Targeted next-generation sequencing and long-read HiFi sequencing provide novel insights into clinically significant KLF1 variants.

BACKGROUND: Krüppel-like factor 1 (KLF1), a crucial erythroid transcription factor, plays a significant role in various erythroid changes and haemolytic diseases. The rare erythrocyte Lutheran inhibitor (In(Lu)) blood group phenotype serves as an effective model for identifying KLF1 hypomorphic and loss-of-function variants. In this study, we aimed to analyse the genetic background of the In(Lu) phenotype in a population-based sample group by high-throughput technologies to find potentially clinically significant KLF1 variants. RESULTS: We included 62 samples with In(Lu) phenotype, screened from over 300,000 Chinese blood donors. Among them, 36 samples were sequenced using targeted Next Generation Sequencing (NGS), whereas 19 samples were sequenced using High Fidelity (HiFi) technology. In addition, seven samples were simply sequenced using Sanger sequencing. A total of 29 hypomorphic or loss-of-function variants of KLF1 were identified, 21 of which were newly discovered. All new variants discovered by targeted NGS or HiFi sequencing were validated through Sanger sequencing, and the obtained results were found to be consistent. The KLF1 haplotypes of all new variants were further confirmed using clone sequencing or HiFi sequencing. The lack of functional KLF1 variants detected in the four samples indicates the presence of additional regulatory mechanisms. In addition, some samples exhibited BCAM polymorphisms, which encodes antigens of the Lutheran (LU) blood group system. However, no BCAM mutations which leads to the absence of LU proteins were detected. CONCLUSIONS: High-throughput sequencing methods, particularly HiFi sequencing, were introduced for the first time into genetic analysis of the In(Lu) phenotype. Targeted NGS and HiFi sequencing demonstrated the accuracy of the results, providing additional advantages such as simultaneous analysis of other blood group genes and clarification of haplotypes. Using the In(Lu) phenotype, a powerful model for identifying hypomorphic or loss-of-function KLF1 variants, numerous novel variants have been detected, which have contributed to the comprehensive understanding of KLF1. These clinically significant KLF1 mutations can serve as a valuable reference for the diagnosis of related blood cell diseases.

A cretaceous frog with eggs from northwestern China provides fossil evidence for sexual maturity preceding skeletal maturity in anurans.

Mesozoic fossils of frogs are rare in the palaeontological record, particularly those exhibiting soft tissues that offer limited insights into early life-history characteristics. Here we report on a skeletally immature frog from the Lower Cretaceous of northwest China, with egg masses in the body and eggs in the oviduct, indicative of a gravid female. CT reconstruction of the specimen allows referral to Gansubatrachus qilianensis and we assign it as a paratype complementing the diagnosis of the type species. The new fossil, which might represent a younger individual than the holotype of Gansubatrachus, shows that sexual maturation occurred before full adulthood in this frog and provides evidence of death linked to mating behaviour. We also discuss other potential sources of variation and life-history traits of Gansubatrachus. The new finding represents the oldest Early Cretaceous frog preserving in situ eggs and provides a glimpse into ancient anuran development during Mesozoic times.

Fully genotyping and screening of clinically important blood-group antigens by MALDI TOF mass spectrometry.

Several molecular biology methods are available for high-throughput blood typing. In this study, we aimed to build a high-throughput blood-group genetic screening system for high-frequency blood-group antigen-negative rare-blood groups in donors and patients. The amplification primers for all blood-type gene fragments involving the selected alleles were designed for detection. Single-base extend primers were also designed based on specific loci. DNA fragments were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) for the last nucleotide identification of amplification products in the extend step. The accuracy was verified by known samples. Thirty-six random samples were detected by serological tests and sequencing to verify the system stability. After verification, according to the collected known rare-blood-type samples, all the alleles designed to be detected matched with the validated single-nucleotide polymorphisms. The verification tests showed that all genotyping results of the random samples were in accordance with the findings of serotyping and sequencing. Then, 1258 random donor samples were screened by the built typing system after the verification. Three Fy(a-) and four s- were screened out in 1258 random blood samples. The multiple polymerase chain reaction-based MS detection system can be used in rare-blood-type screening with good accuracy and stability.

