Study population and sample collection and preparation

The present study on term placentae is executed within the framework of the ENVIRONAGE (ENVIRonmental influence ON AGEing in early life) birth cohort32. The cohort enrolls mothers giving birth in the East-Limburg Hospital (ZOL; Genk, Belgium) and is approved by the Ethics Committee of Hasselt University and East-Limburg Hospital (EudraCT B37120107805). The study is conducted according to the guidelines laid down in the Declaration of Helsinki. All participating women provided informed written consent. Mothers were asked to fill out a questionnaire to get lifestyle information.

The ambient exposure to BC of the mothers was determined, based on their residential address, using a validated spatial and temporal interpolation method19,33. The method uses land cover data obtained from satellite images (CORINE land cover data set) and pollution data of fixed monitoring stations. Coupled with a dispersion model that uses emissions from point sources and line sources, this model chain provides daily exposure values in a high-resolution receptor grid. Overall model performance was evaluated by leave-one-out cross-validation including 16 monitoring points for BC. Validation statistics of the interpolation tool gave a spatiotemporal explained variance of more than 0.74 for BC.

Fresh placentae were collected within 10 min after birth. Biopsies were taken at four standardized sites at the fetal side of the placenta across the middle region, approximately 4 cm away from the umbilical cord and under the chorio-amniotic membrane. The order of the biopsies was clockwise starting at the main blood vessel. Also, one biopsy is taken at the maternal side of the placenta at the equivalent position of biopsy 1 of the fetal side. Accordingly, the biopsies taken at the sides facing towards the fetus and mother are defined as the fetal and maternal side of the placenta, respectively.

The intervariability and intravariability between and within biopsies were assessed using placental tissue from three randomly selected, non-smoking mothers, with average residential BC exposure (between 0.96 and 1.32 µg per m3).

To evaluate the correlation between the BC exposure of mothers (all non-smokers) during pregnancy and accumulation of BC in placentae, 10 mothers with high residential BC exposure and 10 mothers with low residential BC exposure during pregnancy were selected from the ENVIRONAGE biobank. High residential BC exposure during pregnancy was defined as: (i) entire pregnancy and third trimester of pregnancy exposure to residential BC ≥ 75th percentile (1.70 µg per m³ and 2.42 µg per m³, respectively), and (ii) residential proximity to a major road ≤ 500 m. Low residential BC exposure during pregnancy was defined as: (i) entire pregnancy and third trimester of pregnancy exposure to residential BC ≤ 25th percentile (0.96 µg per m³ and 0.63 µg per m³, respectively), and (ii) residential proximity to major road >500 m.

Biopsies of placental tissue from spontaneous preterm births were collected at the East-Limburg Hospital (ZOL; Genk, Belgium). Five biobanked placentae of mothers with spontaneous termination of pregnancy between 12 and 31 weeks of gestation were randomly selected but taking into account the following criteria: (i) non-smoker, (ii) avoiding possible complications that can cause autolysis (mors in utero) or disturb the histological image (infections), and (iii) best possible spread in gestation time, i.e., pregnancy termination at 12, 16, 19, 25, and 31 weeks. The cases were handled strictly anonymously. Accordingly, no personal information is available except for the inclusion and exclusion criteria and, thus, the residential exposure to ambient air pollution is unknown. The use of these tissues for the detection of BC particles was approved by the Ethics Committee of Hasselt University and East-Limburg Hospital (EudraCT B371201938875). Since the employed samples were biobanked, this specific study is not covered by the law of 7 May 2004 on experiments on the human person. Hence, no written consent was needed according to the Ethical Committees. To study the BC loading in the preterm placentae the available biopsy was screened by imaging three regions within five different sections taken in the middle of the tissue (n = 15 images).

Placental biopsies were fixed in formaldehyde for minimal 24 h and paraffin embedded. 4 µm sections were cut using a microtome (Leica Microsystems, UK) and mounted between histological glass slides. To preclude any particulate contamination, particle-free instruments and sample holders were used and all samples were handled in a clean room with filtered air (Genano 310/OY, Finland).

Experimental protocol for BC detection in placentae

BC particles naturally present in the placenta were detected using a specific and sensitive detection technique based on the non-incandescence-related WL generation of the particles under femtosecond illumination as published before19,25. Images of the placental sections were collected at room temperature using a Zeiss LSM 510 (Carl Zeiss, Jena, Germany) equipped with a two-photon femtosecond pulsed laser (810 nm, 150 fs, 80 MHz, MaiTai DeepSee, Spectra-Physics, USA) tuned to a central wavelength of 810 nm with 5 or 10 mW radiant power on average at the sample position using a 10×/0.3 objective (Plan-Neofluar 10×/0.3, Carl Zeiss). WL emission of the BC particles was acquired in the non-descanned mode after spectral separation and emission filtering using 400–410 nm and 450–650 nm BP filters. By employing these two emission filters, the SHG from the placental collagen type I and TPAF of the placental components are collected in the corresponding images. The resulting tile scans had a field of view of 9000 × 9000 µm2 containing 100 images with a pixel size of 0.694 µm and were recorded with a 2.51 µs pixel dwell time. The spatial resolution of the system in the configuration that the measurements were performed (i.e., 10 × /0.3 objective, 810 nm excitation, identical settings): w x = w y = 1.44 µm and w z = 14.8 µm defined as the sizes of the point spread function in the XY-plane (radius of Airy-disk) and along the optical axis (1/e-thickness), respectively. The images were acquired by ZEN Black 2.0 software (Zeiss).

