Human fetal testis: second trimester proliferative and steroidogenic capacities.

The period of Leydig cell hyperplasia (14-18 weeks gestation) in human fetal testis is crucial for normal gonad development. We have studied the spatio-temporal distribution of key developmental and functional markers in human fetal testis between 13-19 weeks gestation. Proliferating cell nuclear antigen-positive cells were immunolocalized to both interstitium and tubules. Image analysis confirmed an increase in positive interstitial cells during Leydig cell hyperplasia (P: < 0.05). c-Myc was localized to the interstitium with no gestational changes. The steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase (protein) and cytochrome P450 17alpha-hydroxylase/C(17-20)-lyase (P450c17; messenger ribonucleic acid and protein) were confined to the Leydig cells. The number of immunopositive cells increased between 13 and 19 weeks (P: < 0.001). P450c17 mRNA (in situ hybridization) and protein were localized to the same population of interstitial Leydig cells. Androgen receptor and Bcl-2 protein (anti-apoptotic) were gradually restricted to the peritubular myoid cells as gestation progressed. Conversely, Bax protein (pro-apoptotic) was predominantly localized to the tubule Sertoli cells, whereas the germ cells were Bax immunonegative. In conclusion, human fetal Leydig cell hyperplasia is characterized by increasing numbers of proliferating cells and increased expression of steroidogenic enzymes. The Bcl-2-positive, Bax-negative status of the peritubular myoid cells may be a strategy for cell survival.

Effect of angiotensin II and 5-hydroxytryptamine on the vessels of the human foetal cotyledon.

1 The actions of angiotensin II (AT II) and 5-hydroxytryptamine (5-HT) on the vessels of the human isolated, perfused, cotyledon were examined in vitro. 1 The cotyledonary vessels were shown to respond to both AT II and 5-HT over the range 10(-8) to (10(-4) M. 3 The preparation was found to be more responsive to AT II than 5-HT. 4 The findings confirm that the responsiveness of the cotyledonary vessels differs from the vessels of the umbilical cord, and that this behaviour does not depend upon the integrity of the endothelium associated with these vessels.

The effect of prostaglandin D2 on the blood vessels of the perfused isolated cotyledon of the human placenta.

Prostaglandin D2 was shown to constrict the blood vessels of the isolated perfused cotyledon of the human placenta. Its potency was less than that of prostaglandin F2 alpha but similar to prostaglandin E2. As it is known to dilate uterine blood vessels it may, by its differential action on the foetal and maternal vascular beds, play a role in the local regulation of utero-placental blood flow.

Melatonin receptors in the human fetal kidney: 2-[125I]iodomelatonin binding sites correlated with expression of Mel1a and Mel1b receptor genes.

Melatonin receptors in the human fetal kidney were identified and characterized by quantitative in vitro autoradiography using the melatonin agonist, 2-[125I]iodomelatonin. Specific binding was localized to cells in the nephrogenic region at the outer perimeter of the developing kidney and was time-dependent, saturable and inhibited in the presence of guanosine 5'-0-(3-thiotriphosphate) indicative of a G protein-coupled receptor. Expression of the Mel1a and Mel1b melatonin receptors in human fetal kidney was determined using RT-PCR. In situ hybridization confirmed the localization of the Mel1a mRNA transcripts. A role for melatonin in development of the human fetal kidney is postulated.

Long-chain polyunsaturated fatty acid transport across the perfused human placenta.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentae (n = 9) from normal pregnancies. The maternal side was perfused ex vivo for 90 min with a modified Krebs Ringer solution containing a physiological mixture of fatty acids and ratio of fatty acid to human albumin. There was no evidence of chain elongation and desaturation of the essential fatty acids. Relative to the value for oleic acid, the rate of transfer to the fetal circulation was: 1.30 +/- 0.02 (P < 0.001) for linoleic acid, 1.61 +/- 0.09 (P = 0.002) for alpha-linolenic acid, 0.67 +/- 0.10 (P = 0.033) for arachidonic acid and 2.10 +/- 0.16 (P = 0.003) for docosahexaenoic acid. For tissue accumulation the values were 1.47 +/- 0.39 (P < 0.001) for linoleic acid, 2.24 +/- 0.37 (P = 0.027) for alpha-linolenic acid, 9.84 +/- 1.03 (P = 0.001) for arachidonic acid, and 3.01 +/- 0.79 (P = 0.064) for docosahexaenoic acid. The order of selectivity for transfer from the maternal to the fetal circulation was docosahexaenoic > alpha-linolenic > linoleic > oleic > arachidonic acid. Such a mechanism would allow the preferential transfer of docosahexaenoic acid and the essential fatty acids to the fetal circulation, thereby protecting the polyunsaturated fatty acid supply to the fetus during a critical period of development.