Preparation of Fe3O4@CA/BNNS/AgNP Magnetic Microspheres and Photocatalysis of Dyes.

In this work, sea urchin-like magnetic Fe3O4@CA/BNNS/AgNP composite microspheres were successfully prepared. The photocatalytic performance of composite microspheres for the organic dye rhodamine B (RhB) was systematically investigated under different conditions, and the catalytic degradation rate of RhB was as high as 95% within 60 min; after three cycles of recycling, the degradation rate of RhB was reduced by only 8%. The main active agents in the reaction are e- and •O2-. Fe3O4@CA/BNNS/AgNP microspheres prepared in this study exhibit photocatalytic and electrochemical properties, making them easy to separate. This work is not limited to the development of Fe3O4-based catalysts but also is expected to provide ideas for the research and progress of photocatalytic composite catalysts with electrochemical properties.

Associations of Bisphenols Exposure and Hyperuricemia Based on Human Investigation and Animal Experiments.

Hyperuricemia is characterized by elevated blood uric acid (UA) levels, which can lead to certain diseases. Epidemiological studies have explored the association between environmental contaminant exposure and hyperuricemia. However, few studies have investigated the role of chemical exposure in the development of hyperuricemia. Here, we sought to investigate the effects of bisphenol exposure on the occurrence of hyperuricemia. Fifteen bisphenol chemicals (BPs) were detected in human serum and urine samples collected from an area with a high incidence of hyperuricemia in China. Serum UA levels positively correlated with urinary bisphenol S (BPS), urinary bisphenol P (BPP), and serum bisphenol F (BPF). The effects of these three chemicals on UA levels in mice were explored at various exposure concentrations. An increase in serum UA levels was observed in BPS- and BPP-exposed mice. The results showed that BPS exposure increased serum UA levels by damaging the structure of the kidneys, whereas BPP exposure increased serum UA levels by disturbing purine metabolism in the liver. Moreover, BPF did not induce an increase in serum UA levels owing to the inhibition of guanine conversion to UA. In summary, we provide evidence of the mechanisms whereby exposure to three BPs disturbs UA homeostasis. These findings provide new insights into the risks of exposure to bisphenol chemicals.

A novel homozygous splice-site mutation of JK gene leads to Jk(a-b-) phenotype.

OBJECTIVES: This study aimed to investigate the molecular mechanism of the Jk(a-b-) phenotype in a Chinese transfusion patient. BACKGROUND: Many different mutation types relating to Jk(a-b-) phenotype have been reported. However, the splice-site mutation is relatively rare and the related functional verification is lacking. MATERIALS AND METHODS: In this study, the blood sample was collected from a transfusion patient with the Jk(a-b-) phenotype. Serotyping was performed using routine serological methods. The exons sequences and coding regions of the JK gene were amplified using polymerase chain reaction and directly sequenced. To perform a minigene splicing assay, the intronic mutation sequences were cloned into a pSPL3 splice reporting vector. The splicing reporter minigene assay was performed in HEK 293T cells. RESULTS: The Jk(a-b-) phenotype of the blood sample was identified through serological testing. Sequencing results revealed that the sample had a novel homozygous splice-site mutation JK*02N (NM_015865.7: c.663+3A>C). Further analysis, including cDNA sequencing and minigene splicing assay, confirmed that the novel splice-site mutation resulted in exon skipping. Interestingly, different numbers of exons being skipped were obtained by the two methods. CONCLUSION: This study revealed a novel homozygous splicing-site mutation associated with the Jk(a-b-) phenotype in Chinese population. Our results emphasise the importance of the in vitro functional method minigene splicing assay, while also acknowledging its potential limitations when compared to cDNA sequencing.

Target-guided isolation and purification of cyclooxygenase-2 inhibitors from Meconopsis integrifolia (Maxim.) Franch. by high-speed counter-current chromatography combined with ultrafiltration liquid chromatography.

