Investigations were undertaken to describe and quantitate the topographical abnormalities which develop in the organ of Corti as a result of acoustic trauma, and to determine their relationship to the associated losses of cochlear function assessed by the compound action Potential (N1) and cochlear microphonics (CM). Thirty guinea pigs were exposed, while anaesthetised, to a tone of 3 KHz at 125 dB SPL for 30 minutes. Both organs of Corti were examined by scanning electron microscopy either immediately or 1,3,7 or 14 days after exposure. In the second study 26 anaesthetised guinea pigs were exposed to 5 KHz at 125 dB SPL for 30 minutes. Cochlear potentials were recorded from the round window of the right cochlea and N1 audiograms (sound Pressure level required to elicit N1 Plotted against frequency) and 10 μv isopotential curves (sound pressure level required to produce 10 μv CM plotted against frequency) were produced for each animal either within one hour or 1,7,14 or 28 days later. The same organ of Corti was examined by scanning electron microscopy. Cochlear potentials were compared to mean values from 16 normal guinea pigs. Most animals developed topographical changes in one or both organs of Corti after exposure to 3 KHz (90%) and in the right organ of Corti after 5 KHz (92%). There was a wide variation in both the length (3 KHz: 0.1-4.15 mm; 5 KHz: 0.5-16.0 mm) of lesions and the number of hair cells affected. Both these indices of damage increased significantly (p <0.01) in the 24 hours following exposure to 5 KHz. Early damage to hair cells included either collapse, fusion or loss of stereocilia and there was an increase in the proportion of affected cells towards the centre of the lesions where supporting cells were damaged also. Subsequent to exposure, collapsed stereocilia appeared to become fused and some hair cells, particularly OHC, with stereocilia abnormalities were lost. However, others, particularly IHC, remained for up to 28 days despite abnormal stereocilia. Early changes occurred around the position of maximum displacement of the basilar membrane and subsequent extension of the lesions occurred equally in both apical and basal directions. Fusion was the only stereocilia change to develop after exposure. Regions of the organ of Corti showing supporting cell damage were replaced within 3-7 days by the proliferation of inner sulcus and Claudius’ cells. The substantial initial loss of both N1 thresholds and CM sensitivity partially recovered during the first 24 hours after exposure, but paradoxically, this was associated with the significant increase in the topographical changes in the organ of Corti. N1 threshold loss occurred over all frequencies and was maximum 1/2 – 1 octave higher than the exposure frequency (5 KHz). All lesions occurred within regions corresponding to changes in N1 thresholds. In the first 24 hours topographical changes occurred over a much smaller area of the organ of Corti than indicated by changes in N1 thresholds. Seven or more days later the longer lesions (30%) reflected the extent of changes in N1 but the remainder were smaller than indicated by this functional loss. This suggests that functionally important damage to the cochlea is more extensive than indicated by hair cell loss, stereocilia abnormality or supporting cell changes in the organ of Corti. Therefore, investigations of the effects of noise should not be based simply on topographical changes in the organ of Corti as these often underestimate the extent of injury to the cochlea.

