Ontogenetic localization and distribution of S-100beta protein in human placental tissues

OBJECTIVE: To investigate whether S-100beta, a brain-specific protein found in amniotic fluid and fetal circulation, is present in fetoplacental tissues throughout gestation. METHODS: S-100beta protein localization and concentration were assessed in placentae, fetal membranes, and cord vessels. Tissues were obtained from 40 pregnant women at different gestational ages: first trimester (n = 10), second trimester (n = 10), early third trimester (n = 10), and late third trimester (n = 10). RESULTS: In the placenta, S-100beta was localized in villous and intermediate trophoblast cells. The intensity of immunostaining and protein concentration increased with advancing gestation. S-100beta protein was also present in amnion, trophoblast, and decidual cells of fetal membranes, and in endothelial cells of umbilical vessels at all gestational ages. CONCLUSION: This study demonstrated that fetoplacental tissues contain S-100beta protein, suggesting that these tissues may, at least in part, be responsible for the high level found in the fetal circulation. Although the significance of placental S-100beta is unknown, this origin should be taken into account when this protein is used as a marker of brain injury in the fetus or infant at birth.     

Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.     

Antibodies to trophoblast in normal pregnant and secondary aborting women

Enzyme-linked immunosorbent assays (ELISA) with the use of chorionic villous plasma membranes prepared from first trimester and term placentae were employed to detect antibodies to trophoblast in normal primigravid women. Normal pregnant women were found to produce IgG antibodies to trophoblast. These antibodies could be eluted from first trimester placentae. This antibody response was observed in the first trimester and gradually decreased as pregnancy progressed. IgM antibody responses were observed only in the third trimester. Antibodies in some primigravid women and secondary recurrent aborters showed allotypic reactivity with individual trophoblast membranes. This finding was confirmed by immunoblotting experiments in which antibodies from some normal pregnant women were shown to recognize the same trophoblast antigens as those recognized by antibodies from secondary recurrent spontaneous aborters.     

Immunohistochemical study of tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125) in the human and cynomolgus monkey placenta,umbilical cord and decidua

Summary Tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125) were studied immunohistochemically by the avidin-biotin immunoperoxidase technique in human and cynomolgus monkey placentae, membranes, umbilical cords and decidua. In early human placentae, TPA was localized mainly in the cell membranes of villous syncytio- and cyto-trophoblast. The cytoplasm of those trophoblastic cells were weakly stained with TPA. The membrane of basal chorionic trophoblast cells was strongly stained with TPA and the cytoplasm stained weakly. In early cynomolgus placentae, similar immunostaining results were obtained. However, the positive stainings for TPA was more marked in the cytoplasm of villous syncytiotrophoblast and basal chorionic trophoblast, and less marked in the cell membrane of villous cytotrophoblast. In early human and cynomolgus placentae, CA125 was not demonstrated immunohistochemically in the villi and basal chorion. In human and cynomolgus term placentae, the villous syncytiotrophoblast and basal and reflected chorionic trophoblast showed similar immunostaining as the early placentae. In addition, TPA was found in the amniotic epithelium in both sorts of placentae. TPA was not detected immunohistochemically in the umbilical cord and decidual cells. While weakly positive stains for CA125 were observed in decidual cells, CA125 was localized mainly in the membrane and cytoplasm of amniotic epithelium in both human and cynomolgus term placentae. TPA and CA125 are thus oncoplacental antigens and the monkey could serve as a model for their investigation.     