Meconopsis integrifolia (Maxim.) Franch. is used extensively in traditional Tibetan medicine for its potent anti-inflammatory properties. In this study, six cyclooxygenase-2 (COX-2) inhibitors were purified from M. integrifolia using high-speed counter-current chromatography guided by ultrafiltration liquid chromatography (ultrafiltration-LC). First, ultrafiltration-LC was performed to profile the COX-2 inhibitors in M. integrifolia. The reflux extraction conditions were further optimized using response surface methodology, and the results showed that the targeted COX-2 inhibitors could be well enriched under the optimized extraction conditions. Then the six target COX-2 inhibitors were separated by high-speed countercurrent chromatography with a solvent system composed of ethyl acetate/n-butanol/water (4:1:4, v/v/v. Finally, the six COX-2 inhibitors, including 21.2 mg of 8-hydroxyluteolin 7-sophoroside, 29.6 mg of 8-hydroxyluteolin 7-[6'''-acetylallosyl-(1→2)-glucoside], 42.5 mg of Sinocrassoside D3, 54.1 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-3''-acetylglucoside, 30.6 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-6''-acetylglucoside and 17.8 mg of Hypolaetin were obtained from 500 mg of sample. Their structures were elucidated by 1 H-NMR spectroscopy. This study reveals that ultrafiltration-LC combined with high-speed counter-current chromatography is a robust and efficient strategy for target-guided isolation and purification of bioactive molecules. It also enhances the scientific understanding of the anti-inflammatory properties of M. integrifolia but also paves the way for its further medicinal applications.

CRISPR/Cas9-Editing K562 Cell Line as a Potential Tool in Transfusion Applications: Knockout of Vel Antigen Gene.

Introduction: The Vel- phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of knockout (KO) cell lines at present. Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the Vel protein was detected using Western blot on single-cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin. Results: The high-efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone. Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin.

Large-scale preparation of five polar polyphenols including three isomers from Phyllanthus emblica Linn. by preparative high-speed counter-current chromatography.

The separation of polar compounds is challenging work due to poor retention and insufficient selectivity. In the present study, an efficient strategy for large-scale preparation of five polar polyphenols including three isomers from Phyllanthus emblica Linn has been established by preparative high-speed counter-current chromatography. Macroporous resin column chromatography was used for the enrichment of the polar polyphenols. However, sugar and other ultra-polar impurities were co-washed out with the targets. Liquid-liquid extraction with ethyl acetate/water (1/1, v/v) solvent system was developed to remove the ultra-polar impurities with a clearance rate of 95%. Finally, the targets were introduced to preparative high-speed counter-current chromatography for separation using ethyl acetate/n-butanol/acetic acid/water (2/7/1/10, v/v/v/v) solvent system. As a result, 191 mg of Mucic acid 1,4-lactone 5-O-gallate, 370 mg of ß-Glucogallin, 301 mg of Gallic acid, 195 mg of Mucic acid 1,4-lactone 3-O-gallate and 176 mg of Mucic acid 1,4-lactone 2-O-gallate with purity higher than 98% were obtained from 1.5 g of sample. Mucic acid 1,4-lactone 3-O-gallate, Mucic acid 1,4-lactone 3-O-gallate, and Mucic acid 1,4-lactone 2-O-gallate are isomers. The results showed that high-speed counter-current chromatography could be well developed for the separation of polar compounds from natural products.

UV-based solvent system screening for high-speed counter-current chromatography fractionation of compounds with similar UV absorption from complex samples followed by preparative HPLC purification: Flavonoids from barley seedlings as sample.

This article proposes a solvent system screening strategy for compounds with similar UV absorption in complex samples by UV spectrophotometer. There is no need to calculate the partition coefficient value of each compound, only the partition coefficient of the whole sample. The partition coefficient value should be close to 1 in order to obtain as many high-speed counter-current chromatography fractions as possible. Then, preparative HPLC was used to purify the high-speed counter-current chromatography fractions. Based on the above strategy, seven c-glycosyl flavonoids and an amino acid were successfully obtained from barley seedlings through high-speed counter-current chromatography fractionation with ethyl acetate/n-butanol/water (8:2:10, v:v:v) system followed by preparative HPLC purification. The research shows that high-speed counter-current chromatography could be well developed as a tool for fractionation before purification, and greatly improves the separation efficiency.

