Sex estimation of skeletons is fundamental to many archaeological studies. Currently, three approaches are available to estimate sex–osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. We present matching osteological, shotgun-genomic, and proteomic data to estimate the sex of 55 individuals, each with an independent radiocarbon date between 2,440 and 100 cal BP, from two ancestral Ohlone sites in Central California. Sex estimation was possible in 100% of this burial sample using proteomics, in 91% using genomics, and in 51% using osteology. Agreement between the methods was high, however conflicts did occur. Genomic sex estimates were 100% consistent with proteomic and osteological estimates when DNA reads were above 100,000 total sequences. However, more than half the samples had DNA read numbers below this threshold, producing high rates of conflict with osteological and proteomic data where nine out of twenty conditional DNA sex estimates conflicted with proteomics. While the DNA signal decreased by an order of magnitude in the older burial samples, there was no decrease in proteomic signal. We conclude that proteomics provides an important complement to osteological and shotgun-genomic sex estimation. Sex and gender are also fundamental in structuring an array of cultural behaviors, including residence patterns, kinship, economic roles, and identity construction and expression How sex interacts with gender and these particular issues is not static and can vary in detail across societies and over time12. It is not surprising that sex is one of the most basic and important measures in bioarchaeological and forensic analyses.Biological sex plays an important role in the human experience, correlating to lifespan, reproduction, and a wide range of other biological factorsos coxae16. However, appropriate markers are not always sufficiently expressed or preserved to estimate sex using morphological criteria17. A lack of sexually-dimorphic markers is especially acute for skeletons of infants and children who have not undergone puberty. Mortuary practices, such as cremation or secondary burial in charnel houses, can also can impose limitations on the utility of osteological sex estimates18.Typically, osteological features are used to estimate sex of skeletal remains, and the most widely used marker is the morphology of the 21. More recently, development of massively parallel DNA sequencing greatly improved genome coverage in archaeological samples25. In addition to providing detailed genetic information, this allows biological sex to be estimated from shotgun sequencing data27. These approaches were an improvement over earlier PCR-based marker methods, which were less sensitive and had a higher risk of contamination32. Even with the application of high-throughput genomic data, confident estimation of biological sex is still restricted by requirements for high levels of DNA preservation27.The advent of DNA sequencing made it possible to use skeletal remains to estimate the sex of very young individuals; it also expanded sex estimations for fragmentary, pathological, and degraded skeletal materials37. Amelogenin genes are well-studied genetic markers of the X and Y chromosomes and have long been a basis of forensic sex determination40. Proteins can be useful targets for analysis in many archaeological settings as their molecular structure is more favorable for preservation relative to DNA44. Moreover, because amelogenin peptides are incorporated within the mineral phase of tooth enamel, the hardest and most durable material in the human body, such peptides may be particularly stable and persistent over long periods of time47.Recently, proteomic analysis of sex-specific amelogenin peptides in tooth enamel has been forwarded as an additional means of sex estimation in archaeological settingsSíi Túupentak  and Rummey Ta Kuččuwiš Tiprectak  48, and another that compared the ratio of X-chromosome reads to all autosomal reads (RX)27. In many cases (n = 55) each method of sex estimation, genomic, proteomic, and osteological, was applied to remains from the same individual.The availability of three independent methods of sex estimation provides an opportunity to compare and cross-check techniques against one another. While recent remains of known sex can be used to validate and estimate the precision of these techniques, such remains do not replicate archaeological conditions. In the current study, we apply three techniques: proteomic analysis of amelogenin peptides, shotgun-sequenced DNA, and standard osteological methods to determine the sex of human remains from two Late Holocene ancestral Ohlone villages in Central California: 27. This allows us to directly compare the respective techniques at a statistical level, and provides a broader framework for interpretation of sex estimation data that employs the strengths and limitations of each approach.To date, this is the largest study using sexually dimorphic amelogenin peptides to estimate biological sex and the largest to estimate sex based on matching shotgun DNA sequencing32. A benefit of shotgun DNA sequencing is that it can detect chemical modifications characteristic of ancient DNA (aDNA) and identify exogenous DNA contamination49. Skoglund and colleagues25 developed a genomic method of sex determination that takes advantage of high-throughput shotgun-DNA sequencing. This method (RY) estimates sex using sequence reads of 30 base pairs (bp) or longer that map to human X- and Y-chromosomes. RY is calculated as the number of Y-mapped reads compared to the total number of X- and Y- mapped reads. The RY method does not filter out homologous portions, but relies on a large number of total sequences to return a robust determination of sex. RY criteria were defined based on published data from 14 modern humans of known sex and 16 archaeological remains that had high-quality, prior PCR-based sex determinations. By artificially down-sampling sequences from these same individuals, Skoglund et al.25 recommended that a minimum of 100,000 total chromosome reads mapped to the human reference genome  were needed for confident sex estimations.Earlier PCR-based approaches that targeted sex-specific molecular markers, usually the amelogenin gene family, were often affected by modern contamination27. To reduce the required number of mapped human sequences, Mittnik and colleagues proposed an alternative method of sex estimation (RX) using high-throughput shotgun-sequenced DNA. The RX method relies on the proportion of reads mapped to the human X chromosome compared to the proportion of reads mapped to each of the autosomal chromosomes. By down-sampling reads from the same high-quality ancient DNA data sets used in Skoglund et al.25, the RX method was able to give confident assignments with as few as 1,000 human genome reads27.This degree of preservation may be problematic for many archaeological remains, as noted by Mittnik et al.52. These genes express distinctive isoforms of amelogenin proteins, AMELX_HUMAN (AMELX) and AMELY_HUMAN (AMELY)53, and detection of these proteins can be used to estimate sex over archaeological time scales55. Nano-liquid chromatography coupled with orbitrap tandem mass spectrometry (nLC-MS/MS) allows peptides to be identified at two levels. The MS1 level measures the precise molecular mass of the intact peptide, and subsequent MS2 data results in a spectrum of fragmented masses that together can be used to statistically match the most likely amino acid sequence to mass fragments of the MS1 peptide56. Signals from peptides with unique amino acid sequences specific to either AMELX or AMELY are identified, while those that are homologous are filtered out. Following removal of these non-specific amelogenin peptides, signals of all peptides unambiguously attributed to either AMELX or AMELY are then combined into a single measure33. This process differs from the methods of Stewart et al.57, Wasinger et al.35, or Froment et al.54, which relied on detection of two or four unique peptide masses only. In contrast, the proteomic method employed here identifies and sums signal intensities of multiple different AMELX and AMELY peptides with various permutations of common post translational modifications (PTMs), such as deamidation or oxidation. The ability to measure a greater number of specific peptides should increase sensitivity. Sensitivity is also likely to be increased in our method by using destructive chemistries as opposed to simple acid-leaching that seeks to preserve gross anatomy58.Amelogenin genes are located on both the X- and Y- chromosomes in humans and play a major role in the biosynthesis of enamelSíi Túupentak (CA-ALA-565/H) and Rummey Ta Kuččuwiš Tiprectak (CA-ALA704/H) are ancestral Native American Ohlone settlements situated in a well-watered valley in the southeast portion of the San Francisco Bay region, Central California, USA , all radiocarbon-dated burials (n = 99) were sampled for DNA, and 55 were sampled for amelogenin proteins. Archaeological mitigation of construction impacts to these archaeological sites, including the discovery, excavation, analysis and reporting of human remains, strictly conformed to all state and local laws and regulations. Members of the Muwekma Ohlone have seen and been provided an opportunity to contribute to the final version of the write-up of this study. In addition to their contributions to this study, the Muwekma Ohlone have advocated for science and genomics as a tool for Indigenous peoples and have strongly supported the Summer internship for INdigenous peoples in USA Fig. . Large-sSíi Túupentak (CA-ALA-565/H) is a large, intensively occupied Late Period village  with both domestic debris and associated cemetery59. Sixty-six burials, comprised of 76 individuals, were recovered. Most (71%) were primary inhumations, along with 21% secondary cremations, and 8% secondary inhumations. The extent and degree of burning varied between cremations, with the vertebrae, sacrum, pelvis, and proximal femora being the most commonly preserved elements. Dentition was generally not present for cremations but those with suitable preservation were analyzed. Seventy burials were dated between 600 to 110 cal BP . Excluding two outliers, most date to a 345-year time span from 525 to 180 cal BP . All dates were calibrated with a mixed marine curve based on established protocols using individual δ13C values, and median intercepts were used to organize the results61. In contrast, nearby Rummey Ta Kuččuwiš Tiprectak (CA-ALA704/H) is a substantial multicomponent settlement with occupation ranging from 2,440–175 cal BP (490 BCE–1775 CE), based on 60 radiocarbon dates from generalized site deposits, features, and burials60. With 88% of dates falling between 2,440–1,610 cal BP, occupation was most intensive in the Early/Middle Transition  and Middle 1 periods 60. Twenty-five burials comprising 29 individuals were recovered. Virtually all (93%) were primary inhumations, with just 7% secondary inhumations. Most interments (n = 26) date from 2,240–1,610 cal BP , but three date later in time, including two that are contemporary with Late Period Síi Túupentak  provided 43 sex estimates (78%). The RX method, that compared the ratio of reads mapping to the X-chromosome with those mapping to each autosomal chromosome, resulted in 50 sex estimates (91%). Osteology provided sex estimates for 28 of the 55 common samples (51%).Data for all burials recovered from both sites (n = 105) is available in supplemental materials Table . Table S63. DNA-based conditional, or “consistent with . . .”, estimates had 95% confidence intervals for the ratios that crossed thresholds for definitive XX or XY karyotype assignment27. Indeterminate samples fell entirely between the two thresholds. Using the genomic RY method, 27 sex estimates (49%) were definitive and 16 were conditional (21%). For the RX method, 26 estimates were definitive (47%) and 24 were conditional (44%). For osteology, conditional estimates were assigned as either “probable”, or “possible”, with the latter having less certainty. Osteology provided 15 definitive estimates (27%) and 13 conditional (24%) estimates  peptides and females having a probability of female sex (Pr(F)) greater than 0.5 (Methods)es Table .Y method agreed with osteological estimates in 18 of 23 cases (82%), and in 20 out of 25 cases (80%) using the RX method. Genomic estimates agreed with proteomic sex estimates in 36 of 43 cases (84%) using the RY, and 41 out of 50 cases (82%) when using the RX method , and 5 out of 16 (31%) individuals when the RX and RY ratios were used, respectively  and 38% (5 of 14) of the time using RY, suggesting that under these conditions DNA-based estimates were close to random. The RY method also resulted in two conflicts among definitive estimates, both of which were below 100,000 reads.Listing all sex estimates by increasing number of total matched sequences shows a similar pattern for both the Rds Table . Among tY and RX methods respectively. In this case, osteological sex estimation included both conditional and definitive assignments.The same is true for conflicts between osteological sex estimation and genomic sex estimates under 100,000 sequence reads. While the numbers were smaller due to a higher indeterminate rate, 4 out of 7 and 5 out of 11 conflicts were obtained with RIt is important to point out that less than half of the 55 common samples met the 100,000 read threshold. Including the samples that failed for DNA reconstruction, only 21 of 55 common samples (38.1%) exceed the 100,000 threshold. Though slightly higher, the proportion of samples exceeding 100,000 human genome sequence reads in the larger set of 99 skeletons sampled for DNA was also below 50%  Table  indicati9 and 9.11 × 109 CI/mg AMELX for RY estimates . Data for all duplicate proteomic samples are listed in Table Overall, there were three conflicts where proteomic sex estimation was female and genomic was male . In each of these cases the genomic assignments were conditionally male, while proteomics detected no AMELY peptides and had abundant AMELX peptides with relatively strong combined intensities corresponding to Pr(F) values of 0.95, 0.97 and 0.99 respectively Table . The low64. No significant linear relationship between the variables was detected in spite of the high power of the sample . This result is consistent with other studies, although sampling from different skeletal locations may have introduced variation65. Results of all statistical analyses can be found in supplemental materials.Since the efficacy of sex estimation is dependent on the quality of DNA and peptide signals, we compared matching signal types taken from each skeleton. Degradation of DNA and protein, particularly from the same sample, would be predicted to affect both signals and result in a positive relationship. Matching AMELX (CI/mg) was plotted as a function of matching total DNA reads Fig. a. In ordp = 0.78). This supports a stable proteomic signal over this timeframe  and is consistent with previously published observations33.Another approach is to compare the values of each variable as a function of archaeological age. With one exception (a more recent sample from CA-ALA-704/H), samples from the two sites fit into two discrete age categories. Late/Historic Period samples from CA-ALA-565/H span 600–100 cal BP, while EMT/Middle 1 Period samples from CA-ALA-704/H date between 2,240–1,610 cal BP . These results support a working hypothesis of independent or orthogonal signals for ancient DNA and amelogenin protein. The practical result is that low signal DNA samples may have high amelogenin signals and vice versa, and that combining information from both DNA and proteomic methods will mutually support concurring estimates and correct for conflicting conditional estimates.The same was not the case for DNA quality. The range of logarithmically transformed total DNA reads averaged 5.13 ± 1.10 orders of magnitude for Late/Historic Period samples and 4.05 ± 1.25 orders of magnitude for EMT/Middle 1 Period samples, a reduction of about an order of magnitude in the older samples , amelogenin protein, and osteological markers were made on the same set of individuals, matching datasets allowed us to make direct comparisons of the performance of the three techniques and develop a framework for managing inevitable conflicting sex estimates. When low values or confidence scores are obtained for any one method, the result can be compared to other methods and help determine the thresholds at which inconsistent sex estimation begin to occur.To the best of our knowledge, this is the largest archaeological study to compare different molecular and osteological methods of sex estimation. Because analyses of shotgun-sequenced DNA , followed by genomic-based sex estimates, and osteology. Overall, there was a high amount of consistency between the different methods. We observed total agreement between the three methods where osteology had definitive sex estimates, when DNA had more than 100,000 total reads, and when Ros coxae and crania. Nonetheless, it is highly reliable when preservation is good. All definitive osteological sex estimates concurred with definitive DNA and proteomic estimates. There were only four and five discrepancies, respectively, with conditional RY- and RX-based sex estimates, and just one of a total of twelve conditional osteological sex estimates disagreed with a proteomic sex estimate.Osteology offers a highly reliable, relatively fast means of estimating sex, although extensive training is required. Osteological methods are especially valuable as there are many contexts where molecular techniques cannot be applied due to cost and preference of the descendent community. However, as shown here, the osteological method is limited to adult skeletons with preserved sexually dimorphic markers, such as 69. It can also be applied to a variety of human tissues, including bone, skin, hair, and teeth. This provides more flexibility than the analysis of amelogenin protein, which is restricted to tooth enamel.A strong benefit of high-throughput shotgun-sequenced DNA-based assignment of sex is that it can piggyback off of analyses performed for other reasons, such as information on the ancestry of an individual or evidence of diseaseX ratio was more sensitive than the ratio based on sex chromosome reads (RY)  Table , with moY) Table  and 2. D25 were supported by proteomic sex estimates and osteological sex estimates. All conflicts with proteomic and osteological sex estimates occurred below this threshold. Thus, caution should be applied to genomic sex estimates when the total number of mapped human sequences is below 100,000. This is particularly so for conditional, or ‘consistent with. . .’, estimates, whether they are made using RY or RX criteria. While no definitive RX sex estimates conflicted with proteomics or osteology, and no conditional estimates above 100,000 total reads conflicted, conditional estimates made on samples below 100,000 reads agreed with proteomics at a rate only slightly better than chance alone (5/14 for RY and 9/20 for RX). While the numbers were smaller the same phenomenon was observed for conflicts with osteological sex estimation.In this study, the limits of 100,000 total reads originally proposed in Skoglund et al.X sex estimates conflicted with proteomics or osteology, it may be possible to increase the total number of confident genomic sex estimates by combining definitive RX estimates below 100,000 reads with RY or RX estimates  that have more than 100,000 total matched human sequences. This would increase the number of confident genomic sex estimates by 16.4% . At what point definitive RX estimates become less reliable, however, remains an open question. The much lower threshold of 1,000 reads originally proposed for the RX method27 could not be confirmed here as no definitive RX sex estimates were obtained below about 5,000 reads.Given that no definitive R70. The use of SNP rates and affirmation or otherwise with proteomic sex estimation is the focus of additional study.Refinements in analysis of shotgun sequenced DNA could also increase confident genomic sex estimates. Researchers may conduct a detailed analysis of sex chromosome sequences to exclude homologous regions and provide a higher confidence of sex chromosome assignment. The use of targeted SNP data to conduct sex estimation helps in this regard. The resulting ratios of average sex chromosome to autosomal coverage based on X and Y rates may reduce chromosome miss-assignment and increase the signal separation between males and females62. Female assignments were more complex, but the calculated probabilities of female sex (Pr(F)) were generally high and lower probabilities were corroborated with high quality DNA data33.In contrast to other methods, sex estimation based on amelogenin proteins was more sensitive, with assignments made on all samples, including those that failed for DNA sex estimation and samples from two cremated individuals Table . All pro71. The proteins are cleaved into peptides in situ, as part of enamel formation during tooth biogenesis73. In order to extract and analyze this peptide population, researchers need to demineralize the enamel and most use acid-based74 approaches. There are two analytical options: a targeted approach focused on a limited number of specific amelogenin peptides54, or a shotgun proteomics approach that seeks to identify all proteins in the proteome and then selectively measure all amelogenin peptides bioinformatically after peptide spectral matching55. This study takes the later approach33. By comprehensively identifying and measuring all unambiguous AMELX and AMELY peptides, the chromosome-specific signal is maximized. Stochastic effects that may result from any one peptide will be minimized. The approach is validated in this study by the high sensitivity of proteomic sex estimation, the stability of proteomic data over time, and the finding that there was no functional correlation