Control Placentas Research Articles

Placental infiltration of inflammatory markers in gestational diabetic women

Gestational diabetes mellitus (GDM) is pathology of glucose intolerance during pregnancy. It is influenced by maternal hyperglycemia and insulinemia through placental circulation. The study was undertaken to investigate the implication of pro-inflammatory factors in the placenta of GDM women. Thirty GDM women have delivered macrosomic babies, and 30 healthy age-matched pregnant women have delivered non macrosomic babies, were recruited in the study. The mRNAs encoding for IL-6, TLR4, TGF-β, CD68, CD14, EMR-1, CCL2, TCR-α, T-bet, GATA-3, leptin and adiponectin were quantified in placental samples by using RT-qPCR. The mRNA expression of the pro-inflammatory factors, i.e., IL-6, TLR4 and TGF-β, was increased in GDM placenta. The mRNA expression of markers of infiltration of macrophage, i.e., CD68, CD14 and EMR-1, was higher in the GDM placenta than the control placenta. The expression of mRNA of TCR-α, an indicator of T-cell infiltration, was significantly higher in the GDM placenta. Interestingly, the expression of mRNA of GATA-3, an indicator of Th2 phenotype differentiation, was unregulated in the GDM placenta. Leptin and adiponectin mRNAs were also significantly increased in the placenta of the GDM group. Our results revealed that there is an increase of inflammation in the GDM placenta which might be involved, in part, in the pathogenesis of macrosomia.

Trophoblast calcyclin is elevated in placental tissue from patients with early pre-eclampsia.

The aetiology of pre-eclampsia is thought to originate from aberrant spiral artery remodelling and invasion evoking cellular oxidative stress. Previously, we discovered differentially expressed proteins in trophoblast cells of pre-eclamptic pregnancies. One of these proteins is calcyclin (S100A6); a Ca(2+)-binding protein associated with cellular stress response. By immunohistochemistry on formalin-fixed paraffin-embedded placental tissue, calcyclin expression was compared between women with early pre-eclampsia (n=72) and non-hypertensive control patients (n=66) (χ(2), p=0.006) blindly by two observers. Significantly more intense staining was seen in trophoblast cells of pre-eclamptic pregnancies compared to control placentas suggesting that trophoblast calcyclin is elevated in early pregnancy.

Differential expression of trophoblast-, endothelial- and embryonic-stem-cell-associated transcription factors in 1st trimester, and 3rd trimester preeclampsia (PE), intrauterine growth factor (IUGR) and control placentae

Stem cells are highly proliferative, undifferentiated cells and stem cell markers are involved in maintaining these characteristics. In the blastocyst, differential expression between embryonic and trophoblast stem cell transcription factors is thought to discriminate cell fate. Although villous cytotrophoblasts are functionally considered a trophoblast stem cell or at least a progenitor cell, which replenishes the outer syncytiotrophoblast layer when necessary, little data is available characterizing the expression of stem cell transcription factors beyond the blastocyst stage. We aimed to describe the expression of trophoblast and embryonal stem cell factors in the placenta between 1st and 3 rd trimester in order to discriminate if these markers might be involved in progenitor cell functions.

Peripheral Insertion of Umbilical Cord

Numerous studies have addressed the significance of marginal and membranous umbilical cord (UC) insertion. Recent reports suggest that an eccentrically inserted UC may also be important. This case-control study assessed the potential relevance of peripheral insertion of UC (PIUC), defined as <3 cm from the nearest margin. Singleton placentas (n = 1418) submitted to the pathology department over an 18-month period were analyzed. Each case of PIUC (n = 119) was matched with a control placenta of the same gestational age. Placentas with marginal or membranous UC and multiple gestations were excluded. The overall prevalence of PIUC was 8.4%, but PIUC frequency was significantly increased in premature births at <28 weeks (21.4%, P < 0.001). There was no association with other adverse pregnancy outcomes. PIUC was associated with decreased placental weight Z-score (-0.69 ± 0.92 versus -0.22 ± 1.3, P = 0.0056), but not fetal weight Z-score, suggesting increased utilization of placental reserve. PIUC was also associated with relatively elongated placentas (length minus width: 2.6 ± 3.2 versus 1.0 ± 3.1, P = 0.006). PIUC tended to be more frequent in young primiparous mothers and was significantly less common in women with a history of prior curettage (66% vs 50%, P = 0.013). These data, together with equivalent rates of prior cesarean section, multiparity, and advanced maternal age, support a primary developmental disorder as opposed to secondary placental migration due to underlying uterine abnormalities ("trophotropism"). Except for a borderline significant association with findings suggestive of maternal malperfusion (P = 0.078), PIUC was not associated with other placental lesions.