Diagnostic value of computed tomography and magnetic resonance imaging in ovarian malignant mesothelioma.

OBJECTIVE: To investigate the diagnostic value of computed tomography (CT) and magnetic resonance imaging (MRI) in ovarian malignant mesothelioma (OMM). METHODS: The clinical and imaging data of 10 pathologically-confirmed OMM patients were analyzed retrospectively. RESULT: (1) The patients were 27 years to 70 years old, with an average age of 57.2 ± 15.4 years. Seven patients reported abdominal distension and pain, 1 reported lower abdominal discomfort and decreased appetite, and 2 patients had no symptoms. (2) Two cases of localized OMM with incomplete semi-annular "capsule" observed around the localized OMM tumors were reported while 8 cases had diffuse OMM in which the tumor parenchyma showed isointense or slightly hypointense on T1WI, inhomogeneous hyperintense on T2WI, and obviously hyperintense on DWI, with obvious inhomogeneous enhancement after enhancement. Diffuse OMM was not mainly composed of ovarian masses and was mainly characterized by mild ovarian enlargement, nodular and irregular thickening of the peritoneum, cloudy omentum, unclear fat gap, and reticular or irregular thickening, which can fuse into a "cake-shape". (3) All 10 patients underwent surgery, while 9 patients underwent systemic chemotherapy or immunotherapy after surgery. All patients with localized OMM survived. Out of the 8 diffuse-type patients, 5 died, 1 was lost to follow-up, and 2 survived. CONCLUSION: OMM has certain clinical and imaging characteristics. There is no liquefaction, calcification, or partition in the tumor. The ovarian enlargement in the diffuse lesion is not significant. The diffuse thickening of the peritoneum and omentum with early appearance of mural nodules and ascites in the upper abdomen, help the diagnosis of OMM.

Integrated strategy for the screening of cyclooxygenase-2 inhibitors from triterpenoid saponins in Clematis tangutica.

INTRODUCTION: Screening of novel cyclooxygenase-2 (COX-2) inhibitors from complex natural products is not an easy task. OBJECTIVES: To establish an efficient and feasible strategy for screening COX-2 inhibitors from triterpenoid saponins (TPSs) in Clematis tangutica. METHODS: Taking TPSs in C. tangutica as example, an optimized macroporous resin (MR) method was established for the enrichment of TPSs. High-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOFMS) was performed to establish the phytochemical profiling of TPSs. Molecular docking was performed to predict the ligand-target interactions and discover the active substances. Chemometric techniques were performed to visualize the structure-effect relationships. High-speed countercurrent chromatography and preparative HPLC were performed to prepare the targets. In vitro activity experiment of COX-2 was performed to verify the virtual screening results. RESULTS: TPSs in C. tangutica were well enriched with the recovery rate of (80.22 ± 2.37)%. Thirty-four kinds of TPSs of oleanane type were deduced by HPLC-QTOFMS. Five TPSs of clematangoside C, clematangoside D, clematangoticoside J, hederoside H1 , and hederasaponin B showed stronger binding abilities with COX-2. The structure with more sugar groups at C-28 may be more conducive to the combination with COX-2. Targets were prepared with purities all above 98%. The IC50 values of target TPSs were 6.03 ± 0.24, 12.44 ± 0.15, 9.36 ± 0.19, 4.78 ± 0.13, and 2.59 ± 0.11 µmol/L, respectively. CONCLUSION: The integrated strategy using MR, HPLC-QTOFMS, molecular docking, chemometrics, target preparation, and in vitro verification was feasible for rapidly screening COX-2 inhibitors from TPSs in C. tangutica.

Target-Guided Isolation and Purification of Antioxidants from Urtica laetevirens Maxim. by HSCCC Combined with Online DPPH-HPLC Analysis.

