In a double-blind, placebo-controlled trial, prothrombin time, activated partial thromboplastin time, international normalized ratio and bleeding time were measured in patients with Alzheimer’s dementia at baseline, weeks 6 and 26. A total of 513 patients were randomized to 120 mg (n = 169) or 240 mg EGb 761® (n = 170) or placebo (n = 174). No relevant changes were found for coagulation parameters and bleeding time. Numbers of bleeding-related adverse events were similar in all groups. Concomitant intake of acetylsalicylic acid was documented for 68 patients in the placebo group and 105 in the EGb 761® groups. Within these groups, the means at baseline and week 26 differed by less than 1 unit for prothrombin time and bleeding time and less than 0.1 unit for international normalized ratio. Data on warfarin treatment in nine patients each taking placebo or EGb 761® did not indicate enhancement of warfarin effects by EGb 761®. No evidence was found that EGb 761® affects hemostasis or increases the bleeding risk. No pharmacodynamic interactions with warfarin or acetylsalicylic acid were found.Following reports of bleeding upon Ginkgo intake, we assessed whether Ginkgo extract EGb 761 Analyses of aggregate spontaneous reports from the US and Australia conclude that the intake of Ginkgo preparations may lead to an increased risk of bleeding due to interactions with warfarin ,2. IndivThe retrospective analysis of a large database  in the US addressed the risk of bleeding in patients on warfarin who concomitantly took “Ginkgo” as a food supplement. The results were interpreted as suggesting that taking Ginkgo preparations concomitantly with warfarin increases the patient’s risk of a bleeding-related adverse event . A descriptive analysis of spontaneously reported adverse drug reactions (ADR) to Ginkgo biloba from 2000 to 2015 was performed in the electronic database of the Therapeutic Goods Administration in Australia . ® to affect blood coagulation or cause bleeding events or pharmacodynamic interactions with warfarin and/or ASA, we analyzed still-unpublished safety data from an already reported double-blind, placebo-controlled trial [To further elucidate the potential of EGb 761ed trial . Data weMethodological details, a description of the patient sample and the main results of the trial have been published elsewhere . The stuOutpatients were recruited by 44 clinical sites including university hospitals, private practices and research institutes specialized in neurology or psychiatry across the United States. The participants were at least 60 years of age and were enrolled if they had probable dementia of the Alzheimer’s type in accordance with the diagnostic criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association, NINCDS/ADRDA , with mi® is a dry extract from Ginkgo biloba leaves ) in compliance with the quality standards of the European Pharmacopoeia. The extract is adjusted to 22–27% Ginkgo flavonoids calculated as Ginkgo flavone glycosides and 5–7% terpene lactones consisting of 2.8–3.4% ginkgolides A, B, C and 2.6–3.2% bilobalide and contains less than 5 ppm ginkgolic acids. Film-coated tablets as well as placebo tablets were manufactured and supplied by Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany, the sponsor of the trial. Drug and placebo tablets were indistinguishable by size, shape and colour.The study drug EGb 761Concomitant treatment with antidementia medications or other drugs acting on the central nervous system was not permitted during the study period. Patients who needed anticholinergic and psychoactive drugs, such as antidepressants, antipsychotics, hypnotics, and tranquilizers, except for occasional doses of short-acting benzodiazepines for restlessness or sleep disturbances, were excluded from the study. The concomitant intake of nonsteroidal anti-inflammatory drugs was permitted at doses that were stable for at least 6 months. Other comedication potentially affecting the coagulation, i.e., warfarin or coagulation disorders, were not excluded. In warfarin-treated patients, the INR was watched closely and kept within the prespecified range , by adapting the dosage regimen of warfarin if necessary. ® (60 mg or 120 mg twice daily) or placebo for 26 weeks. Study drug and placebo were packed and labelled in an indistinguishable manner according to the randomization list, and the investigators as well as the whole study team remained blinded with respect to the treatment assignment until final lock of the database. Follow-up examinations were scheduled at 6, 12, 18 and 26 weeks after baseline. Standard outcome variables were employed to assess efficacy in dementia. Safety was evaluated by means of laboratory tests and by assessment of adverse events (AEs) that occurred after intake of the first dose of study medication. After a medication-free four-week period, patients underwent the baseline examination and were randomized to one of three treatment groups taking 120 mg or 240 mg EGb 761PT , APTT 88 and INRMeasurement of BT was performed at all 44 sites which were able to comply with Clinical Laboratory Improvement Amendments (CLIA) as the US federal regulatory standards for clinical laboratories. ® procedure [® device. The flowing blood was wicked with filter paper at regular intervals of 30 s. The filter paper was brought close to the