between proteomic and genomic signals.Proteomic sex estimation exploits the fact that the highly characterized sex-chromosome-specific amelogenin gene family is expressed as proteins in the most robust tissue in the human body, enamel75. Over time, proteins that are less incorporated in the mineral matrix, such as extracellular matrix proteins, will degrade at a faster rate resulting in a less complex proteome that is relatively enriched with amelogenin peptides35. As a result, remaining amelogenin peptides are more likely to be targeted by the mass spectrometry instrument for fragmentation, increasing the cumulative signal.Because the amelogenin peptide signal appears to be independent from DNA-based sex estimation, confident proteomic sex estimates can occur in samples with low or absent levels of DNA, and vice versa. In this study, amelogenin peptide signal remained stable over approximately 2,000 years while DNA levels significantly decreased in the older samples Fig. b and c. The utility and complementarity of proteomic, genomic, and osteological techniques was related to differences in mortuary treatments and preservation encountered in this study. Proteomics was able to estimate sex in several cases where genomics failed, including skeletal remains from one cremation . On the other hand, not all burials contained teeth with sufficient enamel, which precluded analysis of amelogenin protein. This was particularly true for cremated remains at CA-ALA-565/H, which were secondarily interred and formed a sizeable portion of the burial population (21%). Overall, it was possible for genomic sex estimation to be attempted on a larger number of burials, even though proteomics had greater sensitivity. Combining proteomic, genomic, and osteological data produced highly comprehensive and confident sex estimations for the burial populations analyzed in this study. This allowed detailed male and female survival functions to be constructed, which enabled us to better detect sex-biased mortality patterns among the subadult population at CA-ALA-565/H. These sex-biased mortality patterns are the subject of a forthcoming paper. Future systematic comparisons are needed to understand the relative strengths of these molecular techniques with respect to various mortuary treatments and over a wider range of environmental and temporal contexts.warep (roughly translated as “the earth”), where their loved ones originally placed them with love and respect.Finally, and most importantly for the Muwekma Ohlone Tribe of the San Francisco Bay Area, accurate sex determination provides a greater perspective on the persona of each individual, rather than the nebulous "indeterminate" status of a person or child. Tribal members and representatives of the scientific community are collectively looking into the lives and tragedy of the death of people from the past. If it was not for their sacrifice, struggles, and commitment to their families, Muwekma Ohlone would not survive to this day. Today, the Muwekma Ohlone celebrate the lives of their ancestors by retelling some of their history and stories through archaeology, and ultimately honor them when they are returned to the A large-scale comparison of proteomic, genomic, and osteological methods of sex estimation provides a unique opportunity for contrasting the benefits and limits of each technique. We empirically demonstrate that the thresholds of 100,000 total and 3,000 sex chromosome reads for genomic sex estimation is impactful; all conflicts occur below this threshold and no inconsistencies occur above it. In particular, conditional “consistent with . . .” estimates below this threshold were effectively random with respect to proteomic and osteological determinations.The study showed that osteological sex estimation is reliable , but has a high rate of indeterminate sex assignments when fragmentary and juvenile remains are assessed. Genomic methods help to extend sex estimation to many juvenile or fragmentary remains, but had a high rate of conflict with osteology or proteomic estimates for conditional sex assignments below the 100,000 total mapped read threshold. In the event of a conflict in sex estimation, these conditional DNA-based estimates should be disregarded in favor of other methods. Proteomic sex estimation was the most sensitive technique, providing results in all remains tested, due in part to the stability of the amelogenin peptide signal, but was contingent upon the preservation of dentition associated with each burial. Conflicts between proteomic and DNA based estimates could be attributed to the different level of stability and signal variation between the two types of biomolecules. To obtain the greatest coverage and confidence in sex estimates among archaeological burial populations, proteomic approaches should be combined with osteological and genomic methods.os coxae , six on the cranium and mandible , and five that were quantitatively categorized for robusticity . All assessed traits have previously been shown to contribute to accurate sex estimation77. Due to the complexity of human sexual dimorphism, the scores for these 20 traits were then comprehensively evaluated relative to the local population to best determine the sex of the individuals . All DNA extraction and genomic library preparation rounds included negative controls to account for DNA contamination. Libraries were constructed using the NEBNext Ultra II DNA Library Prep kit and NEBNext Multiplex Oligos  for Illumina, and shotgun-sequenced on a HiSeq 4,000 platform at the UIUC Core Sequencing Facility.Whole genomic DNA extraction was conducted on a total of 99 ancient tooth and bone samples  using Burrows-Wheeler alignment in BWA v. 0. 7.1580. Aligned sequences were transformed to BAM format in SAMtools v. 1.181 and filtered to remove unmapped reads and reads with a quality score less than 30. PCR duplicates were marked and removed with the Picard Toolkit v. 2.10.1 , and index statistics for BAM files were generated using “idxstats” in SAMtools81. RY and RX ratios were calculated following methods described in Skoglund et al.25 and Mittnik et al.27. Mapdamage 2.0 was used to check for DNA damage associated with ancient DNA49.Samples were de-multiplexed and trimmed to have a minimum sequence length of 25 bp using the program FastP v. 0.19.6Síi Túupentak and 16 individuals from Rummey Ta Kuččuwiš Tiprectak; Table 24 with the following changes. Mass spectrometry datasets (.RAW format) were processed with PEAKS (10.0) peptide matching software . Error tolerance for matching peptide spectral assignment was set to 10 ppm for precursor mass and 0.04 Da for fragment ions. AMELX_HUMAN signals (CI/mg) were log transformed and then solved for Pr(F) using the equation Pr(F) = 1.0 + (0.059–1.0)/(1 + (x/7.54)13.99 where “x” is the logarithm (base 10) of the AMELX_HUMAN24. Samples with a Pr(F) < 0.5 were considered indeterminate for proteomic sex estimation. Full details of the proteomic methods are provided in supplemental information84.Amelogenin peptides were extracted and analyzed from the tooth enamel of 55 individuals (39 individuals from https://www.proteomexchange.org85.The mass spectrometry proteomics data, along with customized protein reference library, have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the accession number PXD016076 (Supplementary file


Alveolar hemorrhage (AH) is characterized by the acute onset of alveolar bleeding and hypoxemia and can be fatal. Thrombin has been widely used to achieve coagulation and hemostasis. However, the efficacy of thrombin in patients with AH is unclear. Thus, this study aimed to evaluate the efficacy of thrombin administration in patients with hematological malignancy and AH.This retrospective study included 15 hematological malignancy patients  with AH who were administered intrapulmonary thrombin between March 2013 and July 2018.2/FiO2 (PF) ratio after intrapulmonary thrombin administration was evaluated. Intrapulmonary thrombin was administered a minimum of 3 days after starting mechanical ventilation in all patients, and it immediately controlled the active bleeding in 13 of 15 patients (86.7%). However, AH relapse was noted in 3 of the 13 patients (23.1%). The PF ratio improved in 10 of 15 patients (66.6%), and the mean PF ratio was significantly higher after thrombin administration than before administration (P = .03). No adverse thromboembolic complications or systemic adverse events were observed.All patients received bovine-origin thrombin  via a fiberoptic bronchoscope. A maximum of 15 ml of thrombin was injected via the working channel to control bleeding. The ability of thrombin to control bleeding was assessed. Additionally, the change in the PaOThrombin administration was effective in controlling bleeding in hematological malignancy patients with AH. Intrapulmonary thrombin administration might be a good therapeutic option for treating AH. Conventionally, the diagnosis of AH is based on a finding of abnormally increased blood content in fluid samples obtained during bronchoalveolar lavage (BAL) in the absence of signs of infection. In addition to supportive care, systemic corticosteroids have been used to treat AH in patients with hematological malignancies. So far, however, no prospective, randomized trials have investigated the role of systemic corticosteroids in patients with AH. Furthermore, recent studies have reported similarities in the clinical presentation, management, and high mortality rates in the presence and absence of infection .,5 The difficulty of clinically distinguishing DAH from IAH raises concern that high-dose corticosteroids may increase the risk of infection.Alveolar hemorrhage (AH) is a potentially fatal clinical syndrome that is characterized by acute onset of alveolar bleeding and hypoxemia. It is one of the most common complications among patients with hematologic malignancies, especially patients suffering from leukemia who received an allogeneic hematopoietic stem cell transplantation (HSCT). AH has a reported incidence of 3% to 20% in patients receiving HSCT and a mortality rate that ranges from 50% to 80%.,7 However, a previous study reported that TXA did not improve mortality due to bleeding in patients with acute promyelocytic leukemia. EACA has also been used in addition to corticosteroids in patients with post-transplant AH; however, the effect of EACA in AH is controversial.,9 Recently, the use of recombinant factor VIIa (rFVIIa) in patients with AH has increased. Although rFVIIa was originally developed for patients with hemophilia and acquired coagulation factor inhibitors, the off-label use of rFVIIa has increased and many studies have reported the impact of rFVIIa in the treatment of uncontrolled AH in patients with hematological malignancy.1112131112 However, rFVIIa is prohibitively expensive,,15 and the off-label use of rFVIIa has been associated with major thrombotic complications.,16Traditionally, to enhance pulmonary hemostasis and decrease the dose and duration of corticosteroid