Expression of natural antimicrobials by human placenta and fetal membranes

Preterm birth associated with infection is a major clinical problem. We hypothesized that this condition is associated with altered expression of natural antimicrobial molecules (beta-defensins (HBD), elafin). Therefore, we examined expression of these molecules and their regulation by proinflammatory cytokines in placentae and fetal membranes from term pregnancy. HBD1-3 and elafin were localized by immunohistochemistry in fetal membranes and placenta. Real-time quantitative PCR was used to examine mRNA expression in primary trophoblast cells treated with inflammatory molecules. HBD1-3 and elafin were immunolocalized to placental and chorion trophoblast layers of fetal membranes and placenta. Immunoreactivity was also observed in amnion epithelium and decidua. No differences were noted between samples from women who were not in labour compared to those in active labour. In in vitro cultures of primary trophoblast cells, HBD2 and elafin mRNA expression was upregulated by the proinflammatory cytokine, IL-1beta. These results suggest that the chorion and placental trophoblast layers may be key barriers to the progression of infection in the pregnant uterus. Natural antimicrobial expression may be altered in response to inflammatory mediator expression associated with the onset of labour and/or uterine infection, providing increased protection when the uterus may be particularly susceptible to infection.     

Lysyl oxidases: expression in the fetal membranes and placenta

The cross-linking of the connective tissues in the fetal membranes and placenta is important for their tensile strength and elasticity. We have studied the expression of lysyl oxidase (LOX) because it is the classical enzyme responsible for the cross-linking of collagen and elastin. We have also studied the two recently described, genetically distinct lysyl oxidase-like genes and proteins, lysyl oxidase-like (LOXL) and lysyl oxidase-like 2 (LOXL2), of unknown functions.Specific antisera have been used for immunolocalization in fetal membranes and placentae from early pregnancy terminations and after caesarean section at both preterm and term, prior to labour. In addition, the steady state mRNA levels of the three genes has been quantitated in separated amnion, chorion, decidua and placentae collected at term before labour. The immunocytochemistry shows that the spatial expression of the three lysyl oxidases is similar in early pregnancy in both the fetal membranes and placentae. However, by preterm this pattern had diverged and becomes greatest at term. The expression of the genes found at term was similar to the results of protein expression obtained by immunocytochemistry, with the exception of LOXL which had high placental gene expression, but low levels of immunolocalized protein. Thus by term, LOX was expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL showed highest gene expression in the placenta and lowest expression in the amnion. LOXL2 expression was again different and was expressed predominantly in the chorionic cytotrophoblast of the membranes with low expression in both the amnion and placentae. These results suggest that these three members of the lysyl oxidase family may have similar roles in early pregnancy during the development of the placenta and fetal membranes, but their divergence as pregnancy advances to term, may reflect changes in substrate specificity and connective tissue composition.     

Maternal serum total activin A and follistatin in pregnancy and parturition

Objective To examine changes in maternal serum levels of activin A and follistatin during pregnancy and labour
Design In three cross sectional and three longitudinal studies venous blood was collected from women during pregnancy, spontaneous labour, labour induction and prior to elective caesarean section for the measurement of activin A and follistatin.
Setting Monash Medical Centre, Clayton, Victoria, Australia.
Population One hundred and twenty-three women participated in a cross sectional study in pregnancy, 18 women in two longitudinal pregnancy studies, 36 women in a cross sectional labour study, nine women in a longitudinal study of labour induction. Ten women undergoing elective caesarean section were also studied.
Methods Activin A and follistatin were measured using two sensitive and specific enzyme-linked immunosorbent assays.
Results In the cross sectional study of pregnancy, mean (SEM) maternal serum activin A and follistatin levels increased towards term (2.4 ng/mL (0.3) and 1.8 ng/mL (0.3) in first trimester to 18.9 ng/mL (3.8) and 5.3 ng/mL (0.9) at term, respectively), but the longitudinal study revealed that levels plateau in the last three weeks of pregnancy (16.0 ng/mL (2.6) and 6.2 ng/mL (1.4) at 37 weeks and 16.6 ng/mL (3.5) and 6.2 ng/mL (0.5) before labour for activin A and follistatin, respectively). There was no difference in levels of activin A and follistatin between women delivered by caesarean section and labouring women at term (14.9 ng/mL (2.8) vs 11.0 ng/mL (0.93) and 5.95 ng/mL (0.67) vs 5.71 ng/mL (0.63), respectively) and levels of both proteins did not alter throughout spontaneous or induced labour.
Conclusions We believe that these data argue against activin A playing an acute role in the initiation or regulation of human parturition.     