To count the number of BC particles in the tile scans of each placental section, an automated and customized Matlab program (Matlab 2010, Mathworks, The Netherlands) was used. First, a peak-find algorithm detects pixels above a certain threshold value. Here, threshold values of 0.5% and 45% lower than the highest pixel intensity value of the TPAF and SHG image, respectively, were chosen. These thresholds resulted in highly reproducible values, which were checked manually using Fiji (ImageJ v2.0, Open source software, http://fiji.sc/Fiji). Next, the detected pixels of both images are compared and only the matching ones are used to generate the output image and metrics. In addition, the effectively imaged placental area was determined from the TPAF image using Fiji and the focal volume based on the point spread function of the optical system. Finally, the total relative number, i.e., the number of detected BC particles per cubic millimeter imaged placenta, was defined.

The customized Matlab program is made available upon reasonable request directed to the corresponding author.

Validation experiments of WL from BC in placentae

Validation experiments were performed using a Zeiss LSM 880 (Carl Zeiss, Jena, Germany) and 40×/1.1 water immersion objective (LD C-Apochromat 40×/1.1 W Korr UV-Vis-IR, Carl Zeiss). This setup was used as it allows accurate detection of the emission fingerprint and time correlated single photon counting of the BC particles in placental tissue. All settings were kept identical compared to the measurements performed on the LSM 510 setup unless stated otherwise.

Approximately, 60 images with a pixel size of 0.297 × 0.297 × 0.500 µm3 were acquired throughout the placental section using a pixel dwell time of 4.1 µs. In total, a volume of 300 × 300 × 30 µm3 was imaged. Orthogonal XZ-projection and YZ-projection were made using Fiji.

The emission fingerprints of the BC particles inside the placental tissue sections and TPAF from the placental cells were collected under femtosecond pulsed illumination. Note, for this specific experiment, the gain and laser power were changed to avoid saturation of the emission signal in order to be able to observe the trend of the WL signal over all wavelengths. After spectral separation, the emitted signals ranging between 410–650 nm were collected at an interval of 9 nm using the QUASAR thirty-two channel GaASP spectral detector of the LSM 880 system. The resulting 1024 × 1024 lambda image with a pixel size of 0.104 µm was detected with a pixel dwell time of 2.05 µs. As a reference, the emission fingerprint of commercially available carbon black nanoparticles (US Research Nanomaterials, USA) was recorded using identical settings.

Following femtosecond illumination, the temporal responses of the emitted signals originating from the BC particles in the placental tissue and from the placental cells were detected using the BiG.2 GaASP detector of the LSM 880 system. The detector was connected to an SPC 830 card (Becker and Hickl, Germany) that was synchronized to the pulse train of the MaiTai DeepSee laser. Recordings of 256 × 256 images with a pixel size of 0.346 µm were acquired using a pixel dwell time of 8.19 µs. The instrument response function was determined by detecting the response (IRF) of the laser pulse using potassium dihydrogen phosphate crystals under identical conditions. The IRF value was used in the analysis of all measurements for curve fitting. As a reference, the temporal response of commercially available carbon black particles was recorded employing the same settings. All time-correlated single photon counting measurements were captured and analyzed using the SPCM 9.80 and SPCImage 7.3 software (Becker and Hickl), respectively.

Screening of placental tissue for BC load

Both the intravariability and intervariability in BC loading of the placental biopsies were evaluated. The intravariability (within one biopsy) was examined by screening three regions of 10 × 10 images within five different sections taken in the middle of the examined biopsy (n = 15 images per biopsy). On the other hand, the intervariability (between the four fetal biopsies) was studied by measuring the BC load in three regions of 10 × 10 images within five different sections taken in the middle of the examined biopsy (n = 60 images per mother). The intervariability was solely assessed between the four fetal biopsies since there is already an existing variability between the fetal and maternal biopsies.

To evaluate the BC loading in the placentae of 10 low and 10 high exposed mothers, one biopsy was examined. More specifically, biopsy number 2 was selected with the exception of the following cases: (i) no or too little tissue was available, or (ii) background signal of blood was too high. In the latter cases, biopsy number 3 was chosen. The BC load was measured in three regions within five different sections taken in the middle of the biopsy (n = 15 images).

To study the BC loading in the preterm placentae the available biopsy was screened by imaging three regions within five different sections taken in the middle of the tissue (n = 15 images).

Ultrastructural analysis

Following fixation in 2% glutaraldehyde, the biopsies were gently rinsed and postfixed in 2% osmium tetroxide for 1 h. Subsequently, the biopsies were put through a dehydrating series of graded concentrations of acetone and impregnated overnight in a rotator with acetone:spurr (1:1) (Spurr Embedding Kit, Electron Microscopy Sciences). Next, the samples are placed into molds and fresh spurr solution is added followed by polymerization for 24–36 h at 70 °C. Ultra-thin sections (60 nm) were mounted on 0.7% formvar-coated copper grids and examined in a Philips EM 208 transmission electron microscope operated at 60 kV. Digital images were captured using a Morada camera system and analyzed using SIS analysis software (Germany).

Statistical analysis

All data are represented as means ± standard deviation and were analyzed using the commercially available software Graphpad (Graphpad Prism 6, Graphpad Software Inc., USA) and JMP (JMP Pro 12, SAS Institute Inc., USA). On the intravariability and intervariability data, a two-tailed analysis of variance (ANOVA) was performed followed by the Tukey posttest. To assess the relation between the BC exposure of mothers during pregnancy and accumulation of BC in placentae, the Pearson correlation coefficients were determined. We used the nonparametic Spearman’s Rank test to confirm the results.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.