The introduction to this thesis is a literature review. Kramer, in a study commissioned by WHO, reviewed the literature prior to 1985 on low birthweight. This is extended, mainly in respect to infants who are small for gestational age with emphasis on important findings in relation to birthweight since that time. Work in New Zealand on birthweight is also summarised. The literature is also reviewed in respect to the mechanisms in the pathway between the placenta and the fetus, and in respect to recent work suggesting a link between birthweight and disease in adult life. This thesis examines factors that influence birthweight and placental weight. Birthweight for gestational age percentile curves for the New Zealand population were firstly defined. small for gestational age (SGA) infants could then be categorised. The thesis considers two sources of data, the first a cross-sectional sample of the New Zealand population from 1987 to 1990 (the control subjects of the New Zealand cot Death study, a national case-control study on sudden infant death syndrome), and the second a hospital population in Auckland (National Womens Hospital (l992)). These two datasets are investigated to determine factors that influence birthweight in a univariate situation and then in the multivariate situation. Independent variables are considered using a priori categorisations and where appropriate Quantile-Quantile (Q-Q) derived categorisations determined by producing plots of the quantiles of cases versus controls. A number of variables under the headings of socio-demographic, lifestyle, genetic, obstetric and nutrition are examined and found to be associated with the outcomes of interest at the univariate level. After controlling in multivariate analyses a number of variables are found to be no longer significant, however some show strong relationships. The variable relating to smoking in both datasets shows the greatest detrimental effect on the outcomes considered in respect to birthweight. This confirms that in New Zealand, as in other places in the world, smoking has significant consequences on birthweight. The data is also investigated for the timing of insult to the fetus from smoking, and is found to be most important during pregnancy. comparison of the results comparing those obtained using a binary outcome for SGA, and those obtained using birthweight continuously, show relatively consistent results. The odds ratios and the decreases in birthweight obtained from both datasets show a relatively linear relationship between the two. An examination into whether a distinct group of individuals exists in respect to having large placentae for birthweight, indentified an artefact in the dataset relating to recording of placental weight for twins. After removal of twins from the dataset, examination of factors that influence placental weight showed that the factors that influence placental weight are not the same as those that influence birthweight. In particular smoking is found not to influence placental weight, and haemoglobin, which has no influence on birthweight, is found to be inversely associated with placental weight. other factors such as parity are found to influence placental weight in the same proportion in which birthweight is affected. In conclusion this thesis shows that factors investigated in New Zealand are consistent with findings in the international literature in relation to birthweight. The results on factors that influence placental weight add to the international literature on a topic on which little work has been carried out. The results of this thesis point to areas where future research needs to be carried out, in particular in relation to maternal nutrition during pregnancy and maternal energy expenditure during pregnancy. There is also a need for further research into the relationships of factors on placental weight and the ratio of birthweight to placental weight, and how these relationships affect health outcomes in childhood and adult life.

Over the past decade, a number of epidemiological studies have provided significant evidence that certain major adult noncommunicable diseases, such as hypertension, ischaemic heart disease and non-insulin dependent diabetes mellitus, may be associated with impaired fetal growth. This phenomenon has been termed "programming" which is essentially the term used for persisting changes in structure and function caused by undernutrition or other adverse influences acting during critical periods of early development. Programming has been used as the mechanistic basis to explain the long term sequelae of intrauterine growth retardation (IUGR). The mechanisms underlying the epidemiological observations remain to be elucidated and developed. While it is well established that severe maternal undernutrition during pregnancy leads to IUGR, there has been relatively little well defined animal studies of the somatotrophic axis and postnatal development of growth retarded offspring. The major objectives of this thesis were to establish a model in the rat of IUGR by nutritional restriction of the dam throughout gestation and to examine the effects of fetal growth retardation on endocrine, molecular and growth parameters during postnatal development. In addition, the development of an animal model for IUGR enabled well defined studies testing distinct hypotheses suggested by the epidemiological observations of professor David Barker and colleagues. Timed matings were performed in Wistar rats and dams were randomly assigned to one of two dietary treatment groups. A control group was fed ad libitum throughout pregnancy and a restricted group was fed 30% of ad libitum intake. Restricted fed dams were observed to lose a significant amount of body weight throughout gestation, due to undernutrition, but caught up to the ad libitum group during the lactating period. Maternal undernutrition significantly reduced fetal and