W1503

All powders listed in Supplementary Table S5 were weighted and diluted in water for embryo transfer (Sigma, W1503). The medium was filtered through a 0.22 µm filter and stored at 4°C for up to 3 months. At the morning of IVF experiments, 180 µL drop of pre-incubation medium was placed on the bottom of 35 mm Petri dishes (Falcon 351008) and covered with mineral oil.

For the cryopreservation of sperm gCPA containing 18% raffinose pentahydrate and 3% skim milk supplemented with 100 mM L-glutamine was prepared as published [18 (link), 27 (link)]. Briefly, 0.146 g of L-glutamine (Sigma; G8540) was placed in 10 ml of prewarmed (60°C) water (Sigma; W1503) and vortexed for 3 min. Subsequently, 1.8 g of raffinose pentahydrate (Sigma; R7630) and 0.3 g of skim milk (Becton Dickinson; 232100) was added, the solution vortexed for 3 min and incubated for 90 min at 60°C. gCPA was vortexed every 30 min for 3 min. Next, the solution was centrifuged at 10,000 g for 60 min and the supernatant was filtered through a 0.22 μm filter (PALL; 4652). After osmolality check (500–520 mOsm/kg) aliquots were stored at room temperature for up to 3 months.

All powders listed in Supplementary Table S4 were weighted as indicated and diluted in water for embryo transfer (Sigma, W1503). The medium was filtered through 0.22 µm filter and stored at 4°C for up to 3 months. At the morning of the IVF experiments, 30.7 mg of reduced glutathione (GSH) was added to 1 ml of HTF and mixed. 50 µL from this solution was added to the 5 ml of fresh HTF medium, filtered and used to prepare fertilization dishes. 90 µL drops of HTF medium with GSH were placed on the bottom of 35 mm Petri dishes (Falcon 351008), covered with mineral oil and incubated at 37°C, 5% CO₂ for 20 min.

Embryos were fixed in 2.5% glutaraldehyde in embryo grade water (Sigma, W1503) for 1–2 h at room temperature. Fixed embryos were rinsed with 1x PBS and stored at 4 °C before processing. Processing was performed exactly as described previously^{76 (link)}. Serial blockface sectioning and scanning electron microscopy were performed using a VolumeScope serial block-face EM (Thermo Fisher, Waltham, MA) equipped with a low-vacuum backscatter detector (VS-DBS; Thermo Fisher).

Half an hour before the exposure, emission particles were suspended into DMSO (20 μL mg⁻¹, Merck KGaA, Germany). After that, pathogen-free water (W1503, Sigma-Aldrich Corp., USA) was added to gain a PM concentration of 5 mg mL⁻¹. This suspension was sonicated in an ultrasonic water bath (FinnSonic m03, FinnSonic Ltd., Finland) for 30 min below +35 °C to gain homogenous mixture. Mouse RAW264.7 macrophages were exposed for 24 h to four doses (15, 50, 150, and 300 μg mL⁻¹) of emission particles from wood log combustion. Exposures of the cells to the particulate samples were made at least in three independent experiments. All in vitro experiments contained diesel PM (dose 150 μg ml⁻¹) [63 (link)] and blank substrate (dose 150 μg ml⁻¹) controls. Moreover water (dose 1.7 mM) and DMSO (dose 41.1 μM) served as vehicle controls. Blank sample virtual mass was calculated to be equivalent for PM containing filters average mass.

For the IVF procedure a commercial RVF fertilization medium (Research Vitro Fert, Cook Medical; K-RVFE-50) was supplemented with 1 mM (frozen sperm) or 0.25 mM (freshly harvested sperm) reduced GSH (Sigma; G4251) and the calcium concentration was increased from default 2.04 mM to 5.14 mM (mRVF). Therefore, a 100x CaCl₂ stock solution (310 mM) was prepared by dissolving 0.4558 g of CaCl₂ (Sigma; C7902) in 10 ml of water (Sigma; W1503). The solution was filtered through a 0.22 μm filter and aliquots were stored at -20°C for a maximum of 6 months. On the day of IVF an aliquot of CaCl₂ was thawed at room temperature. Subsequently, 150 μl of 100x CaCl₂ was added to 15 ml of fertilization medium and mixed gently. Next, 1 ml of fertilization medium supplemented with CaCl₂ was placed in a tube containing 30.7 mg of GSH and vortexed. 50 μl (frozen sperm) or 10 μl (freshly harvested sperm) of this solution was added to 5 ml (frozen sperm) or 4 ml (freshly harvested sperm) of fertilization medium supplemented with CaCl₂, mixed gently and filtered using 0.22 μm syringe end filter.