Comparative Proteome Profile of Human Placenta from Normal and Preeclamptic Pregnancies

To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used LC-MS/MS to construct a large-scale comparative proteome profile of human placentas from normal and preeclamptic pregnancies. A total of 2636 proteins were detected in human placentas, and 171 different proteins were definitively identified between control and preeclamptic placentas. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes which occur during pathological changes of preeclamptic placenta. 6 proteins were randomly selected to verify their expression patterns with Western blotting. Of which, 3 proteins’ cellular localizations were validated with immunohistochemistry. Elucidation of how protein-expression changes coordinate the pathological development would provide researchers with a better understanding of the critical biological processes of preeclampsia and potential targets for therapeutic agents to regulate placenta function, and eventually benefit the treatment of preeclampsia.

Function of Survivin in Trophoblastic Cells of the Placenta

BackgroundPreeclampsia is one of the leading causes of maternal and perinatal mortality and morbidity worldwide and its pathogenesis is not totally understood. As a member of the chromosomal passenger complex and an inhibitor of apoptosis, survivin is a well-characterized oncoprotein. Its roles in trophoblastic cells remain to be defined.MethodsThe placental samples from 16 preeclampsia patients and 16 well-matched controls were included in this study. Real-time PCR, immunohistochemistry and Western blot analysis were carried out with placental tissues. Primary trophoblastic cells from term placentas were isolated for Western blot analysis. Cell proliferation, cell cycle analysis and immunofluorescence staining were performed in trophoblastic cell lines BeWo, JAR and HTR-8/SVneo.ResultsThe survivin gene is reduced but the protein amount is hardly changed in preeclamptic placentas, compared to control placentas. Upon stress, survivin in trophoblastic cells is phosphorylated on its residue serine 20 by protein kinase A and becomes stabilized, accompanied by increased heat shock protein 90. Depletion of survivin induces chromosome misalignment, abnormal centrosome integrity, and reduced localization and activity of Aurora B at the centromeres/kinetochores in trophoblastic metaphase cells.ConclusionsOur data indicate that survivin plays pivotal roles in cell survival and proliferation of trophoblastic cells. Further investigations are required to define the function of survivin in each cell type of the placenta in the context of proliferation, differentiation, apoptosis, angiogenesis, migration and invasion.

The majority of variance in birthweight relative to placental weight (as beta=ln(PW)/ln(BW) is accounted for by detailed novel placental measures: implications for fetal origins work

s / Placenta 34 (2013) A1–A99 A14 structural complexity (Figure 2) Microscopic review confirmed differences in the numbers, sizes and shapes of villi (Figure 3 ASD, Figure 4 control). Conclusions: These preliminary data represent our second test of distal villi from ASD and control placentas. However, these subjective assessments must be replaced by quantitative methods. Additional methods to enhance structural detail include tagged cytokeratin and endothelial cell antibodies; analytic methods include fractal analysis, radon transformation and 2D Fourier analysis. These observations appear to be gender independent. Tentatively, these findings support an hypothesis based on angiogenesis driving neurogenesis that relates vascular structure to ASD, a disorder of neuronal connectivity.