placental weights without altering litter size. Postnatally, body weights of offspring from undernourished dams continued to be reduced until at least 18 weeks of age, although they were observed to be growing at the same rate as ad libitum offspring by 2 weeks of age. A cohort of animals from undernourished dams were maintained to measure blood pressure by tail cuff plethysmography. Offspring from undernourished dams were found to have significantly elevated systolic blood pressures from 18 weeks of age. This observation provides direct experimental support for the hypothesis, derived from human epidemiological studies, that the origin of adult hypertension may originate during fetal life as a result of exposure to a sub-optimal intrauterine environment. Parallel reductions in plasma IGF-I and hepatic IGF-I mRNA concentrations before 15 days of age were also observed in growth retarded offspring. Hepatic IGF-I transcription start sites within exon 1 and exon 2 were coordinately reduced with IUGR up to 15 days of age without changes in GHR and GHBP mRNA abundance. The lack of catch-up growth observed in the IUGR offspring despite normalization of their plasma IGF-I and IGF-I mRNA levels from 15 days of age may be due to a state of partial resistance to GH. This observation lead to a series of treatment studies in which neonatal and juvenile offspring from ad libitum and undernourished dams were treated with growth factors to investigate somatic growth responses as a measure for hormone sensitivity. In both treatment studies, ad libitum offspring from both age groups and juvenile IUGR offspring responded to GH treatment However, neonatal IUGR offspring did not exhibit any response to GH treatment.. Analysis of IGF-I gene expression in neonatal offspring showed that GH treatment elevated IGF-I Eb mRNA in ad libitum but not IUGR offspring. These results suggest a possible mechanism for transient GH resistance in that a post-receptor defect in GH action may contribute to the development of temporary postnatal GH resistance as a consequence of IUGR and fetal programming of IGF-I gene expression. In summary, the development of a model of IUGR in the rat using maternal undernutrition throughout gestation has enabled detailed investigation of nutritional regulation of the somatotrophic axis during fetal development and postnatal sequelae. The studies in this thesis have shown that the somatotrophic axis is markedly altered postnatally by nutritional restriction of the dam throughout gestation, leading to prolonged postnatal growth retardation and elevated blood pressure The mechanisms which lead to the induction of such fetal programming and whether these changes may contribute to the development of subsequent adult-onset disease remain to be addressed in future studies.

This thesis is based on two theoretical models of chronic illness: Large, Butler, James, and Peters (1990) introduced a systems model of musculo-skeletal pain which incorporated many of the variables believed to be important in the development and maintenance of pain. Feldman’s model (1974) addressed the difficulties of adapting to chronic illness. Five studies evaluated specific aspects of these models. The epidemiology of pain in New Zealand (NZ) was derived from a psychiatric epidemiology project. Approximately 80% of NZ adults had experienced a life disrupting episode of pain which had required medical consultation. Subjects who reported episodes of pain were more likely to have psychiatric diagnoses of anxiety, depression, and phobia. They were more likely to describe their health as poor and were currently consulting their doctor more than people who did not report an experience of pain. The estimated average cost of health consulting by people attending Auckland Hospital Pain Clinic (AHPC) for the previous year was $1333(NZ). Most people had some subsidy of costs. The health consulting of the AHPC group was higher than that reported in the NZ health literature. Self image and the experience of pain were assessed in two studies. The first asked subjects at AHPC to describe the typical thoughts, feelings, and behaviours, of someone with chronic pain. Subjects described loss of self esteem, alienation from family and friends, fear of the future, frustration and anger. The descriptions focused on psychological aspects of the experience of pain. The second study of self image used repertory grid technique. Two standardised Illness Self Construct Repertory Grids (ISCRG) were evaluated. Issues in the use of standardised grids are discussed and some aspects of ISCRG application are explored. The two ISCRG indicated subjects often identified themselves as a physically ill person and felt isolated from others. People with pain and their "closest other” (CO) completed the ISCRG(A) and questionnaires on the quality of their relationship. Closest others overestimated the role of the physical illness in their partners’ life and believed that they understood them better than the individual with pain thought they did. The personality dimensions of alexithymia and hypnotisability have been hypothesised as pathways for the development of psychosomatic illness. Individuals with chronic pain were tested to establish whether they weremore alexithymic and more hypnotisable than subjects in a general population control group. This was not verified. The constructs of alexithymia and hypnotisability require critical examination. The experience of pain is common and is associated with psychological distress and high health service use. Self construct appears to be a major factor determining response to pain and to treatment programmes. Chronic pain appears to be a particular challenge for individuals who must accept alteration in their lifestyle with perhaps little understanding of what the future may hold. / Whole document restricted, but available by request, use the feedback form to request access.