Activin betaA-subunit and activin receptors in human myometrium at term and during labour

Objective To measure activin A content and to localise and semi-quantitate activin receptors in human myometrium at term and during labour.
Design Myometrium was collected from non-pregnant women (   n = 6  ), pregnant women at term not in labour (   n = 6  ) and at term in labour (   n = 6  ).
Setting Monash Medical Centre, Melbourne, Australia.
Main outcome measures Tissue lysates of myometrium were analysed for activin A content using an enzyme-linked immunosorbent assay and activin receptor proteins IA, IIA and IIB using Western hybridisation. Activin β_A-subunit and activin receptors were localised in myometrium by immunohistochemistry.
Results Activin A was detected by ELISA in non-pregnant, pregnant and labouring myometrium. Levels were significantly higher in labouring myometrium. The three activin receptors IA, IIA and IIB were detected in all myometrial samples by Western hybridisation. Receptor IA was expressed in significantly higher levels in pregnant myometrium. Receptor IIA was very weakly expressed throughout. The expression of receptor IIB was similar in all three groups. Activin β_A-subunit and all three receptors were localised to the endothelial cells of myometrial blood vessels. Neither activin β_A-subunit nor any of the three activin receptors were immunolocalised to myometrial smooth muscle cells in the three groups. This result was confirmed by Western blotting for expression of activin receptors in isolated myometrial smooth muscle and microvascular endothelial cells.
Conclusion The myometrium is not a target for activin A during late pregnancy or labour. However, activin A may have a role in the regulation of microvascular endothelial cell function in the myometrium.     

Inhibin subunits in human placenta: localization and messenger ribonucleic acid levels during pregnancy

This study describes the difference in distribution and levels of inhibin alpha, beta A- and beta B-subunit messenger ribonucleic acids in human placenta during pregnancy. Northern blot analysis indicated that inhibin alpha messenger ribonucleic acid is present in placental extracts collected at the early stage of gestation. Hybridization to inhibin beta A messenger ribonucleic acid was detected in the first trimester but in much lower levels. However, the intensity of the hybridization signal for inhibin alpha- and beta A-subunit messenger ribonucleic acids was greater in extracts prepared from term placentas than in those from the first or second trimester of pregnancy. Low levels of inhibin beta B-subunit messenger ribonucleic acid were observed only in extracts prepared from term placenta. At both early stage and term gestation trophoblast cells showed a positive fluorescent signal with the inhibin alpha-, beta A- and beta B-subunit-specific antisera. However, whereas inhibin alpha-subunit was localized in the cytotrophoblast, inhibin beta B-subunit immunoreactivity was observed in the syncytial layer of the villi, and inhibin beta A-subunit was widely distributed. The different distribution of immunoreactive inhibin subunits was confirmed by in situ hybridization, showing the different localizations of the inhibin messenger ribonucleic acids. These results showed that (1) human placenta produces the inhibin alpha- and beta A-subunits as early as the first trimester of pregnancy, (2) messenger ribonucleic acid levels for each of the three inhibin subunits are highest at term, and (3) immunoreactive inhibin subunits are localized differently in placental villi.     

