Placental transfer and vascular effects of pharmaceutical drugs in the human placenta ex vivo: A review.

At least 80% of pregnant woman in Europe use at least one medication during their pregnancy. The majority of these drugs are prescribed off-label. A better understanding of drug transport and effects in the placenta can provide an improved pharmacological basis to rationalize drug and dose selection for prescription. Here we provide a narrative review of studies that used the ex vivo placenta perfusion model to study placental drug transport and vascular effects of pharmaceuticals. For studies on placental transfer, we found that the methodology used varied substantially between studies as well as the way in which data was reported. Across the different therapeutic groups, ex vivo measurements of transfer generally corresponded well to in vivo findings. Still, further standardization of the perfusion technique would facilitate a broader use of perfusion data, e.g. in the context of quantitative systems pharmacology models as has been explored in recent years. Only few studies investigated the effects of drugs on the vascular tone using the ex vivo dual-side perfusion model. The model was particularly applied to study vasodilatory effects of pharmaceuticals in the fetoplacental circulation. In conclusion, the ex vivo dually perfused human cotyledon provides a relevant system to gain insights in placental drug disposition and study effects on the fetoplacental vasculature.

A massively multi-scale approach to characterizing tissue architecture by synchrotron micro-CT applied to the human placenta.

Multi-scale structural assessment of biological soft tissue is challenging but essential to gain insight into structure-function relationships of tissue/organ. Using the human placenta as an example, this study brings together sophisticated sample preparation protocols, advanced imaging and robust, validated machine-learning segmentation techniques to provide the first massively multi-scale and multi-domain information that enables detailed morphological and functional analyses of both maternal and fetal placental domains. Finally, we quantify the scale-dependent error in morphological metrics of heterogeneous placental tissue, estimating the minimal tissue scale needed in extracting meaningful biological data. The developed protocol is beneficial for high-throughput investigation of structure-function relationships in both normal and diseased placentas, allowing us to optimize therapeutic approaches for pathological pregnancies. In addition, the methodology presented is applicable in the characterization of tissue architecture and physiological behaviours of other complex organs with similarity to the placenta, where an exchange barrier possesses circulating vascular and avascular fluid spaces.

Optimal villi density for maximal oxygen uptake in the human placenta.

We present a stream-tube model of oxygen exchange inside a human placenta functional unit (a placentone). The effect of villi density on oxygen transfer efficiency is assessed by numerically solving the diffusion-convection equation in a 2D+1D geometry for a wide range of villi densities. For each set of physiological parameters, we observe the existence of an optimal villi density providing a maximal oxygen uptake as a trade-off between the incoming oxygen flow and the absorbing villus surface. The predicted optimal villi density 0.47±0.06 is compatible to previous experimental measurements. Several other ways to experimentally validate the model are also proposed. The proposed stream-tube model can serve as a basis for analyzing the efficiency of human placentas, detecting possible pathologies and diagnosing placental health risks for newborns by using routine histology sections collected after birth.

Fetoplacental vascular alterations associated with fetal growth restriction.

INTRODUCTION: Placental functional impairment in pregnancies with fetal growth restriction (FGR) can arise from fetoplacental vascular abnormalities. We aimed to compare the micro and macrovasculature of placentas from normal pregnancies with those showing late onset FGR. METHODS: Placental arterial casts (n = 12 normal, 6 FGR) were prepared. Chorionic arterial number and inter-branch length were examined. Microvascular features were quantified in CD34-stained tissue sections obtained by systematic (n = 12 normal, 12 FGR) and targeted (n = 6 normal, 6 FGR) sampling from the placental periphery and centre. RESULTS: Adjusted for the weight of the placenta or the surface area of the chorionic plate, the number of chorionic arteries was similar in normal and FGR arterial casts. Inter-branch length per unit placental weight was greater in the first generation of arterial branches in FGR (p < 0.05). Villi in FGR placentas were more poorly vascularised, particularly at the periphery and in grossly visible hypovascular regions. Intermediate and terminal FGR villi in these areas exhibited reduced vessel lumens, loss of CD34, and infilling with CD34-negative cells of what appeared to be previously existing vascular spaces. CONCLUSION: Differences in chorionic arterial branching patterns between normal and FGR placentas arise from differences in placental size. FGR placentas show microvascular regression and extreme hypovascularity in peripheral areas. These features may well limit the ability of the placenta to meet fetal nutrient requirements late in gestation. Targeted sampling is more effective than systematic random sampling in revealing vascular defects.