therapy, antifibrinolytic drugs such as tranexamic acid (TXA) and epsilon aminocaproic acid (EACA) have been suggested.,18 Thus far, however, few studies have investigated the effect of thrombin in patients with AH. We present our experience of the intrapulmonary administration of thrombin as a simple and effective technique for the treatment of AH in hematological malignancy patients in the intensive care unit (ICU).Thrombin has been widely used to achieve coagulation and hemostasis. In the setting of hemoptysis, topical application of thrombin has also been employed.22.1In this retrospective study, we reviewed 15 hematological malignancy patients with AH who were treated with intrapulmonary administration of thrombin from March 2013 to July 2018. Patients were identified in 3 different ICUs at Seoul St. Mary's Hospital, Seoul, Republic of Korea. The diagnosis of AH was made based on the occurrence of all of the following clinical criteria:1.acute onset of hypoxemia,2.presence of diffuse pulmonary infiltrates on a chest X-ray or a computed tomography scan, and3.confirmed bronchoscopically after consecutive BAL aliquots yielded an increasingly bloody return.The Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores were calculated based on the vital signs, mental status, and laboratory findings of enrolled patients on the day of ICU admission. The Ethics Committee of Seoul St. Marys Hospital approved this study. The ethics review board waived the need to obtain informed consent due to the retrospective study design (KC18ZESI0804).2.22 and PEEP ≥10 at the time of administration. Non-intubated patients received oxygen supplements by nasal probe or by mouth.Bronchoscopic procedures were performed at the bedside within the ICU by staff members with a minimum of 5 years clinical experience of bronchoscopy in the pulmonary division. Before the procedure, the patient was subcutaneously administered 0.5 mg of atropine sulfate to reduce oral secretions and bronchospasm. Before intrapulmonary thrombin instillation, BAL was performed to diagnose AH and conduct a microbiological investigation. The use of suction was meticulously avoided throughout the procedure. An endoscopic revision of the whole bronchial tree was made once the bleeding had been controlled. For intubated patients, the ventilator settings were generally set on a control mode with 100% FiO2.39/L, and fresh-frozen plasma to normalize prothrombin time and activated partial thromboplastin time. Intrapulmonary instillation of thrombin was performed in patients who did not improve despite receiving conventional treatment for >2 days. Conventional treatment was defined as high-dose corticosteroid (methylprednisolone 500 mg/day) and supportive care . All patients were treated with the instillation of bovine origin thrombin  via a fiberoptic bronchoscope (FOB). The dosing and the frequency of thrombin instillation were at the discretion of the attending physician. Because there are no guidelines for intrapulmonary thrombin instillation, the dosing of thrombin was varied; to control bleeding, a maximum of 15 ml of 1000 IU/ml thrombin was injected at each lobe. The medication was administered by syringe via the working channel.To enhance pulmonary hemostasis, patients received platelet transfusions in an attempt to achieve a level >50 × 102.4P value <.05 was considered statistically significant. All statistical analyses were performed, and a graph was plotted using the R 3.4.1 version .Statistically significant differences between groups were analyzed using the Wilcoxon signed rank test. Data are expressed as the mean ± standard deviations, median (interquartile range), and numbers and percentages. The distribution of continuous variables was determined using the Shapiro–Wilk normality test. A two-tailed 39/L (1.3–18.3), 1.2 × 109/L (0.3–3.0), and 43.9 ± 25.6 × 109/L. The mean APACHE II and SOFA scores were 21.3 ± 7.0 and 8.0 ± 2.0. The underlying hematological diseases of the patients were acute myeloid leukemia (n = 7), acute lymphoblastic leukemia (n = 2), aplastic anemia (n = 1), non-Hodgkins lymphoma (n = 3), and myelodysplastic syndrome (n = 2). The reasons for ICU admission were respiratory failure (n = 10), septic shock (n = 2), massive hemoptysis (n = 2), and severe graft-versus-host disease . Among the 15 patients, 5 received allogeneic HSCT and 10 did not.The characteristics of the 15 hematological malignancy patients with AH who received intrapulmonary thrombin instillation are provided in Table P = .03).Table Acinetobacter baumanii (patient 2) and methicillin-resistant Staphylococcus aureus (patient 4). Co-infections with cytomegalovirus, one of the main infectious diseases that cause AH, and other respiratory viruses were not found in any patient.The etiologies of AH differed among the patients, and microorganisms were isolated from BAL fluid in 2 patients: carbapenem-resistant Among the 15 patients, 11 eventually died. Multiple organ failure was the leading cause of death (n = 7), and 3 patients died of respiratory failure due to AH Table . In the 4,21 In hematological malignancy patients with AH, acute lung injury occurs due to intra-AH and diffuse alveolar damage due to immunologic mechanisms.,22 Therefore, anti-inflammatory and hemostatic therapies should be the cornerstones of therapy.While AH is a rare cause of death in patients with hematological malignancies, it is still a fatal clinical syndrome with a mortality rate of approximately 70%.,7 TXA is a synthetic derivative of the amino acid lysine that binds to plasminogen and blocks its binding to fibrin and subsequent activation to plasmin. TXA in either an aerosolized form or as intrapulmonary injections has been reported to control bleeding in a few cases of pulmonary hemorrhage.,24 EACA is another potent antifibrinolytic agent used to control bleeding by inhibiting the plasmin-plasminogen system.,26 In the case of AH in patients with hematological malignancy, a retrospective study reported that EACA improved the mortality rate by 100 days compared to that in those treated with steroids alone. However, the effect of TXA and EACA in AH remains controversial.To improve pulmonary hemostasis, antifibrinolytic drugs such as TXA and EACA have been used alongside corticosteroids. However, no prospective, randomized controlled trials have confirmed the efficacy of corticosteroids in patients with AH. Previous studies reported that the use of high-dose systemic corticosteroids resulted in transient clinical improvement.,29 However, the use of high-dose corticosteroids has not improved the outcome of AH; previous studies have reported mortality rates of 70% to 100% in AH patients treated with high-dose corticosteroids.,28293031282930In hematological malignancy patients with AH, high-dose corticosteroids have been widely used because the pathogenesis of AH is considered to be an inflammatory response to various insults. Several case reports have described the use of rFVIIa for the management of AH after HSCT.,33,34 However, rFVIIa is prohibitively expensive, and off-label use of rFVIIa has been associated with major thrombotic complications.151615To date, there is no definite treatment for AH in patients with hematological malignancies. Therefore, there remains an urgent need for novel methods to treat AH. Recently, the use of rFVIIa has increased in patients with AH. rFVIIa is an analog of the naturally occurring protease and was originally developed for the treatment of hemophilia patients with acquired coagulation factor inhibitors. Thrombin production is the final coagulation step required to cleave fibrinogen into fibrin, which provides a hemostatic lattice for platelet aggregation and thrombus formation at the site of injury. In addition to the coagulation cascade, thrombin causes vasoconstriction at the smooth muscle cell level and promotes platelet aggregation at the site of the thrombus. Prior studies have reported the effect of thrombin or fibrinogen-thrombin in the treatment of severe hemoptysis with acceptable success rates.,18,37,38 Thus far, however, no studies have investigated the effect of intrapulmonary thrombin instillation in patients with AH. To our knowledge, this is the first study to apply thrombin in the treatment of hematological malignancy patients with AH.Thrombin is a key enzyme in hemostasis and thrombosis, and regulates pro- and anti-coagulant reactions by interacting with other coagulation proteins and cellular receptors.In this study, we have illustrated efficacy of intrapulmonary thrombin instillation in mechanically-ventilated patients with hematological malignancies who developed AH. Our findings indicate that intrapulmonary thrombin instillation can be a useful adjuvant therapy along with supportive care such as corticosteroids or TXA. Although ten patients (66.7%) died in the ICU and 11 patients (73.3%) died within 60 days of the onset of AH, thrombin instillation improved the respiratory status. Bleeding was controlled rapidly in 13 of 15 patients (86.7%) and oxygenation was significantly improved after the procedure. Furthermore, no adverse thromboembolic complications or systemic adverse events were observed. Although the majority of the initial presentations of AH in this study involved respiratory failure (66.7%), the most common cause of death was multiple organ failure (63.6%). This result is consistent with previous studies; respiratory failure with active pulmonary hemorrhage was responsible for <15% of the deaths.In summary, this is the first study to illustrate the efficacy of intrapulmonary thrombin instillation in patients with hematological malignancies who developed AH. Bleeding was controlled effectively and no systemic or thromboembolic adverse events were observed after intrapulmonary thrombin instillation. Intrapulmonary thrombin might be a good therapeutic option for the treatment of AH, particularly when standard treatment fails or bleeding is immediately life-threatening. Several important shortcomings and limitations of our study should be taken into account. First, as this was a retrospective study, it was impossible to compare the effect of intrapulmonary thrombin instillation in AH patients with a control group. Second, the sample size of the present study was small. Further studies with prospective design are needed, with a larger number of patients, to fully assess the risks, benefits, and optimal dosing strategy of thrombin as an adjunctive therapy in the treatment of AH. Despite these limitations, the main advantage of the present study is that it is the first to apply intrapulmonary thrombin in hematological malignancy patients with AH.The authors thank Minyoung Park at Seoul National University, for her helpful advice on the structuring of this manuscript.Conceptualization: Chin Kook Rhee.Data curation: Jongmin Lee.Formal analysis: Seok Lee.Methodology: Seok Chan Kim.Resources: Hee Je Kim.Supervision: Seok-Goo Cho.Validation: Young Kyoon Kim.Writing – original draft: Jongmin Lee.Writing – review & editing: Jong Wook Lee.