Urtica laetevirens Maxim. is used extensively in traditional Chinese medicine (TCM) for its potent antioxidative properties. In this study, three antioxidants were purified from U. laetevirens. using HSCCC guided by online DPPH-HPLC analysis. Firstly, the online DPPH-HPLC analysis was performed to profile out the antioxidant active molecules in U. laetevirens. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the targeted antioxidant active molecules could be well enriched under the optimized extraction conditions. Then, the antioxidant active molecules were separated by high-speed countercurrent chromatography ethyl acetate/n-butanol/water (2:3:5, v/v/v) as the solvent system. Finally, the three targets including 16.8 mg of Isovitexin, 9.8 mg of Isoorientin, and 26.7 mg of Apigenin-6,8-di-C-ß-d-glucopyranoside were obtained from 100 mg of sample. Their structures were identified by 1H NMR spectroscopy.

Association between serum levels of TSH and free T4 and per- and polyfluoroalkyl compounds concentrations in pregnant women.

Many per- and polyfluoralkyl substances (PFASs) may disrupt maternal thyroid hormone homeostasis in pregnancy. Concerns should be raised regarding the PFASs exposure in pregnant women because thyroid hormones are involved in the early development of the fetus. In this study, we measured the concentrations of 13 PFASs, including five novel short-chain PFASs, in serum from 123 pregnant women in Beijing, China. Linear regression models were used to investigate the association between thyroid-stimulating hormone (TSH) or free thyroxine (FT4) levels and PFASs concentrations under consideration of the impacts of pregnancy-induced physiological factors. We found that perfluorobutanoic acid (PFBA) (ß=0.189, 95%CI=-0.039, 0.417, p=0.10) and perfluorodecanoic acid (PFDA) (ß=-0.554, 95%CI=-1.16, 0.049, p=0.071) were suggestive of significant association with TSH in thyroid peroxidase antibody (TPOAb) negative women. No association was observed between all PFASs and FT4 levels after controlling for these confounding factors, such as BMI, gestational weight gain and maternal age. These findings suggest that it should pay more attention to the association between thyroid hormone levels and short-chain PFASs concentrations. Future studies could consider a greater sample and the inclusion of other clinical indicators of thyroid function, such as free T3 and total T3.

Passive Smoking Exposure Modifies Cardiovascular Structure and Function: Beijing Child Growth and Health Cohort (PROC) Study.

Passive smoking exposure in children is prevalent worldwide and exposes children to respiratory and systemic toxins. In this study, we enrolled 568 children to study how secondhand smoke (SHS) might affect children's cardiovascular health in China. The measurement of nicotine and its metabolites in urine showed that 78.9% of children were exposed to SHS. Children exposed to SHS had greater interventricular septum thickness (p = 0.005) and left ventricular mass index (p = 0.008) than nonexposed children. Urinary norcotinine levels were associated with increased ascending aorta diameter (ß = 0.10, 95%CI 0.02-0.17) and decreased left ventricular end systolic diameter (ß = -0.10, 95%CI -0.19 to -0.01). The effects of SHS exposure on cardiovascular function: norcotinine levels associated with lower left ventricular mass index (ß = -0.32, 95%CI -0.59 to -0.05), left ventricular end diastolic volume index (ß = -0.43, 95%CI -0.85 to -0.02), and left ventricular end systolic volume index (ß = -0.20, 95%CI -0.37 to -0.03). Moreover, there no no significant associations of nicotine, cotinine, and trans-3'-hydroxycotinine with cardiovascular health. Overall, SHS exposure in children remains prevalent in Beijing and may affect children's cardiovascular development, in both structure and function. It suggests that stricter and practical measures are needed toward the elimination of tobacco use in children's environments.

Large-scale preparation of two minor polar polyphenols from Phyllanthus emblica Linn. by macroporous resin column chromatography and high-temperature preparative high-speed counter-current chromatography.