Leptin secretion to both the maternal and fetal circulation in the ex vivo perfused human term placenta.

The contribution of placental leptin, if any, to both the fetal and maternal circulation and its role in pregnancy remains to be determined. In an experiment to investigate this, 27 placentae from term pregnancies were perfused ex vivo (gestational age=39.5 s.d. 1.2; range=38-42 weeks: fetal weight=3285 s.d. 482; range=2480-4420; birthweight centile range=4th to the 98th) at both the maternal and fetal interface. Placental leptin was exported into both the maternal and fetal circulations. The log leptin production by the maternal side of the placenta was significantly greater (P=0.001) than that for the fetal side (5.193 s.d.1.049 versus 4.387 s.d. 0.768 ng/placenta/min). There was no significant relationship between maternal and fetal log leptin production and maternal body mass index, birthweight, birthweight centile, ponderal index or gestational age or with cord blood pO(2), pCO(2) and pH. There was however, a significant increase in the maternal log leptin production with increasing fetal to placental weight ratio (P=0.017; r(2)=20.7 per cent) but no corresponding relationship for fetal leptin production. It is proposed that such a mechanism would allow the placenta to modulate fat supply to the fetus in response to the fetal demand relative to placental supply.

Placental nutrient transfer capacity and fetal growth.

The objective of this study was to determine whether the ability of the human placenta to transfer glucose and fatty acids is related to normal fetal growth. The intrinsic nutrient transport capacity of the placenta was measured under standardized conditions during in vitro perfusion of 30 human term placentas and related to birth weight (range 2640-4640g), birth weight centile (8th-99th), ponderal index (2.43-3.69), placental weight (418-1030g) and placental:fetal weight (0.14-0.31). There was no statistically significant change in the rate of nutrient transfer per placenta or per kg fetal weight, with birth weight, birth weight centile, ponderal index, placental weight and placental:fetal weight. There was a weak but significant relationship (P=0.020, r(2)=9 per cent) between the ratio of glucose to fatty acid transport and birth weight centile, largely due to the high ratio found in the lowest birth weight quartile where the babies are thinnest. This study provides no evidence that placental nutrient transport capacity limits fetal growth across a wide range of birth weights in normal pregnancies. It is proposed that the fetus itself may regulate placental nutrient transport in vivo via the fetal cardiac output and the rate of fetal nutrient utilization.

A quantitative study on the effects of maternal smoking on placental morphology and cadmium concentration.

The aim of this study was to quantify the effects of maternal cigarette smoking on placental morphology, paying particular attention to variables known to be influential in facilitating oxygen diffusion. Structural quantities were estimated by stereological analyses of placental samples drawn from non-smoking and smoking women whose smoking habits were assessed both subjectively (from volunteered cigarette consumption) and objectively (by determining levels of plasma cotinine, a major metabolite of nicotine). Concentrations of placental cadmium were also measured. In the smoking group, maternal and fetal haematocrits were elevated and mean birthweight was reduced. Within placentae, the most significant alterations were increases in cadmium levels, the relative volumes of maternal intervillous space, the relative surface areas of fetal capillaries and decreases in the relative and absolute volumes of fetal capillaries. Findings indicate that changes in capillary volume are the result of a decrease in mean capillary diameter rather than total length. The mean thickness of the trophoblast component of the villous membrane was also increased in the smoking group. Although increased haematocrits suggest that fetuses of smoking mothers suffer hypoxic stress, these morphological changes are likely to compromise, rather than assist, transplacental oxygen transfer. This is in marked contrast to the adaptive changes seen in pregnancies associated with preplacental hypoxia and suggests that other factors might be compromising the fetoplacental unit. Finally, although the morphological changes associated with maternal smoking seem to be the result of an all-or-none, rather than dose-dependent, effect, the available evidence is not conclusive.

Uptake of zinc by human placental microvillus border membranes and characterization of the effects of cadmium on this process.

The uptake of Zinc (Zn) by microvillus border membrane vesicles formed from the trophoblast of term human placentae is markedly increased over brief periods of incubation with much slower increases persisting for up to 2 h of incubation. Zinc is both bound to membrane components and transported into intravesicular osmotically active space. Uptake is saturable, temperature dependent from 4 to 37 degrees C with a Q10 of 1.7, and is inhibited by the sulphydryl agent DTNB. About 20 per cent of the uptake is susceptible to inhibition by Cadmium (Cd) at concentrations from 5 to 50 microM, a significant part of the action of this metal being on the transmembrane component of Zn uptake.