A single experiment required 40 younger and older adults to discriminate global shape as depicted by Glass patterns . Such patterns have been widely used for decades, because in order to successfully perceive the depicted shape, the visual system has to detect both locally oriented features (dipoles) and their alignments across extended regions of space. In the current study, we manipulated the number of constituent dipoles in the stimulus patterns (40 or 200), the noise-to-signal ratio , and the pattern size . The observers’ shape discrimination accuracies  decreased markedly as the amount of noise increased, and there were smaller (but significant) effects of both overall pattern size and the number of stimulus dipoles. Interestingly, while there was a significant effect of age, it was relatively small: the overall d’ values for older and younger adults were 2.07 and 2.34, respectively. Older adults therefore retain an effective ability to visually perceive global shape, even for sparsely-defined patterns embedded in noise. Vision scientists have used Glass patterns –2 for deIt is important to investigate aging, because it significantly affects shape perception and other essential visual abilities that support everyday behavior. Any effects of aging upon vision, however, depend greatly on the particular task. For some visual spatial tasks involving the perception of distance, older adults perform well, either better than or the same as younger adults –17. SimiMcKendrick and colleagues –38 have and large Glass Patterns (25 degrees). In addition, in the previous studies by McKendrick and colleagues [One important goal of the current experiment was to evaluate younger and older observers’ performance for small  and lleagues –38, cohelleagues .The Glass patterns were generated by an Apple dual-processor (1.42 GHz) PowerMacintosh G4 computer and displayed using a 22-inch Mitsubishi Diamond Plus 200 color monitor (1280 x 1024 pixels). The stimulus rendering was accelerated by a Radeon 9000 graphics accelerator  and hardware antialiasing was employed.The experimental stimuli were radial and concentric Glass patterns (white points against a black background) like those used in previous research. The global radial and concentric structure  was defiTo ensure that all observers (both younger and older) clearly understood the task, we presented them with block(s) of 40 trials  and asked them to indicate whether each pattern depicted a radial or concentric organization. Auditory feedback (a short beep for correct responses) was only presented during practice trials, and was never presented during the experimental trials. The observers were not allowed to begin the actual experiment until after they had performed at least 90 percent correct during a practice block.Following the practice block, 40 patterns  were judged by each observer for each of the 2 numbers of dipoles (40 and 200), 2 sizes (6 and 25 degrees), and 3 noise-to-signal ratios . Each observer, therefore, made a total of 480 judgments. The trials were blocked by pattern size, thus a block of 240 trials were run for the small patterns (6 deg) and another block of 240 trials were run for the large patterns (25 deg). The order of the size blocks (whether the 6 or 25 degree patterns were judged first) was counterbalanced across observers.There were a total of 40 observers: 20 older adults  and 20 younger adults . The observers’ visual acuity was good . All observers gave written consent prior to participation in the experiment. The experiment was approved by the Western Kentucky University Institutional Review Board. Our research was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).The individual observers’ shape discrimination accuracies  are plo2p = 0.91): the observers’ perceptual sensitivity to shape was very high (average d’ was 3.0 or greater) when there was no noise and deteriorated substantially as more and more noise points were added to the stimulus patterns. There was a slightly larger effect of noise for the 200-dipole patterns than for the 40-dipole patterns (2p = 0.11). There was also an adverse effect of age, particularly when moderate amounts of noise were included in the stimulus patterns . There were small main effects of both the number of dipoles  = 4.6, p = .039, η2p = .11) and pattern size  = 5.8, p = .021, η2p = .13). Finally, as 2 = .084) of the variance in their d’ values.Various aspects of the observers’ results concerning discrimination accuracy are shown in Figs 2p = .43). As 2p = .09). There was no difference in obtained response biases between the younger and older observers  = 1.3, p = .27).The observers’ response biases were calculated in terms of c  and are In our study, while we found an adverse effect of age, it was relatively modest in size . The difference in performance between younger and older adults was small for the no-noise condition, and increased in magnitude when moderate amounts of noise were added to the stimulus patterns see . When noIn addition to the perception of global structure within Glass patterns, aging also adversely affects performance on related tasks –35 that It has been demonstrated  that neuOlder adults retain an effective ability to visually perceive global shape, even for sparsely-defined patterns embedded in noise. In the current study, we found only a small quantitative effect of age upon sensitivity to perceived shape , depite the fact that our older observers were more than 52 years older (on average) than our younger observers.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Malathion-inhalation induced marked systemic toxicity as hepatotoxicity and nephrotoxicity associated with increased serum hepatic and renal enzymes, and hypersensitivity accompanied with increased total IgE serum level. The lung showed severe interstitial pneumonia associated with severe vascular damage and marked eosinophil infiltration. Moreover, the lung showed a marked decrease in the pulmonary surfactant protein, especially SP-D gene expression. While, thymoquinone treatment to malathion-inhaled animals decremented the following; hepatic enzymes and renal function tests, total IgE as well as pneumonia and hypersensitivity pathological features, and augmented the expression of SP-D. In conclusion, thymoquinone could be potentially used in pest control workers to ameliorate the systemic and pulmonary intoxication caused by one of the most field-used pesticides.Malathion is considered one of the vastest pesticides use all over the world. Malathion-inhalation toxicity commonly occurred in many occupational farmers. Therefore, this study aimed to ameliorate the possible malathion-induced pulmonary toxicity through thymoquinone administration. Forty animals were used to conduct our study, divided into five groups; G1 control group, G2 thymoquinone (50 mg/kg) group, G3 malathion group (animals inhaled 100 mg/ml/m About three million poisoning cases and more than 250,000 deaths occur due to pesticides every year2, where the most common pesticides are organophosphates. Organophosphates pesticides (OPs) lead to environmental pollution, causing great public concern1. Moreover, some OPs can be detected as residue in the soil, food, and food products3. So, the human body exposes to pesticides in three ways; dermal and oral in all humans or inhalation by agriculture workers, especially in countries that do not use pesticide safety measures4. When toxic amounts of OPs are inhaled, their first symptoms are usually a respiratory system with a bloody or runny nose, and wheezing in bronchial tubes because of constriction or excess of fluid5. The frequently and widely used organophosphate is malathion ethyl-O,O-di-methyl phosphorodithioate) with high toxicity6. Depending on the above-mentioned information, the metabolic and pathological adverse effects of malathion inhalation were studied in rats. Where Ruckmani et al.7 reported that malathion inhalation adversely affects blood glucose. On the other hand, Hectors et al.8, Rezg et al.9, and Beard et al.10 recorded its adverse effects on the metabolism of carbohydrate and lipid, immune, and nervous systems. Malathion adversely affects the liver, kidney, and hematological parameters11. Histopathologically, malathion increases alveolar macrophages and interstitial neutrophil infiltration and minimal fibrosis1.In agriculture, pesticides have been used in the eradication of insects and controlling vectors of the diseases12, and is a bioactive monomer, chemically is 2-methyl-5-isopropyl-1, 4-benzoquinone14. N. sativa is a plant commonly used for the treatment of various diseases, such as high blood sugar levels, asthma, and eczema in Arabian countries15. TQ characteristic in modern pharmacology varies as it contains active Quinone and has a range of therapeutics effects through anti-inflammatory and analgesic activities 16. Also, Abdel-Daim et al.17 reported that TQ enhances the biochemical and oxidative impacts of malathion, likely by reducing reactive oxygen and nitrogen radicals. Despite current research with animals, it is difficult to predict the degree that pesticide exposure by inhalation affects humans because the rats are resistant rather than human. This protocol examined the possible protective role of different doses of TQ against pulmonary, renal, and hepatic toxicity of malathion inhalation in rats through estimation of some serum biomarkers, pathology, gene expression, and immunohistochemistry.Thymoquinone (TQ) is the primary ingredient (approximately 30–40%) of black seed oil (Nigella sativa) Malathion , and thymoquinone from Sigma Al-drich Co., USA . All biochemical analysis kits were purchased from Biodiagnostics Company .18 and received vehicle distilled water aerosol, malathion group (G3) inhaled 1000 mg /m3 ,  500E, PCP # 4709. Dorchester, Ontario, Canada: Registration and Regulatory) for 15 min for 5 days/week for three weeks) and administered 1 ml of normal saline through the oral route, and the other two groups (G4 and G5) were also inhaled malathion and then given thymoquinone orally at two levels of dosing 25 mg and 50 mg/kg body weight for G4 and G5, respectively. Control and sham groups had a separate cage to avoid possible residues of malathion due to routine successive use. All animal handling procedures are in agreement with the ARRIVE guidelines from the National Center for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs)19 along the experimental period (21 days). The experimental protocol was accepted at Kafrelsheikh University, Egypt, by the institutional animal care and use committee 33516.Forty Sprague–Dawley male rats (220–250 g) were purchased from the laboratory animal house, Faculty of Agriculture, Kafrelsheikh University. Laboratory animals were housed in plastic cages in an air-conditioned room with a 12 h dark/light cycle and temperature 25 ± 2 °C and received standard laboratory balanced commercial diet and water ad libitum. The rats were kept for one week (adaptation period) before starting the experiment. The adapted laboratory animals were divided into , the rats were anesthetized by isoflurane then punctured retro-orbital plexus for collection of blood samples. The blood samples were collected on an anticoagulant for hematological and leukogram estimation. For serum biochemistry, the blood samples were collected without anticoagulant for serum separation. Haemtological parameters were performed by automatic Exigo vet 400 coulter . The activities of the serum enzymes , aspartate aminotransferase (AST), and alkaline phosphatase (ALP)) and serum concentration of urea and creatinine were estimated spectrophotometrically according to the instructions of Bio-Diagnostic Company research kits . Meanwhile, the total IgE level was analyzed by a sandwich ELISA kits at 450 nm wavelength .2 suffocation. All dead rats and remnants of samples and bedding material, were buried in the strict hygienically controlled properly constructed burial pit.The rats from different experimental groups were anesthetized by isoflurane and used for blood sample collection then euthanized using an intraperitoneal injection of an overdose of pentobarbital anesthesia (300 mg/kg). The remaining live rats were euthanized in strong bags by CO20. Histological examination was done using a Leica microscope. The quantitative assessment of the histopathological lesions was estimated within the animals of different groups. The lesions score was estimated according to nine points divided equally on the vascular lesions, pneumonia, and alveolar patency according to Yamanel et al.21 with some modifications. The vascular lesions varied from score 0, which revealed normal blood vessels, score 1 indicated congestion, score 2 indicated congestion, oedema, and hemorrhage, and score 3 indicated all the previous findings with marked loss of the tunica media of the blood vessels and extensive hemorrhage. Pneumonia score was assessed according to the following criteria; score 0 indicated normal lung, score 1 indicated mild interstitial pneumonia with the absence of the perivascular reaction, score 2 indicated a moderate degree of interstitial pneumonia with limited perivascular inflammatory cells infiltration, and score 3 indicated marked interstitial pneumonia associated with the marked perivascular reaction. The alveolar patency was classified into score 0 that indicated normal patent alveoli with thin interalveolar septa lined with type I and II alveolar cells, score 1 indicated mild thickening of the alveolar septa with patent alveoli, score 2 noted moderate thickening of the septa associated with decreasing the alveolar spaces and score 3 that revealed marked thickening of the connective tissue septa with marked decrease and sometimes showed focal obliteration of the