In the present study, an efficient strategy for the large-scale preparation of two minor polar polyphenols from Phyllanthus emblica L. has been established by macroporous resin column chromatography and high-temperature preparative high-speed counter-current chromatography. Macroporous resin column chromatography was used for the enrichment of the target polyphenols. The target polyphenols could be well enriched in the 10% ethanol fraction and the peak area proportion increased from 2.2% in the crude extract to 85.3% in the 10% ethanol fraction. Then, the 10% ethanol fraction was introduced to preparative high-speed counter-current chromatography for separation using an n-butanol-ethyl acetate-water (17:3:20, v/v/v) solvent system. In order to improve the retention of the stationary phase, a high-temperature preparative high-speed counter-current chromatography had been developed by using a 316 L stainless steel pipe as a column. A 50°C of column temperature was used and the retention rate of the stationary phase could reach 75%. Finally, 871 mg of compound Ⅰ and 534 mg of compound Ⅱ with purities higher than 98% were obtained from 4 g of the sample. The chemical structures were identified as corilagin and 1, 6-di-O-galloyl-ß-D-glucoside by 1 H-NMR and 13 C-NMR.

Tetrachlorobisphenol A induced immunosuppression and uterine injury in mice.

Tetrachlorobisphenol A (TCBPA) is used as flame retardant, and it has been widely detected in the environmental and human samples. TCBPA is an endocrine disrupting chemical, but its effects on the immune system remains poorly understood. Here the effects of TCBPA on immune system were studied using combined in vivo and in vitro assays. Results showed that TCBPA could suppress the immune response in BALB/c mice via reducing the ratio of CD3+ T lymphocytes to regulatory T cells. Moreover, TCBPA exposure significantly induced the increasing secretion of four pro-inflammatory cytokines (IL-2, IL-12, IFN-γ, and TNF-α) and four anti-inflammatory cytokines (IL-4, IL-5, IL-10, GM-CSF) in mice serum. Interestingly, uterine edema was observed in over 80% TCBPA-treated mice after 14- day exposure. TCBPA was detected in 18.6% serum samples of 150 female volunteers in this study. Therefore, our findings provided evidence that TCBPA exposure may cause adverse outcomes on immune system and uterus, suggesting that environmental exposure of TCBPA, as well as its adverse effects on human health should be of concern.

Tetrabromobisphenol A Perturbs Erythropoiesis and Impairs Blood Circulation in Zebrafish Embryos.

Tetrabromobisphenol A (TBBPA), a ubiquitous environmental pollutant, has been implicated in developmental toxicity of aquatic animals. However, the impact of TBBPA on development and the related mechanism have not been fully elucidated. In this study, using a live imaging technique and transgenic labeling of zebrafish embryos, we described the toxic effects of TBBPA on hematopoietic development in zebrafish. We demonstrated that TBBPA induced erythroid precursor expansion in the intermediate cell mass (ICM), which perturbed the onset of blood circulation at 24-26 hours postfertilization (hpf). Consequently, excessive blood cells accumulated in the posterior blood island (PBI) and vascular cells formed defective caudal veins (CVs), preventing blood cell flow to the heart at 32-34 hpf. We found that the one-cell to 50% epiboly stage was the most sensitive period to TBBPA exposure during hematopoietic development. Furthermore, our results demonstrated that PBI malformation induced by TBBPA resulted from effects on erythroid precursor cells, which might involve THR signaling in complex ways. These findings will improve the understanding of TBBPA-induced developmental toxicity in teleost.

A pilot study of mothers and infants reveals fetal sex differences in the placental transfer efficiency of heavy metals.

It has been demonstrated that heavy metals cross the placental barrier and exert potentially harmful fetal effects. Although previous studies showed sex differences in response to similar intrauterine environments, little is known about fetal sex-related differences in placental transfer and accumulation of heavy metals. This study aimed to reveal the sex-specific risk of fetal exposure to heavy metals in pregnant women. We detected the exposure levels of eight heavy metals in 64 paired mother-infant maternal blood, cord blood and placental tissue samples. We found that the placental transfer efficiency (PTE) of titanium (Ti) and silver (Ag) was significantly higher in the group with male fetuses than that with female fetuses. The group with male fetuses had a larger placental:maternal blood ratio of Ag levels than the group with female fetuses, indicating fetal sex-related differences in placental transfer and accumulation of Ag. Prospective research should focus on the sex differences of adverse health effects induced by heavy metals and other pollutants.