Maternal uterine secreted mediator, VEGF, enhances embryo development and implantation

Maternal uterine secreted mediator, VEGF, enhances embryo development and implantation

Placental ABCA1 and ABCG1 expression in gestational disease: Pre-eclampsia affects ABCA1 levels in syncytiotrophoblasts

IntroductionTransplacental feto-maternal lipid exchange through the ATP-binding cassette transporters ABCA1 and ABCG1 is important for normal fetal development. However, only scarce and conflicting data exist on the involvement of these transporters in gestational disease. MethodsPlacenta samples (n = 72) derived from common gestational diseases, including pre-eclampsia (PE), HELLP, intrauterine growth restriction (IUGR), intrahepatic cholestasis of pregnancy and gestational diabetes, were assessed for their ABCA1 and ABCG1 expression levels and compared to age-matched control placentas with qRT-PCR and immunohistochemistry. ABCA1 expression was additionally investigated with immunoblot in placental membrane vesicles. Furthermore, placental cholesterol and phospholipid contents were assessed. ResultsABCA1 mRNA levels differed significantly between preterm and term control placentas (p = 0.0013). They were down-regulated in isolated PE and PE with IUGR (p = 0.0006 and p = 0.0012, respectively), but unchanged in isolated IUGR, isolated HELLP and other gestational diseases compared to gestational age-matched controls. Correspondingly, in PE, ABCA1 protein expression was significantly reduced in the apical membrane of the villous syncytiotrophoblast (p = 0.011) and in villous fetal endothelial cells (p = 0.036). Furthermore, in PE there was a significant increase in the placental content of total and individual classes of phospholipids which were partially correlated with diminished ABCA1 expression. Conversely, ABCG1 mRNA and protein levels were stable in the investigated conditions. ConclusionsIn gestational disease, there is a specific down-regulation of placental ABCA1 expression at sites of feto-maternal lipid exchange in PE. At a functional level, the increase in placental lipid concentrations provides indirect evidence of an impaired transport capacity of ABCA1 in this disease.

Preeclampsia Is Associated With the Presence of Transcriptionally Active Placental Fragments in the Maternal Lung

Preeclampsia is associated with increased levels of the circulating antiangiogenic factor sFlt-1 and with an excessive shedding of placenta-derived multinucleated syncytial aggregates into the maternal circulation. However, it remains unclear whether these aggregates are transcriptionally active in the maternal organs and can, therefore, contribute to the systemic manifestations of preeclampsia. In this study, we measured placental soluble fms-like tyrosine kinase-1 (sFlt-1) mRNA levels in preeclamptic- and control placentas and performed RNA in situ hybridization to localize the main placental expression site of sFlt-1 mRNA. Because the maternal lung is the first capillary bed that circulating syncytial aggregates traverse, we studied the presence and persistence of placental material in lungs of preeclamptic and control subjects. To confirm the placental origin of these aggregates in maternal lungs, immunohistochemistry for the placenta-specific marker hCG (human chorionic ghonadotropin) and Y chromosome in situ hybridization were performed. Using human placental tissue, we found that syncytial knots are the principal site of expression of the antiangiogenic factor sFlt-1. In addition, autopsy material obtained from women with preeclampsia (n=9) showed significantly more placenta-derived syncytial aggregates in the lungs than in control subjects (n=26). Importantly, these aggregates still contained the antiangiogenic factor sFlt-1 after their entrapment in the maternal lungs. The current study confirms the important role of syncytial knots in placental sFlt-1 mRNA production. In addition, it shows a significant association between preeclampsia and larger quantities of sFlt-1 containing syncytial aggregates in maternal lungs, suggesting that the transfer of syncytial aggregates to the maternal compartment may contribute to the systemic endothelial dysfunction that characterizes preeclampsia.

Reversal of gene dysregulation in cultured cytotrophoblasts reveals possible causes of preeclampsia.

During human pregnancy, a subset of placental cytotrophoblasts (CTBs) differentiates into cells that aggressively invade the uterus and its vasculature, anchoring the progeny and rerouting maternal blood to the placenta. In preeclampsia (PE), CTB invasion is limited, reducing placental perfusion and/or creating intermittent flow. This syndrome, affecting 4%-8% of pregnancies, entails maternal vascular alterations (e.g., high blood pressure, proteinuria, and edema) and, in some patients, fetal growth restriction. The only cure is removal of the faulty placenta, i.e., delivery. Previously, we showed that defective CTB differentiation contributes to the placental component of PE, but the causes were unknown. Here, we cultured CTBs isolated from PE and control placentas for 48 hours, enabling differentiation and invasion. In various severe forms of PE, transcriptomics revealed common aberrations in CTB gene expression immediately after isolation, including upregulation of SEMA3B, which resolved in culture. The addition of SEMA3B to normal CTBs inhibited invasion and recreated aspects of the PE phenotype. Additionally, SEMA3B downregulated VEGF signaling through the PI3K/AKT and GSK3 pathways, effects that were observed in PE CTBs. We propose that, in severe PE, the in vivo environment dysregulates CTB gene expression; the autocrine actions of the upregulated molecules (including SEMA3B) impair CTB differentiation, invasion and signaling; and patient-specific factors determine the signs.