Systematic review of placental pathology reported in association with stillbirth.

INTRODUCTION: Histopathological examination of the placenta is recommended to determine the cause of stillbirth. Although some reports find causal or contributory placental abnormalities in up to 60% of stillbirths, the significance of such findings in this clinical setting remains uncertain. A systematic review was conducted to i) investigate the likelihood of diagnosing a cause of stillbirth from placental examination and ii) to identify the specific causes of death that can be diagnosed from placental pathology. METHODS: Medline, Embase, Biosis, and Web of Science were searched using the terms "stillbirth", "histopathology", "pathology", and "placenta". Case-reports, narrative review articles and studies that failed to define diagnostic sub-groups were excluded. 473 potential studies were identified. Relevant studies (n = 41) were subdivided into those that investigated causes of stillbirth (n = 13), and those that identified conditions associated with stillbirth (n = 5). The contributory value of placental examination to stillbirth classification was evaluated in 10 studies and the role of specific placental abnormalities in the aetiology of stillbirth in 20 studies. RESULTS: The proportion of stillbirths attributed to a placental cause ranged from 11 to 65%. Classification systems which included multiple placental categories and allowed placental findings to supersede other disorders reported higher rates of placental causes and fewer unexplained stillbirths. Diagnoses were frequently based on qualitative, non-specific terminology. CONCLUSIONS: The utility of histopathological examination of the placenta is affected by the classification system used. International consensus is required for both diagnostic criteria and terminology to describe placental abnormalities and on classification of stillbirths..

IFPA Meeting 2013 Workshop Report III: maternal placental immunological interactions, novel determinants of trophoblast cell fate, dual ex vivo perfusion of the human placenta.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2013 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 placental function, cell turnover and immunology: 1) immunology; 2) novel determinants of placental cell fate; 3) dual perfusion of human placental tissue.

Second and subsequent tumours among 1927 retinoblastoma patients diagnosed in Britain 1951-2004.

BACKGROUND: Retinoblastoma is an eye tumour of childhood that occurs in heritable and non-heritable forms. In the heritable form, there is a predisposition to the development of non-ocular subsequent primary tumours (SPTs). METHODS: This study included 1927 retinoblastoma patients diagnosed in Britain from 1951 to 2004. Ascertainment was through the (UK) National Registry of Childhood Tumours; cases were followed-up for the occurrence of SPTs. Standardised incidence ratios (SIRs) were calculated. RESULTS: We identified 169 SPTs in 152 patients. The SIR analysis included 145 SPTs with cancer registrations from the years 1971 to 2009. These tumours occurred in 132 patients: 112 of the 781 heritable and 20 of the 1075 (presumed) non-heritable cases under surveillance at the start of this period developed at least one registered SPT. The SIRs for all tumours combined were 13.7 (95% confidence interval 11.3-16.5) in heritable cases and 1.5 (0.9-2.3) in non-heritable cases. The main types of SPT in the heritable cases were leiomyosarcoma, (31 cases; SIR 1018.7 (692.2-1446.0)), osteosarcoma (26 cases; SIR 444.6 (290.4-651.4)), and skin melanoma (12 cases; SIR 18.6 (9.6-32.4)). CONCLUSION: The risk of SPTs in heritable retinoblastoma is extremely high. This has important implications for the clinical follow-up and counselling of survivors and their families.

Utero-placental haemodynamics in the pathogenesis of pre-eclampsia.

