57.1%), abruptio placentae 2 (7.14%), intrauterine growth retardation 3 (10.7%), intrauterine fetal death 4 (14.2%) and placenta praevia 4 (14.2%). Four infants (14.2%) had Apgar scores ranging from 0 to 3. Among 48 Himalayan placentas with chorangiosis and chorangioma, incidence of marked subchorionic fibrin was significantly higher (25%) than in the Japanese group (3.57%) (P < 0.05). The incidence of abnormal insertion of the cord was significantly higher (14.2%) in the Japanese group than in the Himalayan group (5%) (P < 0.05). There was no significant difference in the frequencies of intervillous thrombosis and infarction between the two groups. These findings suggest that the incidence of pathological change such as chorangiosis and chorangioma may be increased in placentas at Himalayan altitudes.

Characteristic mucinous ovarian cancer antigen is expressed in malignantly transformed Bloom's syndrome cells.

Using a double-antibody panning procedure, we separated a unique cancer antigen cell line (BS-SHI-4M OVC-MU) expressing a mucinous ovarian cancer (OVC) antigen from a malignantly transformed Bloom's syndrome cell line. In order to gain information concerning a mucinous OVC antigen, we tested this unique cell line in the reaction to sera from patients with various OVCs, Krukenberg (KR) tumor, and signet ring cell cancer of the stomach under immunofluorescence and Western blotting protocols and determined the mucinous OVC antigen band at M(r) 84,000. We also undertook an immune electron microscopic study to gain information concerning the antigen-antibody reaction [BS-SHI-4M OVC-MU cells-sera from patients with mucinous OVC and KR tumor] and concerning the antigenic determinant of the membrane using preembedding methods. Occasional protein A-gold particles were observed along the cell membrane of BS-SHI-4M OVC-MU cells, when treated with sera from mucinous OVC and KR tumor patients, but no labeling was observed in the cell membrane when treated with sera from normal patients and those with other cancers. Results of the immune electron microscopic study strongly support the data from the antigen-antibody reaction obtained by immunofluorescence and Western blotting analyses. The BS-SHI-4M OVC-MU cells separated here would be useful for serodiagnosis of mucinous OVC and KR tumors and for follow-up of patients after therapy.

Enhanced expression of stomach cancer antigen derived from malignantly transformed bloom syndrome cells previously labeled with bromodeoxyuridine.

Bromodeoxyuridine (BrdU) greatly enhanced expression of stomach (ST) cancer antigen (CA) that originated from a malignantly transformed Bloom syndrome (BS) cell line (BS-SHI-4M), although the expression was suppressed with a decrease in sister chromatid exchange (SCE) in the presence of deoxythymidine (dT) or deoxycytidine (dC) and enhanced with an increase in SCE with deoxyguanosine (dG) or deoxyadenosine (dA). Although the exact mechanisms for enhancing CA by BrdU treatment are unknown, these findings appeared to be of special interest because of the parallelism of CA expression and SCE alterations. The finding that BrdU enhancement of the ST CA was effective not only in the immunofluorescence (IF) protocol but also in the band appearance of Western blotting would be worthwhile as a sensitive serodiagnosis of cancer. The 118-kd band obtained from proteins of ST CA cells previously labeled with BrdU was clearly more darkly stained than that from nonlabeled cells and enabled eight weak-positive ST CA to show strong-positive levels retaining complete negativity to nonmalignant sera. Some ST cancer sera (advanced cancer), which originally gave a negative reaction in the nonlabeled condition, still inhibited negative reaction even in BrdU-labeled ST CA cells, however. The inability to detect cancer antibody in our assay might be due to immunocomplexes. Acid dissociation and ultrafiltration of sera from six of seven advanced ST cancers (originally IF negative) have allowed detection of antibody responses to ST CA by Western blot assay with enhanced reactivity as compared with the negativity under native serum conditions. This technique provides a reasonable avenue for study of the mechanisms of CA expression and serodiagnosis.

The characterization of human male-specific 5.7 kb DNA(pKY-1) and its application to fetal sex-determination probe.

A male-specific 5.7 kb DNA(pKY-1) was cloned by screening human genomic libraries. Subsequent to structural characterization, a possibility of its application for prenatal diagnosis probe was tested. The results obtained were as follows: 1) the pKY-1 was specifically hybridized with human Y-chromosome specific repetitive 3.5 kb, but not with female DNA and 2.1 kb, known to be another human Y-specific repetitive-DNA. 2) HaeIII, EcoRI and their double digestion did not produce the Y-specific 3.5 kb fragments from pKY-1. 3) Although the digestion of pKY-1 with BamHI liberated 0.55 kb, ClaI clove remained 5.10 kb into 2.40 and 2.7 kb fragments, while their hybridizabilities with the 3.5 kb DNA were identically conserved. These data suggest that 5.7 kb fragment (pKY-1) should be proposed as a member of Y-chromosome-specific repetitive DNA family in human genome. Thus, this cloned DNA will be not only a useful probe for human fetal sex-determination but also an effective tool for analysis of Y-chromosome with its structure and repetitive DNA.

Clinical experience with trophoblastic diseases in Nepal.