Differential expression of trophoblast-, endothelial- and embryonic-stem-cell-associated transcription factors in 1st trimester, and 3 rd trimester preeclampsia (PE), intrauterine growth factor (IUGR) and control placentae

Objectives: Stem cells are highly proliferative, undifferentiated cells and stem cell-associated markers (SCM) are involved in maintaining these characteristics. Trophoblast progenitor cells are known to express these markers, and are cells which replenish the outer syncytiotrophoblast layer when necessary. Little data is available characterizing the expression of SCMs beyond the blastocyst stage or during pregnancy pathologies. We aimed to describe the expression of trophoblast and other SCMs in the placenta of 1st and 3rd trimester, but also of preeclampsia (PE) and intrauterine growth retardation (IUGR) pregnancies in order to discriminate if these markers might be involved in progenitor cell functions.

SNAT2 expression and regulation in human growth-restricted placentas

Amino acid placental delivery is reduced in human intrauterine growth-restricted (IUGR) fetuses, and the activity of placental amino transporters has been consistently shown to be decreased in in vitro studies. We hypothesized lower placental expression and localization of sodium-coupled neutral amino acid transporter 2 (SNAT2 (also known as SLC38A2)), altered levels of intron-1 methylation, and altered distribution of single-nucleotide polymorphisms in human IUGR vs. normal pregnancies. We studied 88 IUGR and 84 control placentas from singleton pregnancies at elective caesarean section. SNAT2 expression was investigated by real-time PCR and immunohistochemistry. Intron-1 methylation levels were analyzed by pyrosequencing, and single-nucleotide polymorphism distribution was analyzed by allelic discrimination. mRNA levels were significantly decreased in IUGR placentas with reduced umbilical blood flows. Syncytiotrophoblast immunostaining was lower in IUGR placentas than in control placentas. Methylation levels were steadily low in both IUGR and control placentas. SNP genotype and allele frequencies did not differ between the two groups. This is the first study investigating SNAT2 expression and regulation mechanisms in human IUGR placentas. We confirm previous results obtained in rats and cell cultures that support the fundamental role of SNAT2 in fetal growth and well-being, as well as a possible role of oxygen levels in regulating SNAT2 expression, indicating the relevance of hypoxia in IUGR.

Mild Gestational Hyperglycemia in Rat Induces Fetal Overgrowth and Modulates Placental Growth Factors and Nutrient Transporters Expression

Mild gestational hyperglycemia is often associated with fetal overgrowth that can predispose the offspring to metabolic diseases later in life. We hypothesized that unfavorable intrauterine environment may compromise the development of placenta and contribute to fetal overgrowth. Therefore, we developed a rat model and investigated the effects of maternal dysglycemia on fetal growth and placental gene expression. Female rats were treated with single injection of nicotinamide plus streptozotocin (N-STZ) 1-week before mating and were studied at gestational day 21. N-STZ pregnant females displayed impaired glucose tolerance that is associated with a lower insulin secretion. Moderate hyperglycemia induced fetal overgrowth in 40% of newborns, from pregnancies with 10 to 14 pups. The incidence of macrosomia was less than 5% in the N-STZ pregnancies when the litter size exceeds 15 newborns. We found that placental mass and the labyrinthine layer were increased in macrosomic placentas. The expression of genes involved in placental development and nutrient transfer was down regulated in the N-STZ placentas of macrosomic and normosomic pups from pregnancies with 10 to 14 ones. However, we observed that lipoprotein lipase 1 (LPL1) gene expression was significantly increased in the N-STZ placentas of macrosomic pups. In pregnancies with 15 pups or more, the expression of IGFs and glucose transporter genes was also modulated in the control placentas with no additional effect in the N-STZ ones. These data suggest that placental gene expression is modulated by gestational conditions that might disrupt the fetal growth. We described here a new model of maternal glucose intolerance that results in fetal overgrowth. We proposed that over-expression of LPL1 in the placenta may contribute to the increased fetal growth in the N-STZ pregnancies. N-STZ model offers the opportunity to determinate whether these neonatal outcomes may contribute to developmental programming of metabolic diseases in adulthood.