This poststructuralist qualitative study explored the discourses constructing women’s choice for a caesarean section in the absence of clinical indications, in the talk and texts of women, midwives, an obstetrician, professional journals and the media publications. The study affirms inscriptions surrounding choice in childbirth are shaped discursively through a multiplicity of discourses underpinned by social and institutional practices. With advances in technology, childbearing women have a greater variety of options from which to choose. Controversial, is the option of a caesarean section, regardless of clinical need. The issue is depicted in both professional and popular discourse as contentious, complex and contradictory. Its momentum into the 21st century, as a new object of obstetric discourse, has been played out on a number of platforms. In this thesis I draw from the theoretical ideas of French philosopher Michel Foucault, to examine this complex debate. I argue there is a volatile moment in the history of childbirth in which an explosion of discourses have sculptured choice for a caesarean, in the absence of clinical indications, out of a repartee of autonomy, convenience, desire, fear and risk. In this precarious moment, new meanings joust with the old on a shifting terrain awash with rhetoric that co-opts, competes, and contradicts to bring about a caché of mutable ‘truths’. Whether caesarean, as an optional extra, can be explained in terms of a libertarian imperative, an embodiment of lifestyle, the satiation of desire, the attenuation of fear or the avoidance of risk, the democratisation of this choice has exposed a pathologising paradox, whereupon the normal emerges as the abnormal, and the abnormal emerges as the normal. The deconstruction of choice through a poststructuralist lens has enabled insight into how contradiction and contest befall the ‘order of things ’ and in so doing, provides new openings for contemplating the discursive positioning of women through the competing discourses of childbirth.

In the first trimester of human pregnancy specialised placental cells, termed cytotrophoblasts differentiate into extravillous trophoblasts (EVTs), which grow out from the placenta and invade into the maternal decidua, acting to physically attach the placenta to the decidua, and adapt the uterine spiral arteries to support pregnancy. A proportion of these EVTs also temporarily occlude the spiral arteries for approximately the first 10 weeks of gestation, preventing maternal blood flow to the placenta and creating a low oxygen environment in which placental and fetal development occur. The processes of trophoblast outgrowth and invasion, and the establishment of a low oxygen environment, are essential for the success of pregnancy, and inadequate trophoblast invasion into the uterus has been associated with recurrent miscarriage, pre-eclampsia and fetal growth restriction. However, the exact mechanisms that control the differentiation of cytotrophoblasts down the extravillous trophoblast lineage are poorly understood. Since the extent of this invasive process is unique to human implantation, animal models are of limited value in studying trophoblast invasion. Existing in vitro models have major limitations in that many are very difficult to quantify while others many not study the correct trophoblast population. The research in this thesis has focussed on the development of novel models by which to study cytotrophoblast differentiation, and the use of these models to further understand cytotrophoblast differentiation down the EVT lineage, and the regulation of EVT outgrowth by oxygen. Methods Outgrowth from first trimester villous explants was characterized using immunohistochemistry. Explant viability was investigated using dual staining with chloromethylfluorescin diacetate (CMFDA) and ethidium bromide, by examining DNA laddering, and by immunostaining sectioned explants over 96 hours of culture. The extended viability of cytotrophoblasts in multilayered cell islands in villous tips was exploited to isolate these cells using sequential trypsin digests of cultured villous explants. The trophoblast population obtained were characterized by immunohistochemistry. Finally, villous explants were cultured in either 1.5% or 8% oxygen and the frequency and area of outgrowths was quantified in order to determine the effect of gestation and oxygen on EVT outgrowth. Results Approximately 1/4 of explants cultured in 20% oxygen produced EVT outgrowth. Outgrowth formation and expansion resulted from proliferation of cells in the tips of anchoring villi, and EVTs within the outgrowth did not proliferate. The percentage of explants producing outgrowth declined as gestation increased from 8 to 12 weeks. Dual staining with CMFDA and ethidium bromide revealed degeneration of the syncytiotrophoblast by non-apoptotic mechanisms within 4 hours of culture, but this syncytiotrophoblast layer was able to be regenerated. The majority of cytotrophoblasts died within one week of culture, but despite this explants were able to produce EVT outgrowth for up to 3 weeks due to the extended survival of a specific set of cytotrophoblasts located in cell islands in the tips of anchoring villi. These surviving cells were able to differentiate into EVTs, but not regenerate the surrounding syncytiotrophoblast in the villus tip. Trypsinization of first trimester villi after extended explant culture resulted in the isolation of a viable population of ‘putative EVT progenitors’ that did not syncytialise in culture, but were able to proliferate. 20% of these cells differentiated down the EVT lineage within 96 hours of culture. The putative EVT progenitors expressed markers previously localised to cytotrophoblasts in cell islands of anchoring villi, including αvβ6 integrin and FGFR-2. The addition of exogenous FGF-4 did not affect the differentiation of these cells into EVTs, nor did FGF-4 alter the frequency of EVT outgrowth from explants. Culture in 1.5% oxygen significantly reduced the frequency and area of outgrowths in comparison to 8% oxygen. HLA-G and α1 integrin were both expressed throughout outgrowths with no difference in expression of these proteins between oxygen concentrations. Gestation influenced the response of explants to oxygen, with a significant differential response to oxygen concentration in placentae under 11 weeks of gestation but no differential response in placentae of 11 or 12 weeks. Conclusions In the first trimester, oxygen and gestational age regulate extravillous trophoblast outgrowth in both an independent and interdependent manner. The cytotrophoblast population in the first trimester does not consist of one homogenous bipotent population. Rather there are at least two separate populations: 1) EVT progenitors that exist in the tips of potential anchoring villi that are likely to be committed to EVT differentiation and 2) monolayer villous cytotrophoblasts which are likely to be committed to syncytiotrophoblast differentiation. The second population is easily isolated by traditional enzymatic digestion methods whereas the first much smaller population can be isolated by exploiting their prolonged survival in explant culture.