alveoli.The lung samples of different animal groups were trimmed, fixed in neutral buffered formalin (10%), dehydrated, cleared, and embedded in paraffin wax. The hard paraffin blocks were sectioned to make a ribbon of tissue (5–6 µm thicknesses). The tissue ribbon was mounted on a water bath, fixed on a glass slide, and further stained with hematoxylin and eosin (H&E)22. In brief, the lung tissues were mounted on positively charged slides, rehydrated, and then put in EDTA solution PH 8 for antigen retrieval. The slides were treated with 0.3% of hydrogen peroxide for 15 min and protein block solution for 30 min. The slides were then incubated with rabbit polyclonal C-KIT  and polyclonal survivin antibody . The slides were rinsed three times with PBS and then incubated with anti-rabbit IgG secondary antibodies for 30 min . The slides were visualized with diaminobenzidine commercial kits  and counterstained with Mayer's hematoxylin. The primary antibody was replaced by the normal mouse serum as a negative control slide. The labeling index of survivin was expressed as the percent of positive area/mm2 using ImageJ analysis, NIH, USA.Survivin immunohistochemistry was performed according to Khalil et al.24 were designed as mentioned in Table The pulmonary tissue samples were collected from 5 laboratory animals from each group in clean Eppendorf tubes and stored at − 80 °C until used. RNA was extracted from the pulmonary tissues using the Qiagen RNeasy Plus Mini kit. Then, RNA concentration in each sample was detected by Nano Drop ND-1000 Spectrophotometer at 260 and 260/280 nm ratios . The cDNA was developed from the RNA samples using Maxima First Strand cDNA Synthesis Kit . Amplification was done using the Thermo Scientific MAXIA SYBR Green/ROXqPCR Master Mix (2×). Gene-specific PCR primershttps://www.graphpad.com. Variance was considered statistically significant when p ≤ 0.05.All values were expressed as mean ± SD. The data were analyzed by one-way ANOVA followed by Duncan’s post hoc test for multiple group comparisons using Graph Pad PRISM, software, La Jolla, CA, USA. The results of the hemogram were statistically (P ≤ 0.05) decreased in RBCs count, Hb, HCT, MCV, MCH, MCHC, and PLT in the G3 group as a result of malathion inhalation compared to other groups Table . MoreoveRegarding the result of WBCs, there was a statistically increase in WBCs count in the G3 group compared to other groups (P ≤ 0.05). The leukogram demonstrated a noticeable decrease of LYM and an increase of MON, EOS, BAS, and NEU in the G3 group as a result of malathion inhalation compared to the other groups (P ≤ 0.05)  Table . MoreoveThe lungs of control animals showed normal alveoli and bronchi. Moreover, the lungs of normal animals treated with thymoquinone revealed normal pulmonary tissues. The malathion inhalation revealed severe pulmonary tissue injury. The main pathological feature was interstitial pneumonia. The inflammatory lesions were mostly extended from perivascular to interalveolar interstitial tissues. The blood vessels showed a marked decrease in the tunica media and endothelial cell hypertrophy associated with perivascular oedema and hemorrhage. The perivascular areas showed marked inflammatory cell infiltration, mostly eosinophils and mononuclear cells as lymphocytes and macrophages. The alveolar spaces were markedly decreased associated with interstitial fibroplasia and hypertrophy, and hyperplasia of the pneumocyte type 2 cells. The bronchial tree showed the feature of catarrhal inflammation accompanied by severe desquamation of the bronchial lining cells, regenerative hyperplasia of the lining epithelium, and peribronchial infiltration of lymphocytes, macrophages, and with plenty of eosinophils and basophils. The diseased animals treated with thymoquinone showed a marked decrease in the pneumonic and allergic features in a dose-dependent manner. Malathion and a low dose of thymoquinone showed limited perivascular infiltration of inflammatory cells mostly lymphocytes and macrophages. The interstitial reaction was also decreased with a marked increase in the alveolar spaces. The high dose of the thymoquinone noted a marked decrease of perivascular and interstitial reaction, and most of the alveoli and bronchi were patent without any noticeable inflammation. Quantitative scoring of the histopathological lesions revealed a marked decrease in the lesion score in both thymoquinone-treated groups (p ≤ 0.005)  antibody was used for detection of mast cells within the pulmonary tissues Fig. . The conThe survivin immunostaining of the lungs of the control animal showed slight within the alveolar cells and moderate expression with the bronchial lining epithelium. A similar reaction was noticed with the same group. In the lungs of malathion-inhaled animals, marked expression of survivin within the alveolar and bronchial lining epithelial cells consistent with the hypertrophied pneumocyte type 2 alveolar cells and the regenerative bronchial lining cells. Diseased animals treated with thymoquinone showed a marked decrease in the percent of survivin immunostaining within the pulmonary tissues in a dose-dependent manner in comparison with the malathion treated group (P ≤ 0.001) Fig. .Figure 5As shown in Fig. 25. Haematological blood profiles may provide valuable information on the organism's internal environment26. In the present study, malathion inhalation badly affected the blood hematology of the rats. These results are compatible with Kanu et al.27 and Ghazy et al.28 who reported that other OPs are associated with a significant decrease in the hematological parameters, especially RBCs, hemoglobin, and platelet counts, and with a significant increase in the WBCs count. Moreover, Ghazy et al.28 reported decreases in erythrocytes could be as a result of either direct erythrocytes destruction by the pesticide or indirect through its bad effects on the bone marrow. Coles29 stated that low packed cell volume promotes reticulocyte released from bone marrow, which increases the MCV. The increment in the MCV and reduction in the MCHC post malathion exposure suggested either haemolytic or hemorrhagic anemia. The obtained results suggested the incidence of macrocytic hypochromic anemia, Where insecticide can initiated erythrocytes destruction as it enhanced the production of reactive oxygen species (ROS), which had an unfavorable effect on erythrocytes membrane. Kalender et al.30 attributed the occurrence of macrocytic hypochromic anemia to either interference of the malathion with hemoglobin synthesis or reduction in the RBC life span. The platelet count reduction may be caused by the bone marrow depression by free radicals, which decrease PLT production or depress thrombopoiesis31. These adverse effects on the hematological parameters were greatly ameliorated by TQ supplementation. These results are consistent with Ghazy et al.28, the alleviation of anemia induced by diazinon toxicity by TQ administration. The improved hematological parameters may be attributed to the ameliorative effect of TQ against free radicals caused by malathion inhalation33. Malathion was also associated with the inflammation either directly through pro-inflammatory cytokines or subsequently due to the production of the free radicals that leads to the formation of MDA34. Inflammation was manifested by a significant increase of leukogram in the malathion inhalation group. These results were correlated well with the histopathology results that appeared by marked perivascular and peribronchial inflammatory cell infiltration. This inflammatory process improved by TQ, which has anti-inflammatory properties35.It is noteworthy that respiratory failure is the prime mover of deaths caused by acute OPs toxicity37. On the other hand, there are several parameters related to renal diseases, most of them deliver information only when renal damage is well established, as is the case for serum creatinine38. The results of serum biomarkers of the liver and kidney tissue damaged were statistically affected by malathion inhalation. These results may be attributed to the oxidative damage caused by malathion inhalation on the liver and kidney tissues. These results are in agreement with Al-Attar39 and Jabbar et al.40 who reported a significant increase in the serum activities of AST, ALT, and ALP and concentration of the urea and creatinine. Moreover, Kanu et al.27 recorded the toxic effects of dichlorvos on the liver and kidney of the rats. Abouelghar et al.41 reported hematological and biochemical alterations induced by sub-acute exposure to the fipronil in albino mice. These results were confirmed by the results of the liver and kidney histopathology. The histopathological results of the hepatic and renal tissues as a result of malathion inhalation are constant with the result of Mamun et al.42 in the mice. Moreover, Selmi et al.43 reported severe damages in hepatic and renal tissue in prepubertal male mice treated with malathion. Thus, malathion inhalation induced deterioration in the liver and kidney function, which improved by TQ supplementation. This improvement may be a result of the antioxidant effect of TQ32. These results are in harmony with El-Sheikh et al.44 who reported that TQ has hepatorenal protection against methotrexate-induced toxicity in the rats through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms. Moreover, Ghazy et al.28 reported the protective effect of TQ against the hepatotoxic effect of diazinon in male rats. In addition, Aboubakr et al.45 and Farid et al.46 recorded the antioxidant protective effect of the natural substances against tilmicosin and Carbon tetrachloride toxicity in the rats, respectively.Liver damage as a result of toxicity or alterations in the membrane architecture of the liver cells can be diagnosed by measuring ALT and AST activities47. Interestingly, malathion is increased basophils, activated mast cell degranulation process, and potentiated macrophages48. Moreover, it was noticed that most of the pulmonary blood vessels showed atrophy and loss of the media and endothelial cells, respectively. This might clarify the thrombotic activity of the organophosphorus compounds49. The adverse effect of the malathion inhalation was mitigated by TQ supplementation in a dose-dependent manner. The present results are going with Al-Gabri et al.50 that noticed that TQ alleviates the lipopolysaccharides-induced lung injury through the reduction of inflammatory edema, thickening of interalveolar septa, hypertrophy of the smooth muscles around the blood vessels and airways, and hyperplasia of the bronchial associated lymphoid tissue. Moreover, TQ has potent anti-inflammatory, spasmolytic, and bronchodilator effects51. These explained to increase the pulmonary alveolar spaces in diseased rats treated with TQ. Malathion was associated with marked regenerative hyperplastic changes of type 2-alveolar epithelial cells. Most of the pulmonary toxicants are accompanied by hypertrophy and hyperplasia of type 2 epithelial cells53. Where the survivin expression increased in the malathion inhalation group. Survivin is a member of the inhibitor of apoptosis protein (IAP) family, regulates mitosis, and chromosome segregation55. Survivin is mostly undetectable in the normal adult differentiated tissues56 and highly expressed in the undifferentiated cells as a notice in many human tumors58. Although, increase tissue damage may be associated with suppression of antiapoptotic genes including survivin60. However, it was reported an interesting dignity of the survivin expression associated with a chronic pulmonary injury, which mostly associated with regenerated alveolar cells and infiltrated inflammatory cells. In addition, the results support the cytoprotection role of the survivin against pulmonary61.All the hematological results in malathion and/or TQ groups were co-related with the results of the histopathology, immunohistochemistry, and gene expression in the lung tissues. The lung of the malathion group showed bronchial and alveolar obliterative lesions associated with marked inflammatory cell infiltration either peribronchial or interstitial. The interesting finding was the allergic features associated with inflammatory cell infiltration as eosinophils and mast cells, macrophages, and lymphocytes. There was a great relationship between the development of asthma and eosinophils infiltration, which may be associated with the production of cytokines responsible for the development of type II sensitivity62. Malathion group revealed a significant decrease of SP-D mRNA. While treatment of the diseased rats with TQ markedly elevated the SP-D mRNA. It is noteworthy that SP-D acts allergy antagonist through decreased IgE secretion, controlled cytokines produced by Th2, and decreased eosinophilia that mostly abrogates pulmonary inflammation64. From the aforementioned results, it had been concluded that chronic exposure of malathion inhalation resulting in pulmonary lesions mimicked allergic pneumonia that associated with high serum IgE level, eosinophils, and mast cell tissue infiltration, and low pulmonary surfactants. The administration of TQ markedly ameliorated the malathion-induced pulmonary allergy and obstructive lesions. Therefore, thymoquinone administration could be beneficial for insecticides applicators in crop fields to season.Alveolar type II epithelial cells were the main source of different pulmonary surfactant SP-A, SP-B, SP-C, and SP-D. There was a marked association of increased serum SP-D and progression of chronic obstructive pulmonary disease (COPD), therefore, it has been committed as a biological indicator of COPD statusThe current study was approved by the institutional animal care and use committee Kafrelsheikh University, Egypt, 33516.