Maternal cigarette smoking and oxygen diffusion across the placenta.

The aim of this study was to test whether or not adaptations in partial, total and specific oxygen diffusive conductances occur in the placentae of women who smoke cigarettes during pregnancy and help to compensate for intrauterine fetal hypoxic stress. Tissue sections were randomly sampled from human term placentae divided into two groups (non-smokers and smokers) according to maternal smoking status. In smokers, status was expressed as either declared smoking rate or level of plasma cotinine (the major metabolite of nicotine). Sections were analysed stereologically to estimate key structural quantities (vascular volumes, exchange surface areas, tissue diffusion distances). These were combined with previously-published physicochemical quantities (oxygen-haemoglobin reaction rates and tissue oxygen diffusion coefficients) in order to estimate the partial conductances of six tissue compartments of the oxygen pathway: maternal erythrocytes and plasma, villous trophoblast, villous stroma (including fetal capillary wall), fetal plasma and erythrocytes. From partial conductances and birthweights, total and specific conductances were calculated for each placenta. Results were assessed statistically by analyses of variance and t -tests. Despite apparent improvements in the partial conductances of the maternal erythrocytes and plasma, total and specific conductances did not alter significantly in smoking groups. However, the relative biases affecting these estimates may be different in smokers and non-smokers. We conclude that total conductance does not increase in placentae associated with maternal smoking. However, given that the fetus suffers chronic hypoxic stress as a consequence of smoking (evidenced here by elevated haematocrits), even a constant diffusive conductance implies a reduced transplacental partial pressure gradient. This could be a contributory factor to the reduced birthweight.

Characterization of an antibody to the human melatonin mt1 receptor.

Melatonin acts via high affinity, G-protein coupled, seven transmembrane domain receptors. To precisely localize these receptors, antibodies were raised in chickens against a 15 amino acid fragment at the intracellular C-terminal region of the human melatonin receptor subtype mt1 (DSSNDVADRVKWKPS, mt(1338-352)). A chimeric form of the receptor with a hydrophilic Flag peptide (DYKDDDDK) in sequence with the extracellular N-terminus (Flag-mt1) was generated by polymerase chain reaction and expressed in mammalian cell lines. An IgY antibody (Y31), which gave high antibody titres by enzyme-linked immunosorbent assay, was used to localize Flag-mt1 in stably transfected cells by immunofluoresence. Flag-mt1 localization with Y31 was identical to that obtained with the M5 antibody directed against the Flag epitope and was mainly localized to the Golgi apparatus with some staining at the cell surface. No staining was seen in untransfected cells with either antibody. Y31 staining was abolished using antibody preabsorbed with peptide antigen. Y31 immunofluorescence in fetal human kidney sections was restricted to nephrogenic regions and matched that of 2-((125)I)iodomelatonin binding and mt1 gene expression by in situ hybridization. Y31 was used to immunoprecipitate biotinylated membrane proteins from Flag-mt1 stably transfected and untransfected CHO cells. Western blotting of immunoprecipitated proteins revealed two major bands specific to stably transfected cells, one at 63 kDa and one at 86 kDa. The first band almost certainly corresponds to the glycosylated form of Flag-mt1 and the second band to receptor dimers. Thus, Y31 antibody is suitable for use in detecting the human mt1 receptor subtype in tissues and in transfected cells.

Fetal swallowing and voiding in relation to hydramnios.

Fetal swallowing and voiding were measured using colloidal gold and an ultrasonic scanner, respectively. Subjects in the study were in their 38-40th week of pregnancy. Cases were divided into 3 groups: normal, hydramnios, and oligohydramnios. The normal group had a mean swallowing rate of 198 ml/day and a mean voiding rate of 23.6 ml/hr. No significant differences were found among these rates and the corresponding rates in the other 2 groups. It is concluded that mechanisms other than fetal swallowing and voiding can contribute to the control of amniotic fluid volume at term.

Dietary intake and tissue concentration of fatty acids in omnivore, vegetarian and diabetic pregnancy.