Trophoblast Inclusions Are Significantly Increased in the Placentas of Children in Families at Risk for Autism

Gestation is a critical window for neurodevelopmental vulnerability. This study examined whether the presence of trophoblast inclusions (TIs) in the placenta could serve as a predictor for children at elevated risk for autism spectrum disorder (ASD). Placentas were obtained from 117 births in the MARBLES (Markers of Autism Risk in Babies-Learning Early Signs) cohort of families who have one or more previous biological children with ASD, placing their newborn at elevated risk for neurodevelopmental compromise. Control samples were obtained from 100 uncomplicated term pregnancies of multiparous women with one or more typically developing biological children. Frequency of TIs was compared across the two groups. Placentas from at-risk pregnancies had an eightfold increased odds of having two or more TIs compared with control samples (odds ratio: 8.0, 95% confidence interval: 3.6-18.0). The presence of≥2 TIs yielded a sensitivity of 41% and a specificity of 92% for predicting ASD risk status, whereas≥4 TIs yielded a sensitivity of 19%, a specificity of 99.9%, and a positive likelihood ratio of 242 and conservatively predicted an infant with a 74% probability of being at risk for ASD. Our findings suggest that the placentas from women whose fetuses are at elevated risk for autism are markedly different from control placentas. These differences are manifested histologically as TIs. Their identification has the possibility of identifying newborns at risk for ASD who might benefit from targeted early interventions aimed at preventing or ameliorating behavioral symptoms and optimizing developmental outcomes.

OP005. Preeclampsia is associated with the presence of transcriptionally active placental fragments in the maternal lung

Preeclampsia is associated with increased levels of the circulating anti-angiogenic factor sFlt-1 as well as with an excessive shedding of placenta-derived multinucleated syncytial aggregates into the maternal circulation. However, it remains unclear whether these aggregates are transcriptionally active in the maternal organs and can therefore contribute to the systemic manifestations of preeclampsia. We hypothesized that in preeclampsia, syncytial knots are the primary placental site of sFlt-1 production and that increased numbers of sFlt-1-containing syncytial aggregates are retained in the maternal lungs. In this study, we measured placental sFlt-1 mRNA levels in preeclamptic- and control placentas and performed RNA in situ hybridization to localize the main placental expression site of sFlt-1 mRNA. Because the maternal lung is the first capillary bed that circulating syncytial aggregates traverse, we studied the presence and persistence of placental material in lungs of preeclamptic women and control subjects. To confirm the placental origin of suspected syncytial aggregates in these lungs, immunohistochemistry for the placenta-specific marker hCG and Y-chromosome in situ hybridization were performed. Using human placental tissue, we found that syncytial knots are the principal site of expression of the anti-angiogenic factor sFlt-1. In addition, in autopsy material obtained from women with preeclampsia (n=9), we observed significantly more placenta-derived syncytial aggregates in the lungs than in control subjects (n=26). Importantly, these placental aggregates still contained the anti-angiogenic factor sFlt-1 following their entrapment in the maternal lungs. The current study confirms the important role of syncytial knots in placental sFlt-1 mRNA production. Additionally, it shows a significant association between preeclampsia and larger quantities of sFlt-1 containing syncytial aggregates in maternal lungs, suggesting that the transfer of syncytial aggregates to the maternal compartment may contribute to the systemic endothelial dysfunction that characterizes preeclampsia.