Intervertebral disc degeneration (IDD) is the main contributor to low back pain, which is a leading cause of disability worldwide. Although substantial progress has been made in elucidating the molecular mechanisms of IDD, fundamental and long‐lasting treatments for IDD are still lacking. With increased understanding of the complex pathomechanism of IDD, alternative strategies for treating IDD can be discovered. A brief overview of the prevalence and epidemiologic risk factors of IDD is provided in this review, followed by the descriptions of anatomic, cellular, and molecular structure of the intervertebral disc as well as the molecular pathophysiology of IDD. Finally, the recent findings of intervertebral disc progenitors are reviewed and the future perspectives are discussed. Pathomechanism of intervertebral disc degeneration. Several risk factors including genetic factors as well as mechanical stress, trauma and smoking, attributed to the development of intervertebral disc degeneration. These biological and environmental factors induce the reduction of cell numbers and transformation of intervertebral disc cells through several molecular mechanisms, resulting in decreased production of extracellular matrix due to increased catabolic activity and decreased anabolic activities. Thereafter, the structured integrity of intervertebral disc is lost and intervertebral disc degeneration is further accelerated. In 1995, the prevalence of IDD, based on magnetic resonance imaging (MRI) findings, was described in a population‐based cohort in Finland.3Before mid‐ to late 1990s, repetitive mechanical loading or wear and tear were believed to cause IDD. However, recent family and twin studies have suggested that the occurrence of IDD is determined largely by genetic factors, with environmental factors having an important role.3.1ACAN,CLIP,COL1A1,COL9A2,COL11A1,GDF5,IGF1R,IL‐1,IL‐6,MMP2,MMP3,MMP9,SKT,THBS2,VDR.PARK2CHST3.Genetic influences predominate among the reported risk factors for IDD. In 1995, a population‐based cohort study was performed using 115 male monozygotic twin pairs, in which IDD was evaluated by MRI.3.2Excessive mechanical stress is thought to induce IDD, considering that IDD is more frequently observed in the lower lumbar spine, where IVDs suffer higher mechanical stress,3.3A study involving well‐matched cohorts, including 50 subjects who underwent discography and 52 control subjects, revealed that compared to matched controls, subjects who underwent discography showed accelerated IDD at 7 to 10 years of follow‐up; the progression of disc degeneration assessed by MRI was observed in 54 discs (35%) in the discography group compared to 21 discs (14%) in the control group.3.4Smoking is the only chemical exposure known to be associated with the onset of IDD. On investigating the cohort of monozygotic twin pairs with different smoking exposures, Battie et al. found slightly greater IDD scores in the lumbar spine of smokers than in the lumbar spine of nonsmokers.4The IVD is composed of different but interrelated tissues, including the central highly hydrated gelatinous nucleus pulposus (NP), surrounding elastic and fibrous annulus fibrosus (AF), and cartilaginous end plates (CEP), which provides connection to the vertebral bodies Figure .40 The NThe AF consists of a series of concentric rings, or lamellae, with collagen fibers lying parallel within each lamellae, providing tensile strength and the ability to withstand forces applied from any direction.The CEP is a layer of hyaline cartilage that covers the caudal and cephalic ends of the disc, which plays an important role in the transport of fluids and solutes in/out of the disc.5IDD can be attributed to several factors, including genetic factors as well as aging, mechanical stress, and injury. These biological and environmental factors induce the reduction of cell number and transformation of IVD cells, resulting in decreased production of ECM of IVD owing to increased catabolic activity and decreased anabolic activities. Thereafter, the structural integrity of IVD is lost and IDD is further accelerated.5.1In humans, a general decrease in type II collagen production and a shift to type I collagen synthesis by NP cells or inner AF cells is observed as IDD progresses.5.2Similar to the changes in the expression of collagens, a decrease in the proteoglycan content of human IVD is observed during degeneration.5.3During the process of development of IDD in humans, several histological findings are observed in NP, including loss of demarcation between NP and AF owing to the shift of synthesis from type II collagen to type I collagen, dehydration caused by the decrease in proteoglycan production, presence of fissure, and cell cluster formation Table .55 With 5.4Organismal aging results from time‐dependent accumulation of molecular and cellular damage, which leads to impaired tissue homeostasis, and eventual physiological and functional decline.Ercc1−/Δ mice, in which ERCC1‐XPF is deficient (involved in DNA damage repair), showed typical features of disc aging, including loss of proteoglycan, decreased IVD height, and an increase in the number of senescent cells.With recent progress in aging research, some of the molecular pathways leading to disc aging have been elucidated. In particular, genomic instability and resulting cellular senescence have been determined to be important drivers of IDD.INK4A, were increased in human IVD samples. These markers were positively correlated with the expression of matrix metalloproteases including matrix metalloproteinase 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5).There are two types of cellular senescence, namely replicative senescence and stress‐induced premature senescence (SIPS). Replicative senescence is characterized by cessation of cell proliferation due to critical telomere shortening after consecutive replicative cell cycles.5.5Various kinds of inducers have been used to reproduce IDD in experimental animals, including compression,6The presence of local progenitors or the recruitment of appropriate cells into the damaged sites is required for tissue maintenance and repair.6.1Considering the harsh microenvironments in IVD, activation of endogenous progenitor cells could be a promising therapeutic strategy for IDD. In 2007, using an explant culture to isolate progenitors from degenerate human discs, Risbud et al. identified cells from both NP and AF, expressing typical marrow mesenchymal stem cell markers such as CD105, CD166, CD63, CD49a, CD90, CD73, p75 low‐affinity growth factor receptor, and CD133/1, and these results were also confirmed in rat IVDs.+GD2− cells were found to be precursors of Tie2+GD2+ cells, and CD24, which was previously reported to be a specific marker of the NP,+GD2+ cells. Using these markers, NP cells were classified into four subtypes: dormant stem cells (Tie2+GD2−CD24−), self‐renewing stem cells (Tie2+GD2+CD24−), committed NP progenitor cells (Tie2−GD2+CD24+), and mature NP cells (Tie2−GD2−CD24+).+ cells, enabling the development of a strategy to stimulate ANG‐1 to enhance Tie2+ progenitor cells for prevention of IDD.Sakai et al. identified NP progenitors with novel NP‐specific cell markers, and demonstrated that these progenitors are exhausted with aging and degeneration.6.2Some of the aforementioned progenitor cells maintain their potential multipotent differentiation and self‐renewal in vitro; however, knowledge regarding their in vivo characteristics, such as development, localization, or functional role in the maintenance of IVD homeostasis, is lacking.The methods to identify progenitors in vivo include label retention assay and lineage‐tracing experiments. In label retention assay, synthetic nucleic acid analogs, such as 5‐bromo‐2‐deoxyuridine (BrdU) or 5‐ethynyl‐2′‐deoxyuridine (EdU), are used to detect slow‐cycling cells, which are thought to be potential progenitor cells. A prolonged chase period results in dilution of the incorporated nucleic acid analogs, although the slow‐cycling cells remain labeled.loxP‐STOP‐loxP sequence.STOP sequence.Lineage tracing provides more direct evidences, allowing us to follow cell fate decisions of progenitor cells and their descendants within a living organism without any perturbation.7Recent population‐based cohort studies showed that IDD is very common and is associated with the presence of LBP and suggested that the degeneration of CEP has an important role in the pain associated with IDD. Previous epidemiologic studies have clearly shown that IDD is highly heritable. Although candidate gene approaches, family linkage analyses, and recent genome‐wide association studies have revealed several IDD‐sensitive genes, further studies are warranted to elucidate the molecular mechanisms through which each candidate gene causes IDD. The pathomechanisms of IDD have been elucidated using several animal models. However, caution should be exercised while interpreting the information obtained from animal models, as there are many differences between species, including disc size, cell type, nutrition, and mechanical forces.The authors declare no conflicts of interest.T.O., Y.T., Y.O., S.T., and T.S. contributed the concept of the paper and wrote the manuscript. All authors have read and approved the final submitted manuscript.