Expression of GLUT12 in the fetal membranes of the human placenta

The aim of this study was to characterize the expression of the novel glucose transporter GLUT12 in the fetal membranes of the human placenta. RT-PCR and Western blotting of extracts of amnion and choriodecidua from four normal term placentas identified GLUT12 mRNA and protein expression. In all four samples the signals for GLUT12 were markedly stronger in the choriodecidua than in the amnion, whereas the signals for GLUT1, a glucose transporter know to be expressed in fetal membranes, were similar for the two tissues. In further studies, paraffin sections of fetal membranes were analyzed by immunohistochemistry with GLUT12 and GLUT1-specific polyclonal antibodies. GLUT12 immunoreactivity was localized predominantly to the trophoblast cells in the chorion and to a lesser extent to decidual cells and to epithelial and fibroblast cells of the amnion. GLUT1 was localized to chorionic trophoblast cells and amniotic epithelial and fibroblast cells. GLUT12 expression was predominantly cytoplasmic, whereas GLUT1 was associated with the membrane of the cells. These results show that GLUT12 is expressed in cells of human fetal membranes and suggest that GLUT12 may play a role in the facilitation of glucose transport into these cells.     

Placentae of light for dates infants born to underweight mothers at term: a morphometric study

Fifteen light for dates infants and their placentae were compared to 15 well-grown infants and their placentae. The former were born to thin, underweight women while the latter were born to women of normal weight. The light for dates infants were symmetrically growth retarded but not wasted at delivery and their placentae had a reduced weight, volume, chorionic surface area, percentage parenchyma and total villous surface area. The peripheral villous surface area and volume of peripheral villous trophoblast, fetal capillaries and connective tissue was also reduced in the placentae of light for dates infants, suggesting retarded placental growth in the latter half pregnancy. In contrast, the stem villous surface area and volume of stem villous trophoblast, fetal capillaries and connective tissue was similar in both groups of placentae, suggesting the same rate of growth in early pregnancy. There were no differences in the volume of fibrin or infarcts. The ratio of total villous surface area to infant weight, length and head circumference was reduced in the light for dates infants. This may restrict the materno-fetal oxygen exchange, and thereby increase the risk of fetal hypoxia during labour. It is concluded that the placentae of light for dates infants born at term to underweight women are both absolutely and relatively small with a reduced villous surface area.     

Inhibin A, inhibin B and activin A concentrations in umbilical cord artery and vein.

Activin A and inhibins (A and B) are growth factors expressed during pregnancy by the human placenta, decidua and fetal membranes, and by several fetal organs. They are secreted in both the maternal and the fetal circulations, but the net contribution of the fetus to inhibins/activin A production is still unclear. In the present study we determined whether there was a difference in the serum concentration of activin A, inhibin A and inhibin B between the artery and vein of the umbilical cord. Arterial and venous umbilical cord blood was obtained immediately before elective Cesarean section of 16 term infants from uncomplicated pregnancies. Inhibins and activin A levels were assayed by specific enzyme-linked immunosorbent assays. The paired t-test and linear regression analysis were used to calculate statistical significance. Inhibin A levels did not differ between the artery and vein of the umbilical cord. In contrast, arterial inhibin B levels were significantly (p < 0.001) lower, and activin A concentrations significantly (p < 0.05) higher than the respective venous concentrations. A significant correlation between arterial and venous levels of inhibin A (r = 0.591; p < 0.05), inhibin B (r = 0.749; p < 0.0001) and activin A (r = 0.571; p < 0.05) was found. The present findings suggest that the human placenta is the main source of inhibin B, and the fetus of activin A, in the umbilical cord. In light of the possible roles played by inhibin and activin in erythroid differentiation, protection of neurons against brain injury and modulation of adrenal and pancreatic hormone release, the present data may be of help in evaluating their changes in the umbilical cord when gestational diseases occur.     