Fetal exposure to high levels of corticosterone in a low birth weight rat model

Fetal exposure to maternal steroids is suggested as a potential mechanism contributing to the developmental programming of adult CVD risk. 14‐day timed pregnant SD Dams were used for the RUPP while matched control SD females were subjected to sham surgeries. Corticosterone levels in Dam plasma were significantly higher in RUPP compared to E19 Control Dams. At P14, the corticosterone levels remained significantly higher in the RUPP compared to the Control Dams. Pups were sacrificed at E19, E20, and P14. E19 and E20 fetuses showed significant decrease in weight compared to the control fetuses. P14 IUGR showed significantly decreased weight compared to control pups. E19 and E20 placental weights significantly decreased in the RUPP compared to the control group. E19 and E20 placental efficiency significantly increased in the RUPP compared to control fetuses. No significant difference in plasma corticosterone between the E19, E20, and P14 IUGR compared to the Control groups. qPCR showed increased 11betaHSD2 message at E20 compared to Control placentas. Western blot analysis with kidney homogenates indicated significantly increased 11betaHSD2 in the P14 RUPP compared to Control Dams. The preliminary data suggests that while there is significant increase in plasma corticosterone in the RUPP compared to the Control Dams, the corticosterone is not crossing the placenta. The indication is that 11betaHSD2 may be part of the fetal protective mechanism; however, not the major mechanism.

Placental protein tyrosine nitration and MAPK in type 1 diabetic pre-eclampsia: Impact of antioxidant vitamin supplementation

AimTo examine the role of placental protein tyrosine nitration and p38-Mitogen-Activated Protein Kinase α (p38-MAPKα), Extra Cellular-Signal Regulated Kinase (ERK) and c-Jun NH2-Terminal Kinase (JNK) activity, in the pathogenesis of type 1 diabetic pre-eclampsia, and the putative modulation of these indices by maternal vitamin C and E supplementation. MethodsPlacental samples were obtained from a sub-cohort of the DAPIT trial: a randomised placebo-controlled trial of antioxidant supplementation to reduce pre-eclampsia in type 1 diabetic pregnancy. Placenta from placebo-treated: normotensive (NT) [n=17], gestational hypertension (GH) [n=7] and pre-eclampsia (PE) [n=6] and vitamin-treated: NT (n=20), GH (n=4) and PE (n=3) was analysed. Protein tyrosine nitration was assessed by immunohistochemistry in paraffin-embedded tissue. Catalytic activities of placental p38-MAPKα, ERK and JNK were measured by enzyme-linked immunosorbent assay (ELISA). ResultsNitrotyrosine immunostaining was present in placebo-treated NT, GH and PE placentae, with no significant difference observed between the groups. There was a non-significant trend towards decreased p38-MAPKα activity in PE vs NT control placentae. ERK and JNK were similar among the three outcome placebo groups and vitamin supplementation did not significantly alter their activity. ConclusionNitrotyrosine immunopositivity in normotensive diabetic placentae indicates some degree of tyrosine nitration in uncomplicated diabetic pregnancy, possibly due to inherent oxidative stress and peroxynitrite production. Our results suggest that p38-MAPKα, ERK and JNK are not directly involved in the pathogenesis of type 1 diabetic pre-eclampsia and are not modulated by vitamin-supplementation.

The proprotein convertase furin is required for trophoblast syncytialization

The multinucleated syncytial trophoblast, which forms the outermost layer of the placenta and serves multiple functions, is differentiated from and maintained by cytotrophoblast cell fusion. Deficiencies in syncytial trophoblast differentiation or maintenance likely contribute to intrauterine growth restriction and pre-eclampsia, two common gestational diseases. The cellular and molecular mechanisms governing trophoblast syncytialization are poorly understood. We report here that the proprotein convertase furin is highly expressed in syncytial trophoblast in the first trimester human placentas, and expression of furin in the syncytiotrophoblast is significantly lower in the placentas from pre-eclamptic patients as compared with their gestational age-matched control placentas. Using multiple experimental models including induced fusion of choriocarcinoma BeWo cells and spontaneous fusion of primary cultured cytotrophoblast cells or placental explants, we demonstrate that cytotrophoblast cell fusion and syncytialization are accompanied by furin expression. Furin-specific siRNAs or inhibitors inhibit cell fusion in BeWo cells, as well as trophoblast syncytialization in human placental explants. Furthermore, type 1 IGF receptor (IGF1R) is indicated in this study as a substrate of furin, and processing of IGF1R by furin is an essential mechanism for syncytialization. Finally, using lentivirus-mediated RNAi targeting to mouse trophectoderm, we demonstrate that furin function is required for the development of syncytiotrophoblast structure in the labyrinth layer, as well as for normal embryonic development.