Pre-eclampsia is associated with insufficient adaptations of spiral arteries which theoretically alter haemodynamics within the intervillous space. Such changes could damage the syncytiotrophoblast and release factors which instigate maternal endothelial dysfunction. We tested this hypothesis using an in vitro dual perfusion model of the human placenta, representing putative changes in flow arising from these spiral artery maladaptations. Whilst fetal-side flow rates remained constant (6 ml/min) perfusion rates on the maternal side were increased from 14 ml/min to 45 ml/min. As well as increasing placental derived intervillous hydrostatic pressures, and changes in flow dynamics observed by colour Doppler, these elevated flow rates resulted in morphologic damage, vacuolation and shedding of the syncytiotrophoblast, focal features previously defined in pre-eclampsia. The collected maternal perfusates recovered under high flow conditions also contained significantly elevated levels of biochemical markers of syncytial damage, including lactate dehydrogenase, alkaline phosphatase and human chorionic gonadotrophin. There were also significant elevations in chemokines GROalpha and RANTES, compared with the low flow perfusions. The soluble components of the maternal high flow rate perfusions decreased the number and proliferation of HUVECs after 24h exposure. These results could not be attributed to GROalpha or RANTES alone or in combination. This study provides evidence that alterations in intervillous flow have the potential to influence both the integrity of the syncytiotrophoblast and the liberation of potentially pathogenic soluble factors. This therefore offers a putative link between utero-placental maladaptations in pregnancy and the vascular endothelial complications of pre-eclampsia.

In vitro dual perfusion of human placental lobules as a flow phantom to investigate the relationship between fetoplacental flow and quantitative 3D power doppler angiography.

Flow phantoms have been used to investigate and quantify three-dimensional power Doppler data but this is the first study to use the in vitro, dual perfused, placental perfusion model. We used this model to investigate and quantify the effect of variation in fetal-side flow rates and attenuation on 3D power Doppler angiography. Perfusion of a placental lobule was commenced within 30 min of delivery and experimentation was successful in 8 of the 18 placenta obtained. Fetal and maternal perfusate was modified Earle's bicarbonate buffer which, following equilibration, was supplemented on the fetal side with whole heparinised cord blood. Imaging was performed with a Voluson-i ultrasound machine. A 'vascular biopsy' the thickness of the placental lobule was defined and signal quantified within using VOCAL (GE Medical Systems, Zipf, Austria). Three vascular indices are generated: vascularisation index (VI) defined as the percentage of power Doppler data within a volume of interest; flow index (FI), the mean signal intensity of the power Doppler information; and vascularisation flow index (VFI), a combination of both factors derived through their multiplication. Attenuation was investigated in this model with the addition of tissue mimic blocks. Our results showed a predictable relationship between flow rates and the vascular indices VI and VFI. However the FI was a less reliable predictor of flow; thus it should be interpreted with caution. The power Doppler signal was markedly affected by attenuation leading to a complete loss of information at a depth of 6 cm in the model used. In conclusion this model can be adapted to provide a phantom to analyse and quantify 3D power Doppler signals and demonstrates that vascular indices within a tissue remain related to volume flow. This model provides further evidence that depth dependent attenuation of signal needs to be accounted for in any in vivo work where the probe is not in direct contact with the tissue of interest.

Vasoactivity to and endogenous release of vascular endothelial growth factor in the in vitro perfused human placental lobule from pregnancies complicated by preeclampsia.

Vascular endothelial growth factor (VEGF) is a potent, physiologically relevant, vasodilator of the human term fetoplacental vasculature of placental lobules from normal pregnancy. There is evidence that VEGF and its receptors are dysregulated in preeclampsia (PE). Here, we used dual perfusion of the human placental lobule to test the hypothesis that the VEGF vasodilatory effect on the fetoplacental circulation is altered in PE and examined how vascular responsiveness relates to circulating levels of free VEGF in fetal sera in this disease. Umbilical cord sera and fetal venous perfusate concentrations of free VEGF from pregnancies complicated with PE were significantly lower compared to the normal group (P<0.05 and P<0.01, respectively). There was elevated in vitro placental release of the sequestrating soluble receptor, sVEGFR-1, into the fetal-side perfusate with PE compared to the normal group (P<0.05). The umbilical sera PlGF-1 level was higher by an order of magnitude in the fetal circulation in PE compared to normal pregnancy (P<0.0001), with the placenta appearing to contribute appreciably to these levels. Placental net contribution to maternal systemic free VEGF levels appeared to be negligible in both groups. sVEGFR-1 levels were elevated in the maternal-side venous perfusate with PE compared to the normal pregnancy (P<0.01). Perfused lobules from PE pregnancy exhibited an enhanced fetoplacental vasodilatory response to exogenous VEGF (P<0.001), with a longer recovery time (P<0.05), compared to the normal control group. Extrapolation of our combined functional and biochemical data suggests that a decrease in the in vivo circulating levels of free VEGF in PE is likely to contribute to compromised fetoplacental vascular patency in this disease.