Hypoxia induced activin secretion by the fetoplacental unit: differential responses related to gestation

OBJECTIVE: To determine whether activin A levels reflect oxygen availability in basal and hypoxic conditions in the late pregnant fetus and newborn lamb. DESIGN: In vivo animal experimental study. SETTING: Department of Physiology, Monash University. POPULATION: Chronically catheterised fetal sheep in late gestation. METHODS: Fetal hypoxia was induced at 125 (n = 4), 135 (n = 4) or 145 days ('term'; n = 3) gestational age by maternal nitrogen exposure, for 4 hours, during which maternal and fetal arterial, and amniotic fluid samples were collected. Lambs (age one, five and eight days; n = 3) were exposed to 1 hour of hypoxia via nitrogen exposure. MAIN OUTCOME MEASURES: Activin A, prostaglandin E2 (PGE2) and cortisol were analysed in plasma and amniotic fluid, and whole blood was used to determine Pao2, Paco2, %O2, lactate and pH. RESULTS :Basal activin A concentrations in the fetal arterial circulation remained unchanged between 125 days (0.230 [0.10] ng/mL) and term (0.28 [0.10] ng/mL), as did fetal oxygen saturation (59.11% [4.74%] to 52.25% [4.84%]) and pH (7.35 [0.02] to 7.37 [0.02]). Moderate fetal hypoxia (50% fall in fetal arterial %O2) produced a significant increase in circulating activin A (2.05 [0.67] ng/mL) and a significant decrease in pH (7.27 [0.03]) at 125 days of gestation, however, at 135 and 145 days, activin A and pH remained unchanged. Fetal activin A concentration was significantly correlated with pH (P = 0.036) but not %O2 (P = 0.072). Hypoxia in the lambs did not alter circulating activin A. CONCLUSIONS: In response to hypoxia, activin A is increased in the circulation of 125-day-old fetuses, but not in older fetuses. Fetal arterial activin A levels sensitively reflect pH but not oxygen saturation, with increasing activin A in conditions of metabolic acidosis.     

Cellular localization of vascular endothelial growth factor in ovine placenta and fetal membranes

To further understand the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during development in the sheep placenta and fetal membranes, we examined the localization of VEGF mRNA and protein in placental, chorionic and amniotic tissues by in situ hybridization and immunohistochemistry in ovine fetuses at 62, 102 and 141 days gestation (term=150 days). In the placenta, VEGF mRNA expression and VEGF protein immunostaining were strong in cytotrophoblasts surrounding the villi. In addition, VEGF protein was localized in smooth muscle cells around fetal and maternal blood vessels and in the maternal epithelium. There was no apparent difference in placental VEGF mRNA or protein levels associated with advancing gestation. In the fetal membranes, VEGF mRNA was detected in the amniotic epithelium and the chorionic cytotrophoblastic cell layer. The intensity of the hybridization signals in both amnion and chorion appeared low at 62 days, moderate at 102 days and high at 141 days gestation. VEGF protein was detected in amniotic epithelium and chorionic cytotrophoblasts at all gestational ages studied. The increase in VEGF gene expression in fetal membranes as term approaches suggests that during fetal development VEGF may promote the vascularity and permeability of the microvessels which perfuse the fetal membranes, as well as permeability of the amniotic membrane itself. Thus VEGF may participate in the regulation of amniotic fluid volume.     

Macrophage inhibitory cytokine-1 in gestational tissues and maternal serum in normal and pre-eclamptic pregnancy

Macrophage inhibitory cytokine-1 (MIC-1), a divergent member of transforming growth factor-beta superfamily, has been recently shown to be produced by the human placenta with detectable levels in maternal serum. In this study, using immunohistochemistry, we have localized MIC-1 in placenta, decidua and foetal membranes across pregnancy and, using an enzyme-linked immunoassay, measured MIC-1 in maternal serum in normal pregnancy, in association with labour and pre-eclampsia. In the placenta MIC-1 was principally localized to the syncytiotrophoblast while in the foetal membranes MIC-1 was present in the amniotic epithelium, chorionic trophoblasts and adherent decidual cells. There were no differences in MIC-1 staining distribution or intensity in the placentae between women in labour and not in labour, or between healthy and pre-eclamptic pregnancies. MIC-1 staining in the foetal membranes was slightly stronger after a labour and delivery compared to those delivered by elective Caesarean section. MIC-1 levels in the maternal serum increased with advancing gestation but there were no significant differences in maternal serum levels associated with either labour or pre-eclampsia.These observations would be consistent with MIC-1 having roles at the maternal-foetal interface, perhaps in the establishment and/or maintenance of pregnancy. Our data argue against MIC-1 having a significant role in the regulation of labour or in the pathophysiology of pre-eclampsia.     