In the first trimester of human pregnancy specialised placental cells, termed cytotrophoblasts differentiate into extravillous trophoblasts (EVTs), which grow out from the placenta and invade into the maternal decidua, acting to physically attach the placenta to the decidua, and adapt the uterine spiral arteries to support pregnancy. A proportion of these EVTs also temporarily occlude the spiral arteries for approximately the first 10 weeks of gestation, preventing maternal blood flow to the placenta and creating a low oxygen environment in which placental and fetal development occur. The processes of trophoblast outgrowth and invasion, and the establishment of a low oxygen environment, are essential for the success of pregnancy, and inadequate trophoblast invasion into the uterus has been associated with recurrent miscarriage, pre-eclampsia and fetal growth restriction. However, the exact mechanisms that control the differentiation of cytotrophoblasts down the extravillous trophoblast lineage are poorly understood. Since the extent of this invasive process is unique to human implantation, animal models are of limited value in studying trophoblast invasion. Existing in vitro models have major limitations in that many are very difficult to quantify while others many not study the correct trophoblast population. The research in this thesis has focussed on the development of novel models by which to study cytotrophoblast differentiation, and the use of these models to further understand cytotrophoblast differentiation down the EVT lineage, and the regulation of EVT outgrowth by oxygen. Methods Outgrowth from first trimester villous explants was characterized using immunohistochemistry. Explant viability was investigated using dual staining with chloromethylfluorescin diacetate (CMFDA) and ethidium bromide, by examining DNA laddering, and by immunostaining sectioned explants over 96 hours of culture. The extended viability of cytotrophoblasts in multilayered cell islands in villous tips was exploited to isolate these cells using sequential trypsin digests of cultured villous explants. The trophoblast population obtained were characterized by immunohistochemistry. Finally, villous explants were cultured in either 1.5% or 8% oxygen and the frequency and area of outgrowths was quantified in order to determine the effect of gestation and oxygen on EVT outgrowth. Results Approximately 1/4 of explants cultured in 20% oxygen produced EVT outgrowth. Outgrowth formation and expansion resulted from proliferation of cells in the tips of anchoring villi, and EVTs within the outgrowth did not proliferate. The percentage of explants producing outgrowth declined as gestation increased from 8 to 12 weeks. Dual staining with CMFDA and ethidium bromide revealed degeneration of the syncytiotrophoblast by non-apoptotic mechanisms within 4 hours of culture, but this syncytiotrophoblast layer was able to be regenerated. The majority of cytotrophoblasts died within one week of culture, but despite this explants were able to produce EVT outgrowth for up to 3 weeks due to the extended survival of a specific set of cytotrophoblasts located in cell islands in the tips of anchoring villi. These surviving cells were able to differentiate into EVTs, but not regenerate the surrounding syncytiotrophoblast in the villus tip. Trypsinization of first trimester villi after extended explant culture resulted in the isolation of a viable population of ‘putative EVT progenitors’ that did not syncytialise in culture, but were able to proliferate. 