Vasoactive and permeability effects of vascular endothelial growth factor-165 in the term in vitro dually perfused human placental lobule.

Vascular endothelial growth factor (VEGF) is an important vasodilator and effector of permeability in systemic blood vessels. Molecular and tissue culture techniques have provided evidence for its placental synthesis and release. Using an in vitro dual-perfusion model of the term placental lobule from normal pregnancy, we report here the relative secretion of total VEGF, soluble VEGF receptor (VEGFR)-1, and free VEGF into the maternal and fetoplacental circulations of the placenta. We tested the hypothesis that VEGF has vasomotor and permeability effects in the fetoplacental circulation of the human placenta, and we examined the broad intracellular pathways involved in the vasodilatory effect that we found. We show that total VEGF is released into the fetal and maternal circulations in a bipolar fashion, with a bias toward maternal side output. Soluble VEGFR-1 was also secreted into both circulations with bias toward the maternal side. Consequently, free VEGF (12.8 +/- 2.4 pg/ml, mean +/- se) was found only in the fetoplacental circulation. VEGF-165 was found to be a potent vasodilator of the fetoplacental circulation (maximum response: 77% of previous steady-state fetal-side inflow hydrostatic pressure after preconstriction with U46619; EC(50) = 71 pm). This vasodilatory effect was mediated by the VEGFR-2 receptor and nitric oxide in a manner-independent of the involvement of prostacyclin and the src-family tyrosine kinases. However, nitric oxide could explain only 50% of the vasodilatory effect. Finally, we measured the permeability of the perfused placenta to inert hydrophilic tracers and found no difference in the presence and absence of VEGF.

Patterns of care and survival for patients aged under 40 years with bone sarcoma in Britain, 1980-1994.

The purpose of the study was to calculate population-based survival rates for osteosarcoma (OS) and Ewing's sarcoma (ES) in Great Britain during 1980-1994, determine proportions of patients treated at specialist centres or entered in national and international clinical trials, and investigate effects of these factors on survival. Data on a population-based series of 1349 patients with OS and 849 with ES were compiled from regional and national cancer registries, UK Children's Cancer Study Group, regional bone tumour registries and clinical trials. Follow-up was through population registers. Survival was analysed by actuarial analysis with log-rank tests and by Cox's proportional hazards analysis. Five-year survival rates during 1980-1984, 1985-1989 and 1990-1994 were 42% (95% CI: 37, 46), 54% (95% CI: 50, 59) and 53% (95% CI: 48, 57), respectively, for OS and 31% (95% CI: 26, 37), 46% (95% CI: 40, 51) and 51% (95% CI: 45, 57) for ES. Proportions of patients treated at a supraregional bone tumour centre or a paediatric oncology centre in the three quinquennia were 36, 56 and 67% for OS and 41, 60 and 69% for ES. In 1983-1992, 48% of OS patients were entered in a national trial; for ES, 27% were entered in 1980-1986 and 54% in 1987-1994. Survival was similar for trial and nontrial patients with OS. For ES, trial patients had consistently higher 5-year survival than nontrial patients: 1980-1986, 42 vs 30%; 1987-1992, 59 vs 42%; 1993-1994, 54 vs 43%. During 1985-1994, patients with OS or ES whose main treatment centre was a nonteaching hospital had lower survival rates. In multivariate analyses of patients diagnosed during 1985-1994 that also included age, sex, primary site, surgical treatment centre, the results relating to main treatment centre for both OS and ES retained significance but the survival advantage of trial entry for ES became nonsignificant. For both OS and ES diagnosed since 1985, patients whose main treatment centre was a nonspecialist hospital had a lower survival rate.

Regulation of transplacental water transfer: the role of fetoplacental venous tone.