The epidemiology of trophoblastic disease in Nepal remains unknown. In this study, the first data regarding patients with trophoblastic disease were collected from two hospitals in Kathmandu, Nepal. We found 68 molar pregnancies during 1986 to 1987 at the Maternity Hospital, there. The incidence of molar pregnancies during two years ranged 2.84 per 1000 pregnancies and 3.24 per 1000 deliveries, while the total number of trophoblastic diseases from 1985 to 1988 at Teaching Hospital, Tribhuvan University, Kathmandu, numbered 26. The incidence of trophoblastic disease ranged from 7.07 per 1000 pregnancies to 8.04 per 1000 deliveries. As a result, the rate of trophoblastic disease in Nepal seems to be higher than expected, and it indicates a incidence similar to that other Asian countries at sea level.

[Total replacement of the urinary bladder using an ileal pouch bladder].

We have used the continent ileal bladder as a bladder replacement after radical cystectomy. The ileal bladder is an ileal pouch which is anastomosed to the urethral stumps. The ureters are implanted by a free end ureteroileostomy. The long term results with 26 patients who underwent this procedure are reported. In the early postoperative period, urodynamic and radiographic studies revealed small capacity and high intravesical pressure of the ileal bladder. However, it became a low pressure reservoir with increased capacity gradually. The average bladder capacity was about 250 ml and average residual urine was 30 ml. Most of the patients were continent in the daytime if the voiding intervals were less than 3 hours at night, some patients were incontinent. Urinary leakage was the most frequent complication. VUR and hydronephrosis were still the problems to be solved. The ileal pouch bladder is a valuable procedure in properly selected cases.

Cancer antigens are expressed in a carcinogen-transformed Bloom syndrome B-lymphoblastoid cell line.

We have cloned malignant cells carrying specific antigens associated with ovarian cancer (OVC) and malignant lymphoma (ML) from BS-SHI-4M cells, a line derived from a 1-methyl-3-nitro-1-nitrosoguanidine-treated B-lymphoblastoid cell line isolated from a patient with Bloom syndrome. Since BS-SHI-4M cells react with sera from various individual cancer patients at relatively low frequencies (2-9%), as detected by an indirect immunofluorescence technique, cell clones that specifically react with sera from patients with OVC and ML were separated by the "panning" method in which polystyrene dishes were coated with sera from OVC and ML patients and cells with the corresponding antigens bound to the dishes. Subsequent cloning by limiting dilution provided cell clones highly enriched for OVC- and ML-associated antigens. Karyotype analyses revealed that cell clones with OVC and ML antigens had common marker chromosomes, der(14)t(14;14) (p11;q11),t(6;?)(p25;?) and t(9;?)(q34;?), besides t(17;?) (q25;?) found in the OVC-antigen-positive clones and t(5;?) (p13;?),t(7;?)(q36;?) found in the ML-antigen-positive clones. Interestingly, in cell clones with a strong OVC antigen response, the distal part of the Y chromosome (Yq11) was missing in 100% of the cells. Therefore the cell line BS-SHI-4M appears to be a reservoir of cell clones each of which carries a specific tumor antigen and thus provides a potential tool for rapid serological diagnosis of cancer.

Placental protein 21. Localization in human placenta and concentrations in the body fluids of men and nonpregnant and pregnant women.

The immunohistochemical localization of placental protein 21 (PP21) was marked in the syncytial brush border and basal membrane during the 1st and 2nd trimesters of pregnancy and also in the chorionic epithelial brush border and basement membrane at term. A weaker stain was found in the cell membranes of amniotic epithelial and chorionic trophoblast cells. Neither heparin nor changes in temperature significantly influenced PP21 concentration. Relatively high serum PP21 concentrations were measured during the follicular and luteal phases in healthy nonpregnant women and in healthy men whose seminal plasma also showed a high PP21 concentration. Serum PP21 levels in normal pregnancy rose from a median of 29.1 ng/ml at 6-7 weeks of gestation to 82.0 ng/ml at 36-37 weeks of gestation. Although maternal urine showed low PP21 levels during pregnancy, amniotic fluid PP21 levels were higher at 7-21 weeks of gestation than at term. Cord blood sera showed almost the same PP21 concentration as maternal sera, but retroplacental blood showed much higher levels. Maternal serum PP21 levels in hydatidiform mole patients did not differ from the normal pregnancy range, although their molar vesicular fluids contained higher PP21 concentrations. These results suggest an extraplacental source for PP21.

Characteristic differences in immunohistochemical localization of new placental proteins (PP1, PP19, PP21) in the human placenta.

The immunohistochemical localization in the human placenta of new placental proteins PP1, PP19, and PP21 was clarified using modified indirect enzyme-labeled antibody method and compared with that of pregnancy-specific beta 1-glycoprotein (SP1). The major results are as follows: positive staining for PP1 was seen at the nucleus and cytoplasm of villous cytotrophoblasts, the X cells at the basal plate, and of chorionic trophoblasts, while the decidua cells and amnion were not stained. PP19 was characteristically seen in the nucleus and cytoplasm of syncytiotrophoblasts. X cells in basal plate, chorionic trophoblasts, and maternal leukocytes. The villous cytotrophoblasts, decidua cells, and amnion were not stained. PP21 localization was found at the microvilli and basal membrane of syncytiotrophoblasts and at the cytotrophoblast plasma membrane of the chorionic villus in early gestation. In late gestation, increased staining was seen at the syncytiotrophoblast microvilli and the villous basement membrane, and moderate staining at plasma membrane of the amniotic epithelium and chorionic trophoblasts. SP1 was found only at the syncytiotrophoblast cytoplasm of chorionic villi. Studies using these four placental proteins simultaneously may therefore provide a new key learning about unknown metabolic functions of trophoblasts.