Association of placenta organotin concentrations with congenital cryptorchidism and reproductive hormone levels in 280 newborn boys from Denmark and Finland

Is the placental burden of organotin compounds (OTCs) associated with congenital cryptorchidism in infant offspring from Finland and Denmark? Increasing concentrations of OTCs had a negative association with cryptorchidism in Finland, whereas a positive association was found in Denmark. The rapid increase in the prevalence of cryptorchidism suggests that environmental factors, such as endocrine disruptors, may be involved. OTCs are endocrine disruptors at very low concentrations due to activation of the retinoid X receptor (RXR). Between the years 1997 and 2001, placentas from mothers of cryptorchid boys and from healthy controls were collected from Denmark (39 cases, 129 controls) and Finland (56 cases, 56 controls). In Denmark 33 and 6 boys, and in Finland 22 and 34 boys had mild or severe cryptorchidism, respectively. The association between concentrations of four OTCs [monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT) and triphenyltin (TPhT)] and case-control status was estimated. In both countries, placenta samples were selected from larger cohorts. In Finland placenta samples were collected from boys with cryptorchidism at birth and matched controls (nested case-control design). Matching criteria were parity, maternal smoking (yes/no), diabetes (yes/no), gestational age (±7 days) and date of birth (±14 days). Numbers of controls per case was 1. In Denmark, all available placentas from cryptorchid boys were chosen and control placentas were selected randomly from the total Danish cohort (case-cohort design). The average number of controls per case was 3.3. OTCs in placenta samples were analysed with liquid extraction, ethylation and gas chromatography-mass spectrometry determination and coded by country-specific tertiles. Generally, the concentrations of OTCs were very low. For most analytes, a large proportion of samples (29-96% depending on the country and case-control status) had OTC concentrations below the limit of quantification (LOQ). As an exception, the concentration of TBT was >LOQ in 99% of Finnish placentas. The mean concentrations of DBT and TBT were 1.5 and 7 times higher in Finland than in Denmark, respectively. For DBT in Danish placentas, the odds ratio (OR) for cryptorchidism in the second tertile (0.10-0.14 ng/g) when compared with the first tertile (<0.10 ng/g, <LOQ) was 3.13 (95% CI 1.19-8.26) and the OR for the third tertile (≥0.15 ng/g) when compared with the first tertile was 4.01 (95% CI 1.42-11.33). For TBT in Finnish placentas, the OR for cryptorchidism in the second tertile (0.10-0.39 ng/g) when compared with the first tertile (<0.1 ng/g) was 0.61 (95% CI 0.18-2.01) and the OR for the third tertile (≥0.40 ng/g) when compared with the first tertile was 0.13 (95% CI 0.03-0.54). The main limitation of the study was the relatively small number of mother-boy pairs that limits the extrapolation of the study results to the general population. Also misclassification of exposure is a reason for caution for two reasons: because the concentrations of most OTCs were below or only barely above the LOQ in a large proportion of samples and because it is not known how well OTCs measured from placenta represent exposure at the time window that is relevant for cryptorchidism occurrence. This is the first study to measure the concentrations of OTCs from human placenta samples, and to associate these concentrations to cryptorchidism. As opposite results were obtained with regard to OTC concentration in placenta and cryptorchidism status in Finland and Denmark, and no mechanism is known at the moment by which OTCs could affect testicular descent, these results cannot be generalized to other populations. However, some animal tests described in the literature show opposite effects of OTCs on fat deposition at different ranges of exposure. It is also clearly shown in the literature that TBT has an impact on sexual development of gastropods through RXR. As TBT is known to activate human RXR, further laboratory studies should be designed to explore the potential impact of TBT on male sexual development.