The aim of this study was to determine the effect of fatty acid intake and insulin dependent diabetes on the fatty acid composition of maternal erythrocytes, the placenta and cord. Fatty acid intake (from food frequency questionnaire) and the fatty acid composition of maternal erythrocytes, the placenta and cord from pregnant vegetarians (n = 4) and insulin dependent diabetics (n = 5) was compared with pregnant omnivores (n = 10). There was a significantly lower intake of n-6 long chain polyunsaturated fatty acid (LCPUFA) (-75% P < 0.01) and n-3 LCPUFA (-92% P < 0.01) and increased ratio of n-6/n-3 LCPUFA in the vegetarians (103%; P < 0.001). The concentrations of 22:4 n-6 (+28%; P < 0.05) and 22:5 n-3 (+40%; P < 0.05) were higher in vegetarian erythrocytes. Placental 18:2 n-6 (+26.9%; P < 0.05) 18:3 n-3 (+139%; P < 0.05) and 22:5 n-3 (+24%; P < 0.05) were increased while 20:5 n-3 (-36%; P < 0.05), 22:6 n-3 (-16%; P = 0.059), and the ratios of 20:4 n-6/18:2 n-6 (P < 0.01) and 22:6 n-3/18:3 n-3 were reduced. 22:6 n-6 (-49%; P < 0.05) and total n-3 LCPUFA (-11%; P < 0.01) were reduced in vegetarian cord. For the diabetic mothers, all of the n-6 LCPUFA and n-3 LCPUFA were reduced in the maternal erythrocytes; 22:4 n-6 (-42%; P < 0.05), 22:5 n-6 (-46%; P < 0.05) and 22:6 n-3 (-41%; P < 0.05). For the diabetic placenta and cord the general pattern of n-3 LCPUFA was the same as that in the vegetarians. In the vegetarian mothers, the PUFA profiles in the maternal erythrocytes, placenta and cord are consistent with an elevation in the rate of LCPUFA synthesis in order to make up the relative deficit in LCPUFA intake. However, it may be that the higher level of desaturase activity is not able to overcome the dietary deficit of 22-6 n-3 and 22:6 n-6. Despite the fact that the dietary LCPUFA intake in the pregnant diabetic was comparable with that in the pregnant 'normal' omnivore mothers, the pattern of PUFA in the tissues resembled that of the vegetarian mothers.

Identification and characterisation of 2-[125I]iodomelatonin binding and Mel1a melatonin receptor expression in the human fetal leptomeninges.

Melatonin binding sites were identified over the leptomeninges surrounding the human fetal brain using quantitative in vitro autoradiography and the melatonin agonist, 2-[125I]iodomelatonin. Binding was found to be saturable and of high affinity (dissociation constant (Kd) = 54 pM and maximal theoretical binding (Bmax) = 13 fmol/mg protein), and inhibited by guanosine-5'-o-(3-thiotriphosphate) (GTPgammaS) suggesting that these binding sites represent G protein-coupled melatonin receptors. RT-PCR performed on mRNA isolated from the human fetal leptomeninges detected expression of the G protein-coupled melatonin receptor Mel1a, but not Mel1b. In situ hybridisation confirmed the localisation of Mel1a mRNA transcripts over the leptomeninges of the fetal brain. The identification of 2-[125I]iodomelatonin and Mel1a melatonin receptor expression in the fetal leptomeninges implies that melatonin may play a role in the early growth and development of the human brain.

The role of melatonin in the human fetus (review).

Melatonin, an indole amine, primarily derived from the pineal gland is secreted during the hours of darkness. Melatonin acts as a hormonal transduction of photoperiod influencing the timing of seasonal and daily (circadian) physiological rhythms. Maternal melatonin crosses the placenta and enters the fetal circulation providing photoperiodic information to the fetus influencing the subsequent circadian and seasonal rhythms of the offspring. The function of melatonin in humans is more obscure. However, melatonin has attained prominence as a treatment for disturbed circadian rhythms and sleep patterns which occur as a result of transmeridian travel, shift work or blindness. The biological clock, the hypothalamic suprachiasmatic nuclei (SCN), possesses melatonin receptors, in both the adult and fetal human. This concurs with the reported influence of melatonin on human circadian rhythmicity and indicates that this influence may begin in utero. Melatonin receptors are widespread in the human fetus and occur in both central and peripheral tissue from early in fetal development. Thus, the influence of melatonin on the developing human fetus may not be limited to entraining circadian rhythmicity. Considering the transplacental availability of melatonin to the fetus the ingestion of melatonin by pregnant women may be inadvisable.

NMR imaging in human pregnancy: a preliminary study.