We used the in vitro dually perfused human placental lobule to test the hypothesis that known vasoconstrictors of the fetal placental circulation, angiotensin II and the thromboxane mimetic U46619 could induce fetomaternal water transfer. Secondly, we used a combination of vasoconstrictor and mechanically induced increases in fetal placental circulatory pressure to examine the role of the venous system in this context. Fetal-side administration of angiotensin II (A-II) and U46619 (n=6 and n=9, for A-II and U46619, respectively) induced dose dependent, recoverable elevations in fetal inflow hydrostatic pressure (HP; A-II: maximum contractility=83 mmHg, EC50=22.0 nM; U46619: maximum contractility was not achieved, but exceeded the A-II effect) and loss of perfusate from the fetal side (A-II: EC50=70.2 nM, maximum fetal-side solvent loss=1906 microl/min; U46619: maximum fetal-side solvent loss was not achieved, but exceeded the A-II effect). Fetal-side solvent loss, for both agonists, was correlated linearly with fetomaternal inflow HP (FMIHP) in a biphasic manner (between 0 and 30 mmHg the slopes (+/-S.E.) were 6.4+/-2.2 and 17.1+/-5.8 microl/(min mmHg) for A-II and U46619, respectively; between 30 and 70 mmHg the slopes (+/-S.E.) were 35.6+/-6.5 and 43.7+/-15.9 microl/(min mmHg) for A-II and U46619, respectively). Increasing fetal-side lumenal pressure (n=3) by raising the fetal outflow catheter caused a loss of perfusate from the fetal side which was reduced in the presence of U46619 (fetal solvent loss per unit increase in fetal-side inflow HP: slopes were 1.198+/-0.123 and 0.783+/-0.085 microl/(min mmHg mmHg), respectively). Notwithstanding the possibility of fetoplacental arterial constriction, we conclude that vasoconstrictive agonists in the fetoplacental circulation affect venous resistance, causing fetomaternal fluid loss. These observations could be relevant to the oligohydramnios associated with intrauterine growth restriction, a condition associated with increased resistance in the umbilical circulation.

Neurokinin B is a paracrine vasodilator in the human fetal placental circulation.

Neurokinin (NK) B is a member of the tachykinin family of neurotransmitters, exerting hypotensive or hypertensive effects in the mammalian vasculature through synaptic release from peripheral neurons, according to either NK(1) and NK(2) or NK(3) receptor subtype expression, respectively. There is recent evidence that NKB is expressed by the syncytiotrophoblast of the human placenta, an organ that is not innervated. We hypothesized that NKB is a paracrine modulator of tone in the fetal placental circulation. We tested this hypothesis using the in vitro perfused human placental cotyledon. Our data show that NKB is a dilator of the fetal vasculature, causing a maximal 25.1 +/- 4.5% (mean +/- SEM; n = 5) decrease in fetal-side arterial hydrostatic pressure (5- microM NKB bolus; effective concentration in the circulation, 1.89 nM) after preconstriction with U-46619. RT-PCR demonstrated the presence of mRNA for NK(1) and NK(2) tachykinin receptors in the placenta. Using selective receptor antagonists, we found that NKB-induced vasodilation is through the NK(1) receptor subtype. We found no evidence for the involvement of either nitric oxide or prostacyclin in this response. This study demonstrates a paracrine role for NKB in the regulation of fetal placental vascular tone.

Expression of BCL-2, BAX and BAK in the trophoblast layer of the term human placenta: a unique model of apoptosis within a syncytium.

The regulation of apoptosis in the syncytiotrophoblast is of particular interest because this is the only true syncytial epithelium in human cell biology. Nuclei characteristic of apoptotic cells have been localized to this syncytium especially in association with fibrin-containing fibrinoid deposits. The factors responsible for regulating cell death-like features in the trophoblast syncytium are unknown. We tested the hypothesis that fibrin was required for trophoblast apoptosis. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP end-labelling) staining to detect DNA fragmentation typical of apoptosis was performed in term human placentae revealing labelled nuclei associated with fibrin-type fibrinoid, as well as labelled nuclei in discrete areas of syncytiotrophoblast without fibrin. We also hypothesized that members of the BCL-2 family of apoptosis-associated proteins contribute to the regulation of syncytiotrophoblast apoptosis. To identify members of this protein family that might regulate trophoblast apoptosis, we assessed expression of three important members of the bcl-2 gene family. We used immunohistochemistry with monoclonal antisera against human BCL-2 and polyclonal antisera against human BAX and BAK to study paraffin-embedded sections of human term placentae (n=5) from uncomplicated pregnancies. The anti-apoptotic BCL-2 protein was expressed throughout the syncytium of normal villi with much less staining in cytotrophoblast. Staining was also seen adjacent to fibrin deposits and in syncytium overlying fibrin deposits. Expression of the pro-apoptotic BAX protein was undetectable in the syncytiotrophoblast, was expressed in rare cytotrophoblast and was prominent in connective tissue and perivascular cells within the villous core. Localization of a second pro-apoptotic protein, BAK, revealed immunoreactivity in isolated areas of intact syncytium of normal villi. Additionally, fibrin deposits were associated with intense BAK staining in both syncytiotrophoblast and cytotrophoblast. From these data, we speculate that modulation of BAK expression is one factor regulating apoptosis in human trophoblast.