Spatial and temporal patterns of expression of messenger RNA for insulin-like growth factors and their binding proteins in the placenta of man and laboratory animals

To better understand the role of the insulin-like growth factors (IGF-I and -II) and their binding proteins (IGFBPs 1-6) in placental development and function, it is important to review similarities and differences between species in expression of the respective mRNAs. In human placenta, IGF-II mRNA is expressed in chorionic mesoderm and first trimester villous cytotrophoblast, but not in syncytiotrophoblast. In contrast, in rhesus monkey placenta, IGF-II mRNA is expressed in syncytiotrophoblast but not in chorionic mesoderm. IGFBP-3 mRNA is present in the chorionic mesoderm of placental villi from both these species and may modulate IGF-II action through a paracrine mechanism. In rodent placentae, IGF-II mRNA is expressed both in fetal mesoderm and in the trophoblast of the placental labyrinth. In guinea pig, where IGFBP-5 mRNA is expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth, interaction between IGF-II and IGFBP-5 mRNA may be involved in vascularization of the placenta by fetal vessels. In sheep placenta, IGF-II mRNA is expressed, not in the trophoblast layer, but in the fetal mesoderm immediately adjacent to it. In the basal plate of human, rhesus monkey and baboon placentae, extravillous trophoblasts express IGF-II mRNA and uterine decidual cells IGFBP 1-6 mRNAs. The inference is that there is interaction between IGF-II and IGFBPs at the maternal-fetal interface of the primate placenta during trophoblast invasion and decidualization. IGFBP-1 expressed by the decidua may also interact with alpha(5)beta(1)integrin expressed by the extravillous trophoblast. The placentae of rodents are also of the invasive type. Glycogen cells of the mouse placenta are analogous with human extravillous trophoblast and express IGF-II mRNA. However, expression of IGFBP mRNAs in the mouse, as in the guinea pig, is confined to non-decidualized endometrium and myometrium. IGF-II mRNA is strongly expressed by trophoblasts invading uterine vessels in human and guinea pig placentae. Interactions probably occur between IGF-II expressed by these trophoblasts and IGFBPs expressed in the vessel walls. However, it is possible that IGFBPs expressed by maternal vessels are associated with processes that are independent of trophoblast invasion. Thus, IGFBP-3 mRNA is highly expressed in the maternal blood vessels of the non-deciduate sheep placenta. Findings to date highlight the diversity in the expression of the IGF system among placentae of man and different laboratory animals, and even between closely related species. Comparative studies will continue to be required to understand the functional role of IGFs and IGFBPs in each species.     

Comparative study of 15 lysosomal enzymes in chorionic villi and cultured amniotic fluid cells. Early prenatal diagnosis in seven pregnancies at risk for lysosomal storage diseases

A large number of chorionic villi samples obtained from women undergoing elective first trimester termination of pregnancy was analysed by enzyme assays similar to those applied to cultured amniotic cells. The levels of 15 lysosomal enzymes were compared to those observed in tissue cultures of amniotic cells obtained through amniocentesis at 16-18 weeks of pregnancy and the results were discussed in order to assess the usefulness of trophoblast biopsy for first trimester diagnosis of hereditary lysosomal diseases. The data suggest the applicability of this source of fetal cells for prenatal diagnosis of fifteen respective genetically determined enzyme deficiencies with the probable exception of alpha-L-iduronidase deficiency. Enzyme determinations were performed on chorionic villi samples of two pregnancies at risk for Tay-Sachs disease, three pregnancies for GM1 gangliosidosis type 1, one for mucopolysaccharidosis type VI and one for Wolman's disease.