In an effort to demonstrate the potential of nuclear magnetic resonance imaging (NMR) in the management of pregnancy, 15 patients in the first trimester and one in the third trimester of pregnancy have been investigated in the Aberdeen University NMR imager. Simple measurements of biparietal diameter and crown-rump length were made from both the NMR images and the ultrasound images on the same day, with good correlation between the two. The NMR data was displayed as inversion recovery, calculated T1, proton density and S1-S2 images. The proton density and S1-S2 images were found to be the most useful for the demonstration of the fetus and for discriminating between placenta and uterus. The calculated T1 data provided accurate quantification of the proton-spin lattice relaxation times of the different tissues, indicating that this measurement may be of use in the study of fetal brain development and placental function. The inversion recovery images showed poor tissue discrimination and were found to be of limited value. The unique information available using NMR and the non-invasive nature of the technique indicated that it should provide a useful method for the investigation of both fetal development and placental function in addition to making basic measurements of fetal size.

Human placental cytochrome P450 and quinone reductase enzyme induction in relation to maternal smoking.

Components of cigarette smoke such as cadmium and polycyclic aromatic hydrocarbons have been shown to induce quinone reductase (QR) activity in placental explants. This study examines the relationship of maternal smoking habit and maternal plasma cotinine concentration with the activities in vitro of both QR and the cytochrome P450 (CYP1A) marker ethoxyresorufin O-deethylase (EROD) in placental tissue. Maternal plasma samples were taken at Week 34 of gestation, and placental tissues were obtained at term. Plasma cotinine concentrations were determined by high-performance liquid chromatography. Trophoblast cytosolic QR and microsomal EROD activities were measured by resazurin reduction and ethoxyresorufin O-dealkylation respectively. QR activity was inhibited 70% by a mixture of dicoumarol (1 microM) and rutin (20 microM). Plasma cotinine concentrations correlated significantly (P < 0.001) with both declared smoking rate (r = 0.67, N = 37) and placental EROD activity (r = 0.63, N = 36), but not with QR activity, whether measured as total QR activity or specifically as either DT-diaphorase or carbonyl reductase. It is concluded that smoking up to 40 cigarettes per day induces EROD but does not affect QR activity in the placenta at term.

Transfer of immunoglobulin G across the isolated perfused human placental lobule.

This study demonstrates that IgG transfer in vitro across the isolated perfused human placental lobule can be successfully studied by using natural forms of IgG. The transfer of anti-RhD IgG (anti-D) was measured in the presence and absence of intravenous immunoglobulin (IVIgG). When anti-D and IVIgG were present alone each crossed the placenta at about the same rate, but when both forms were present at the same time the movement of one interfered with the movement of the other. This pattern of transfer is consistent with receptor-mediated transcytosis. The interactions of IgG with trophoblastic transporters may therefore be studied without the complications that might arise from the use of conventionally labelled molecules.

Sexual differentiation of the human midtrimester brain.

It is firmly believed that sexual differentiation of the brain is linked with external genital differentiation in timing as an in utero event in the human. An extensive search for oestrogen, androgen and progestin receptors failed to show their presence despite adequate controls in cytosols from human fetal brain of gestational ages 14-20 weeks. It is possible that the receptors are present in levels so low that they are undetectable by present-day methods. Our results would indicate that hormonally influenced in utero brain sexual differentiation is most unlikely to occur as a mid-trimester event.

Admission-discharge policies for hysteroscopic surgery: a randomised comparison of day case with in-patient admission.

OBJECTIVE: To study the effectiveness and acceptability of day case hysteroscopic surgery. DESIGN: Prospective randomised controlled trial. SETTING: Aberdeen Royal Infirmary. SUBJECTS: One hundred and ninety four consecutive women who underwent hysteroscopic endometrial ablation. INTERVENTION: Seventy three women were allocated to day case surgery and 37 to inpatient admission; 84 women though otherwise fit for day case surgery were scheduled for in-patient admission as they lived more than 20 miles away. All women completed a questionnaire 24 h after their operations. Readmission rates were obtained from case notes. Satisfaction rates 12 months after the operation were recorded by means of a follow-up questionnaire. RESULTS: Post-operative pain was absent or slight in 48 (75%) of the women in the day case group 27 (84%) of women in the in-patient group, and 55 (82%) in the non-randomised in-patient group. Post-operative analgesia was necessary in 34 (52%) women in the day case group, 24 (75%) women in the in-patient group and 36 (53%) women in the non-randomised in-patient group. Hospital costs were significantly less in the day case group. Satisfaction with stay 92% in the day case group, and 100% in the other two groups. CONCLUSION: In this setting, day care is a safe acceptable and less expensive alternative to in-patient care for hysteroscopic endometrial ablation.