Denudations as paracellular routes for alphafetoprotein and creatinine across the human syncytiotrophoblast.

We tested two hypotheses: 1) that fibrin-containing fibrinoid-filled denudations of the syncytiotrophoblast may provide a route for paracellular diffusion and 2) that placentas from women who had elevated maternal serum alphafetoprotein (MSAFP) in midgestation had raised permeability to AFP and greater denudation than in normal pregnancy. We measured AFP and creatinine clearance across term placental cotyledons from the above groups and used light microscope morphometric analysis to determine the volume density of fibrin-containing fibrinoid deposits. There was no significant difference between the two groups in terms of AFP and creatinine clearance or volume density of fibrin-containing fibrinoid deposits. The combined data showed a significant (P < 0.05) positive correlation between creatinine clearance, but not AFP clearance, and volume density of fibrin-containing fibrinoid. We conclude that syncytiotrophoblast denudations, with associated fibrinoid, do provide a route for diffusion of small hydrophilic solutes, but that other anatomic features of the placenta are rate limiting for transfer of AFP and similarly sized molecules.

Case-control studies of relation between childhood cancer and neonatal vitamin K administration.

OBJECTIVE: To investigate the possible link between neonatal administration of intramuscular vitamin K and childhood cancer. DESIGN: Matched case-control study. SETTING: Selected large maternity units in England and Wales. SUBJECTS: Children with cancer born 1968-85, diagnosed 1969-86; controls matched for sex, month of birth, and hospital of birth. MAIN EXPOSURE MEASURES: Neonatal administration of vitamin K, length of gestation, birth weight, type of delivery, admission to special care baby unit. RESULTS: After exclusion of cases with missing notes or unknown hospital vitamin K policy, 597 cases and matched controls were studied. Written records on the use of vitamin K were available for only about 40% of these, and to avoid possible bias from selective recording it was assumed that the stated hospital policy was followed. The association between cancer generally and intramuscular vitamin K was of borderline significance (odds ratio 1.44, P = 0.05); the association was strongest for leukaemia. There was, however, also an effect of abnormal delivery, which could explain some of the findings. CONCLUSIONS: The lack of consistency between the various studies so far published, including this one, and the low relative risks found in most of them suggest that the risk, if any, attributable to the use of vitamin K cannot be large, but the possibility that there is some risk cannot be excluded. A comparison of the predicted consequences of various policies shows that even a 10% increase would imply that prophylaxis using the commonly recommended 1 mg intramuscular dose should be restricted to babies at particularly high risk of vitamin K deficiency bleeding; alternatively a lower dose might be given to a larger proportion of those at risk.

Ecological studies of relation between hospital policies on neonatal vitamin K administration and subsequent occurrence of childhood cancer.

OBJECTIVE: To investigate the possible link between neonatal administration of intramuscular vitamin K and childhood cancer. DESIGN: Ecological studies comparing incidence of cancer in groups of children classified by the vitamin K policy in operation at their hospital of birth. SETTING: Selected large maternity units in England, Scotland, and Wales. SUBJECTS: Children born in these units in varying periods between 1966 and 1991. MAIN OUTCOME MEASURES: Cancer occurring among these children before age 15 years identified by using the National Registry of Childhood Tumours. Ratios of observed to expected numbers of these conditions calculated for hospitals where the policy was to give all babies intramuscular vitamin K (non-selective) and where the policy was to use this treatment only for a selected minority of babies at increased risk of vitamin K deficiency bleeding (selective). RESULTS: These ratios were calculated for children born in 94 hospitals with varying vitamin K policies. A raised risk was occasionally associated with vitamin K, but the overall results were not significant, and there was no evidence to support the previously suggested doubling of the risk of childhood cancer. CONCLUSIONS: On the basis of the results reported here it is unlikely that there is a greatly increased risk of childhood cancer attributable to intramuscular vitamin K given to newborns, if indeed there is any.