incision, but care was taken not to touch the wound and the forming platelet clot. BT was determined as the time when blood no longer stained the filter paper, rounded up to the nearest 30 s time point.BT to test platelet function was measured in an area on the lateral aspect of the volar surface of the forearm, approximately 5 cm distal to the antecubital fossa, employing the established Simplaterocedure . After lDescriptive statistics were calculated for the blood coagulation parameters and BT at baseline, after 6 and 26 weeks of treatment. These sample characteristics together with the patients’ individual values were assessed for relevant changes during treatment at the level of treatment groups as well as individual patients. The analyses are based on the intention to treat (ITT) analysis set. If blood coagulation parameters or BT of patients who dropped out prematurely were available at the end of the individual treatment period, these values were used for the analyses. Missing values were not replaced. This is a conservative approach for safety analyses since it can be expected that treatment effects are not underestimated.(1)Those taking neither ASA nor warfarin as concomitant medication;(2)Those taking ASA concomitantly;(3)Those taking warfarin concomitantly.Separate analyses were performed for three groups of patients:® and 240 mg EGb 761® for each timepoint separately using t-tests. Since the third group was very small, the outcomes were compared between patients treated with EGb 761® (120 or 240 mg) and placebo using t-tests. Two-sided p-values < 0.05 were considered statistically significant; p-values ≥ 0.20 were regarded as a hint that there were no relevant differences between the compared treatment groups. The threshold of 0.2 is used since this indicates that the type-one error rate for concluding an increased risk is at least 20%. Within the first two groups, coagulation parameters and BT were compared between active treatment groups and placebo and between 120 mg EGb 761® high-dose group) took both ASA and warfarin; thus, the group was too small to be evaluated separately. No bleeding event was observed in the individual patient taking EGb 761®. This patient’s values for PT, APTT and INR showed some fluctuation around the upper limits of the reference ranges. As a conservative approach, these three patients were added to both subgroups per concomitant medication. Three patients  or 240 mg EGb 761® (n = 170) or placebo (n = 174). At baseline, blood coagulation tests  of 499 patients could be analysed. BT measurements at baseline were available from 278 patients. ® 120 mg: 20.1% and EGb 761® 240 mg 17.6%) dropped out prematurely and 80% completed the trial as scheduled. The following reasons were documented in the treatment groups (placebo/EGb 761® 120 mg/EGb 761® 240 mg): withdrawal of informed consent (n = 13/9/10), AEs (n = 7/8/10), SAEs (n = 3/3/5), lack of efficacy (n = 3/8/2) and other reasons (n = 13/6/3).In total, 513 patients were randomized to take either 120 mg , selective serotonin reuptake inhibitors (SSRIs) or serotonin and noradrenaline reuptake inhibitors (SNRIs), which might affect platelet aggregation or bleeding time, was similar in all treatment groups: 60, 59 and 66 cases of NSAID use and 2, 2 and 1 cases of SSRI/SNRI use in the placebo, 120 mg and 240 mg EGb 761® (both doses)  . At bothh doses) . At baseon or BT .® groups took concomitant ASA, mostly for stroke prevention or as an analgesic. The median dose of ASA was 325 mg per day (range 81–700 mg) in the placebo group at baseline (n = 67), 200 mg (range 81–1000 mg) in the group treated with 120 mg EGb 761® (n = 53), and 325 mg (range 81–650 mg) in the group taking 240 mg EGb 761® (n = 48). Overall, more than 90% in each group were taking less than 500 mg ASA per day. Overall, 68 of the patients in the placebo group and 105 in the EGb 761® groups showed no relevant differences. Obviously, EGb 761® did not enhance the antiplatelet effects of ASA . The means of APTT were slightly lower (1.1 and 1.5 s) in the active treatment groups than in the placebo group (p = 0.196 and 0.094). At baseline, the mean/median value of 98 patients with available values showed a prolonged bleeding time as expected from the pharmacological action of ASA . After 6s of ASA . At basep = 0.189 for the comparison of PT between 120 mg EGb 761® and placebo with a higher mean in the placebo group, p ≥ 0.200 for the other comparisons at week 26). At the end of the active treatment period, the mean APTT of patients treated with 120 mg and 240 mg EGb 761® were 1.06 and 1.03 s shorter, respectively, than in the placebo group (p = 0.083 and p = 0.127). These differences were not clinically relevant, and they were already present at baseline. In the high-dose group, the bleeding time, which was slightly higher than in the low-dose group already at baseline, increased somewhat until week 6, but returned to baseline level until week 26. Minor differences between the two doses of EGb 761® were observed after 6 weeks of treatment for PT and INR which were statistically significant but not clinically relevant. There were no consistent dose- or time-dependent changes, and after 26 weeks of treatment there was no dose-dependent effect of EGb 761® on any parameter of blood coagulation or BT  and placebo  in the 240 mg EGb 761® group. There