20% of these cells differentiated down the EVT lineage within 96 hours of culture. The putative EVT progenitors expressed markers previously localised to cytotrophoblasts in cell islands of anchoring villi, including αvβ6 integrin and FGFR-2. The addition of exogenous FGF-4 did not affect the differentiation of these cells into EVTs, nor did FGF-4 alter the frequency of EVT outgrowth from explants. Culture in 1.5% oxygen significantly reduced the frequency and area of outgrowths in comparison to 8% oxygen. HLA-G and α1 integrin were both expressed throughout outgrowths with no difference in expression of these proteins between oxygen concentrations. Gestation influenced the response of explants to oxygen, with a significant differential response to oxygen concentration in placentae under 11 weeks of gestation but no differential response in placentae of 11 or 12 weeks. Conclusions In the first trimester, oxygen and gestational age regulate extravillous trophoblast outgrowth in both an independent and interdependent manner. The cytotrophoblast population in the first trimester does not consist of one homogenous bipotent population. Rather there are at least two separate populations: 1) EVT progenitors that exist in the tips of potential anchoring villi that are likely to be committed to EVT differentiation and 2) monolayer villous cytotrophoblasts which are likely to be committed to syncytiotrophoblast differentiation. The second population is easily isolated by traditional enzymatic digestion methods whereas the first much smaller population can be isolated by exploiting their prolonged survival in explant culture.

The developmental origins of health and disease hypothesis suggests that exposure to adverse prenatal environmental influences can determine an individual’s susceptibility to obesity in adult life. However, the specific causal mechanisms which underlie this hypothesis have yet to be identified. Focusing on the potential mechanistic role of the leptin endocrine axis, the main objective of this thesis was to investigate the long term effects of prenatal undernutrition and different levels of postnatal nutrition on leptin sensitivity and the development of diet-induced obesity (DIO) in the Wistar rat. A well established animal model of maternal undernutrition during pregnancy was used to induce prenatal undernutrition in experimental offspring. To investigate the interaction between prenatal nutrition and postnatal diet, and its effects on obesity development, female offspring were placed on three different diets: standard chow, a high fat diet or a calorie restricted diet. The effects of prenatal undernutrition and postnatal diet on leptin sensitivity were investigated, in adult offspring, by measuring the response to 14 days of peripheral leptin treatment. Changes in gene expression in the liver, retroperitoneal adipose tissue and soleus muscle were then characterised by custom microarray and quantitative real-time RT-PCR (QPCR) analysis. Adult female offspring exposed to prenatal undernutrition (UN offspring) were found to exhibit leptin resistance in adulthood, independent of postnatal DIO. This result demonstrates for the first time that exposure to prenatal undernutrition has a long term effect on adult leptin sensitivity. In UN offspring fed on a high-fat diet, leptin resistance significantly accelerated the development of DIO while in contrast, offspring maintained on calorie restriction remained lean. These findings suggest that prenatal nutrition can shape future susceptibility to DIO by altering postnatal leptin sensitivity. An analysis of gene expression suggests that prenatal undernutrition causes the development of peripheral tissue-specific leptin resistance, and may also further enhance an offspring’s susceptibility to DIO by altering the regulation of peripheral tissue lipogenesis, mitochondrial function, glucocorticoid metabolism and insulin sensitivity. In conclusion, these studies identify peripheral leptin resistance as a key mechanism that can influence postnatal susceptibility to DIO in female offspring exposed to prenatal undernutrition. Furthermore, the identification of specific changes in peripheral gene expression highlights four additional metabolic mechanisms which may also facilitate the development of DIO in leptin resistant UN offspring.