Adequate antenatal care (ANC) services are key for early identification of pregnancy related risk factors and maintaining women’s health during pregnancy. This study aimed to assess the influence of ANC provided by the Medical Mission Primary Health Care Suriname (MMPHCS) and of ethnicity on adverse birth outcomes in Tribal and Indigenous women living in Suriname’s remote tropical rainforest interior.≤1 vs. > 1) and antenatal visits utilization (≥8 vs. < 8) in 15 interior communities were retrospectively analyzed using descriptive statistics, crosstabs and Fisher’s exact tests.From April 2017 to December 2018 eligible Tribal and Indigenous women with a singleton pregnancy that received ANC from MMPHCS were included in the study. Data on low birth weight (LBW < 2500 g), preterm birth (PTB < 37 weeks), low Apgar score (< 7 at 5 min), parity .A total of 204 women were included, 100 (49%) were Tribal, mean age was 26 ± 7.2 years and 126 women (62%) had 8 or more ANC visits. One participant had a miscarriage; 22% had adverse birth outcomes: 16 (7.9%) LBW and 30 (14.8%) PTB; 7 women had a child with both PTB and LBW; 5 women had stillbirths. None of the newborns had low Apgar scores. Maternal age, ethnicity, ANC and parity were associated with PTB (χDespite an almost 100% study adherence over one fifth of women that received ANC in the interior of Suriname had adverse birth outcomes, in particular PTB and LBW. Younger nulliparous Indigenous women with less than the recommended 8 ANC visits had a higher risk for PTB. The rate of adverse birth outcomes highlights the need for further research to better assess factors influencing perinatal outcomes and to put strategies in place to improve perinatal outcomes. Exposure assessment of this sub-cohort and neurodevelopment testing of their children is ongoing and will further inform on potential adverse health effects associated with environmental exposures including heavy metals such as mercury and lead. Antenatal care (ANC) services provided by healthcare professionals are key for early identification of pregnancy related risk factors and maintaining women’s health during pregnancy. During 2010–2015, ANC coverage, defined as the percentage of pregnant women aged 15–49 years who attended at least one ANC visit with a skilled health care provider, was approximately 85% globally and 77% in the least developed countries . AdequatSuriname is an upper middle income country in northeast South America with an estimated population of 575,991 ; 10% of This study aimed to evaluate prenatal care, as measured by the quality and number of ANC services provided by MMPHCS on birth outcomes among pregnant women in Suriname’s interior. We hypothesized that adequate prenatal care would have a positively influence on birth outcomes. Also, we assessed potential differences in birth outcomes between Tribal and Indigenous communities.In this prospective cohort study a review of the ANC services was conducted to identify the quality of ANC provided by the MMPHCS. ANC was defined as the total package of care that pregnant women receive from an organized health facility. The three components considered for quality of ANC were: 1) the timing of first ANC visit 2) the number of ANC visits and 3) health services provided during each visit. According to the WHO these three components ensure timely risk identification, prevention and management of pregnancy-related or current diseases and health education and health promotion.MMPHCS provides primary healthcare services 24/7, including emergency care, maternal and childcare, family planning, management of communicable and non-communicable diseases, school health program, dental care, home visits to elderly and health education and promotion activities. The ANC guidelines include: monitoring of weight, blood pressure and blood sugar, deworming, toxoid immunization, malaria testing and treatment, counseling and testing for Human Immunodeficiency Virus, Syphilis and Hepatitis B, blood and urine testing, iron and folic acid supplementation, uncomplicated delivery at MMPHCS healthcare facility, early identification and referral of high risk pregnancies, health education regarding pregnancy and breastfeeding, and home visits. Adequate ANC services was defined as at least eight ANC contacts with the MMPHCS healthcare provider starting from the first trimester of pregnancy.There are no specialized neonatal care facilities in the interior. Newborns that need special care are immediately transported to the hospitals in the city. Preferably pregnant women in premature labor are referred in time to the capital city. Reasons for referral to the gynecologist would be risk factors such as grand multiparous women, adolescent nulliparous, hypertension, obesity, anemia or previous C-sections.The study population is a subset of the overall Caribbean Consortium of Environmental and Occupational Health (CCREOH) environmental epidemiologic cohort study that examines the effects of chemical and non-chemical stressors on birth outcomes and pediatric neurodevelopment in mother/child dyads in Suriname. To date 1069 mothers and their infants are included [Women aged 16 years and older with a singleton pregnancy who had registered at one of 15 randomly selected MMPHCS health centers were eligible to enroll in the study  for continuous variables and numbers/proportions for categorical variables including age, ethnicity, parity, number of antenatal visits, preterm birth, birth weight, and Apgar score at 5 min.Categorical variables were assessed using crosstabs. Socio-demographic variables were re-coded for the logistic regression models as follows: age at delivery as < 20 and ≥ 35 vs. 20–34 years; parity as nulliparous or one previous live birth vs. more than one previous live birth; and ethnic group as Tribal vs. Indigenous.The dependent variables preterm birth (PTB), low birth weight (LBW) and Apgar score (AS) were recoded as binominal variables and defined as follows: PTB as < 37 vs. ≥37 weeks, LBW as < 2500 vs. ≥2500 g, and Apgar score as < 7 vs. ≥7 at 5 min. Gestational age was calculated using the last menstrual period (LMP). Newborns < 28 weeks were excluded from the analyses. Crosstabs were used to explore associations between each of the outcome variables. Differences in proportions between variables were tested with the Pearson’s chi-squared test and if assumptions were not met, Fisher’s exact test was used. Multiple logistic regression was used to develop predictive models for the outcome variables. Variables included in the logistic model were based on the results of the bivariate associations. All analyses were conducted using IBM SPSS version 20 and EPI INFO™ version 7.2.2.6.Written informed consents in the native languages Saramaccan and Trio were obtained. Ethical approval was granted by the Institutional Review Board of the Government of Suriname (VG 023–14) and the Tulane University Institutional Review Board. Participants were enrolled from April 2017 through December 2018.Of the 206 eligible pregnant women, 203 were ultimately included in the study  of the participants was 26 years (7.2); 46 (22.5%) were <  20 years of age of which 16 (34.8%) were from a Tribal community and 30 (65.2%) were Indigenous (Table 116 (72%) participants’ first ANC visit was within the first 4 months of pregnancy. 201 (99%) deliveries were facility-based with a skilled birth attendant present; two women delivered at home. 126 (61.8%) women had eight or more ANC visits, data on ANC visits were missing for 17 (8.3%) of cases. 60% of the Indigenous women had less than 8 ANC visits compared to 48% of the Tribal women (Table 77 (38%) of the women delivered at one of the health facilities in the interior, 127 (62%) were referred to the hospital for delivery. 76 (59%) of the referred participants were of the Tribal and 52 (41%) of the Indigenous community. Reasons for referral for Tribal and Indigenous women were respectively: teenage pregnancy and nulliparity 11 (14.5%) vs. 16 (30.8%), grand multiparity 25 (37.9%) vs. 7 (11.5%) and other  40 (31.2%) vs. 29 (22.7%).10 (4.9%) participants from the Tribal community had pregnancy-induced hypertension of which 3 women developed pre-eclampsia. One participant in the Tribal community had diabetes gravidarum. None of the Indigenous women had either hypertension or diabetes. 187 (92%) of the participants had vaginal delivery. A total of 198 (97%) women had live births, five participants had a stillbirth , and one Tribal participant had a miscarriage.The distribution of birth outcomes is presented in Table 2 = 11,83, p = 0.005, χ2 = 4,97, p = 0.026, χ2 = 17,45, p < 0.001, χ2 = 11,47, p < 0.001 respectively. Multivariate analysis for place of birth as dependent and PTB, age, ANC, ethnicity, parity and BW as independent variable showed an association for ANC  and ethnicity  for women who were referred to the hospital.Maternal characteristics associated with LBW and PTB are presented in Table We aimed to assess the utilization of antenatal care and its effect on birth outcomes, specifically PTB and LBW, and potential differences between Tribal and Indigenous pregnant women from Suriname’s interior receiving ANC services from the MMPHCS. ANC adherence of the women enrolled in the study was appropriately high: nine out of ten women returned for follow up visits until the end of their pregnancy. 97% of women had a live birth, 62% were referred to the hospital, 22% had adverse birth outcomes of which 56.5% in the referral group. PTB was positively associated with less number of ANC visits, young age, Indigenous ethnicity, and nulliparity. LBW was not associated with any of these risk factors.Despite the high ANC adherence, the 14.8% PTB rate observed in this study population was higher compared to the rate of 12% in lower income countries and the 9% rate in high-income countries  and was 8% of the women in this sub-cohort had a newborn with low birth weight, lower than the incidence of LBW in the total cohort  and the country-wide rate of 14.7% , these wNeither maternal age, parity nor ANC were associated with birth weight in our study. Several factors that were not included in the analyses of this sub-cohort study could be of influence such as maternal pre-pregnancy weight and weight gain during pregnancy , hyperteTwo-thirds of our Indigenous study population was younger than 20 years compared to 35% of the Tribal women. Frequent ANC attendance is particularly important in pregnant adolescents aged 15–19 as complications have been shown to be higher among these teenage girls . In thisParity was associated with preterm birth: women with first time pregnancies had a higher risk of preterm labor. Studies on the association between parity and preterm birth are inconclusive in their findings: in a meta-analyses no association was found , while oIn our study utilization of ANC services were 100% covered for at least one visit during the pregnancy. In 2016, the World Health Organization (WHO) updated the Antenatal Care model with recommendation as follows: ANC should commence within the first 4 months of pregnancy and women should have at least eight ANC visits during the course of an uncomplicated pregnancy . AlthougWomen living in remote interior communities in Suriname, where mercury is abundantly used in artisanal and small-scale goldmining activities, are known to have high concentrations of mercury in hair . These hOne of the strengths of our study is that all pregnant women in our study population received a standardized package of ANC care which covered all the recommendations indicated by WHO. In addition, the data were abstracted from accurately documented medical records. This is also the first study to evaluate the impact of ANC utilization with an almost 100% adherence and frequency of adverse birth outcomes in the interior of Suriname.The study included some limitations: we did not assess the quality and content of ANC as this may influence the ANC across the different MMPHCS health facilities; secondly, we did not examine the mother’s history of PTB and LBW; mothers with previous PTB or LBW are more likely to have babies with LBW . Also, gDespite the high attendance of ANC, over one fifth of women living in the interior of Suriname that received antenatal care from the Medical Mission Primary Health Care Suriname had adverse birth outcomes, particularly preterm birth and low birth weight. Younger nulliparous Indigenous women with less than the recommended 8 ANC visits were particularly at risk for PTB. The high rate of adverse birth outcomes highlights the need for further research to better assess the factors influencing perinatal outcomes and also strategies to put in place to improve perinatal outcomes. Exposure assessment of this sub-cohort and neurodevelopment testing of their children is ongoing and will further inform other potential adverse health effects associated with environmental exposures. A client satisfaction survey is recommended to evaluate the ANC services provided by the MMPHCS.