In the first trimester of human pregnancy specialised placental cells, termed cytotrophoblasts differentiate into extravillous trophoblasts (EVTs), which grow out from the placenta and invade into the maternal decidua, acting to physically attach the placenta to the decidua, and adapt the uterine spiral arteries to support pregnancy. A proportion of these EVTs also temporarily occlude the spiral arteries for approximately the first 10 weeks of gestation, preventing maternal blood flow to the placenta and creating a low oxygen environment in which placental and fetal development occur. The processes of trophoblast outgrowth and invasion, and the establishment of a low oxygen environment, are essential for the success of pregnancy, and inadequate trophoblast invasion into the uterus has been associated with recurrent miscarriage, pre-eclampsia and fetal growth restriction. However, the exact mechanisms that control the differentiation of cytotrophoblasts down the extravillous trophoblast lineage are poorly understood. Since the extent of this invasive process is unique to human implantation, animal models are of limited value in studying trophoblast invasion. Existing in vitro models have major limitations in that many are very difficult to quantify while others many not study the correct trophoblast population. The research in this thesis has focussed on the development of novel models by which to study cytotrophoblast differentiation, and the use of these models to further understand cytotrophoblast differentiation down the EVT lineage, and the regulation of EVT outgrowth by oxygen. Methods Outgrowth from first trimester villous explants was characterized using immunohistochemistry. Explant viability was investigated using dual staining with chloromethylfluorescin diacetate (CMFDA) and ethidium bromide, by examining DNA laddering, and by immunostaining sectioned explants over 96 hours of culture. The extended viability of cytotrophoblasts in multilayered cell islands in villous tips was exploited to isolate these cells using sequential trypsin digests of cultured villous explants. The trophoblast population obtained were characterized by immunohistochemistry. Finally, villous explants were cultured in either 1.5% or 8% oxygen and the frequency and area of outgrowths was quantified in order to determine the effect of gestation and oxygen on EVT outgrowth. Results Approximately 1/4 of explants cultured in 20% oxygen produced EVT outgrowth. Outgrowth formation and expansion resulted from proliferation of cells in the tips of anchoring villi, and EVTs within the outgrowth did not proliferate. The percentage of explants producing outgrowth declined as gestation increased from 8 to 12 weeks. Dual staining with CMFDA and ethidium bromide revealed degeneration of the syncytiotrophoblast by non-apoptotic mechanisms within 4 hours of culture, but this syncytiotrophoblast layer was able to be regenerated. The majority of cytotrophoblasts died within one week of culture, but despite this explants were able to produce EVT outgrowth for up to 3 weeks due to the extended survival of a specific set of cytotrophoblasts located in cell islands in the tips of anchoring villi. These surviving cells were able to differentiate into EVTs, but not regenerate the surrounding syncytiotrophoblast in the villus tip. Trypsinization of first trimester villi after extended explant culture resulted in the isolation of a viable population of ‘putative EVT progenitors’ that did not syncytialise in culture, but were able to proliferate. 20% of these cells differentiated down the EVT lineage within 96 hours of culture. The putative EVT progenitors expressed markers previously localised to cytotrophoblasts in cell islands of anchoring villi, including αvβ6 integrin and FGFR-2. The addition of exogenous FGF-4 did not affect the differentiation of these cells into EVTs, nor did FGF-4 alter the frequency of EVT outgrowth from explants. Culture in 1.5% oxygen significantly reduced the frequency and area of outgrowths in comparison to 8% oxygen. HLA-G and α1 integrin were both expressed throughout outgrowths with no difference in expression of these proteins between oxygen concentrations. Gestation influenced the response of explants to oxygen, with a significant differential response to oxygen concentration in placentae under 11 weeks of gestation but no differential response in placentae of 11 or 12 weeks. Conclusions In the first trimester, oxygen and gestational age regulate extravillous trophoblast outgrowth in both an independent and interdependent manner. The cytotrophoblast population in the first trimester does not consist of one homogenous bipotent population. Rather there are at least two separate populations: 1) EVT progenitors that exist in the tips of potential anchoring villi that are likely to be committed to EVT differentiation and 2) monolayer villous cytotrophoblasts which are likely to be committed to syncytiotrophoblast differentiation. The second population is easily isolated by traditional enzymatic digestion methods whereas the first much smaller population can be isolated by exploiting their prolonged survival in explant culture.

Human mammary tissue was incubated in vitro with [7-3H] dehydroepiandrosterone sulphate (DHA-sulphate) and in agreement with other investigators this steroid conjugate was metabolized to DHA and other steroid products. Sulphatase activity was greater in the malignant than the non-malignant tissues and was found to be a function of the tissue cellularity. One of the major products, a "polar steroid" necessitated identification. The "polar steroid" was identified principally as 7a-hydroxy DHA by chemical modification techniques and co-crystallization of the purified steroid metabolite with carrier 7a-hydroxy DHA. This carrier required synthesis and characterization.