is no hint of an increased risk of bleeding events for patients treated with EGb 761® .In total, 37 bleeding events affecting 30 patients were documented in the treatment groups . Fourtee® groups  were affected by bleeding events, for whom no concomitant anticoagulant drug exposure was documented. However, in six of them, evident other causes such as invasive cancer or fall- or accident-related injuries were present, and they were not assessed as causally related to the study medication. Of these events, the majority occurred in patients who took at least one concomitant drug with known anticoagulant side effects. Only eight patients in the EGb 761n = 4 in the EGb 761® groups, n = 1 in the placebo group) as potentially related to the study medication. Of these, only the individual patient in the placebo group did not take any concomitant medication with a known anticoagulant effect. The investigator assessed seven bleeding events in five patients  these events were due to lab errors. In one patient who had discontinued 120 mg of EGb 761® long before, this was due to a serious adverse event (hepatitis) not related to the study drug; and two cases  the event was related to warfarin treatment.A few test results that were above the reference range were assessed by the investigators as being clinically relevant. One patient taking 120 mg EGb 761® and four bleeding events in 3 of 48 patients taking ASA and 240 mg EGb 761®. Bleeding-related adverse events were not documented in any of the patients in the warfarin group. Slight changes in coagulation parameters or bleeding time were considered to be not clinically relevant by the investigators. Eleven bleeding events in 7 of 68 patients taking ASA and placebo were recorded, two bleedings in 2 of 57 patients taking ASA and 120 mg EGb 761® is approved in many countries for the treatment of dementia. In randomized, placebo-controlled trials and meta-analyses, EGb 761® was found to improve cognition, activities of daily living, overall condition and neuropsychiatric symptoms in patients with dementia [The defined Ginkgo biloba leaf extract EGb 761dementia ,12,13. ® on the extrinsic and intrinsic coagulation system in 499 elderly patients with probable dementia of the Alzheimer’s type. PT determines the clotting tendency of blood by measuring the time until coagulation after addition of tissue factor and evaluates the extrinsic pathway. The INR was devised to standardize the results of test systems for PT and is used to monitor the anticoagulant effect of warfarin. The APTT measures the time for plasma to coagulate after activation and evaluates the intrinsic pathway. Laboratory tests of blood-clotting parameters  with well-established methods revealed no influence of EGb 761®. Overall, the group comparisons of laboratory parameters and adverse events revealed no antiplatelet or other inhibitory activity of EGb 761® on the haemostatic system as potential for pharmacodynamic interactions with anticoagulant drugs. In healthy persons, the BT measured with the Simplate® method is up to 4 min. Under ASA therapy at steady state, the bleeding time increases by about 30% [Our data from 278 patients also showed that bleeding time remained unaffected by EGb 761bout 30% , which i® when taken alone or in combination with ASA or warfarin. They are consistent with results of two studies with 50 and 32 volunteers, respectively, in which no clinically significant effects of EGb 761® on parameters of blood coagulation, platelet function or BT were found [® or placebo [® [® involving 3323 patients, the number of bleeding events documented as adverse events was not statistically different from that in the placebo group [®. Overall, the clinical database does not provide evidence that EGb 761® causes pharmacodynamic effects on the coagulation system. However, drugs which are not coagulation-inhibiting themselves may affect the metabolism or elimination of anticoagulant substances and thereby lead to bleedings. This was discussed for EGb 761® as well. However, no evidence for clinically relevant pharmacokinetic interactions was found so far [These findings corroborate and add to the evidence derived from other studies and meta-analyses addressing the safety of EGb 761re found ,16. Anot placebo . Likewisacebo [® . In a sabo group . In two bo group ,21. Cumud so far . A systematic review of the safety of concurrent use of food, herbal or dietary supplements and warfarin found 149 articles reporting 78 herbs (including Ginkgo biloba) which interact with warfarin . From th®-treated patients, which indicates that the Ginkgo extract did not enhance the effects of warfarin. In line with these observations, another study reported no effect of EGb 761® on the clotting status or pharmacokinetics or pharmacodynamics of warfarin [®. PAF is characterized—among other effects—by an induction of rabbit platelet aggregation and is therefore detected in an assay with rabbit platelets. Of note, at least a 200-fold higher PAF concentration is required to induce aggregation of human platelets compared with rabbit platelets. This alone makes the involvement of PAF in human physiological platelet aggregation highly unlikely; nor has it been scientifically demonstrated to date.In our study, only very few patients took warfarin concomitantly, which does not allow us to draw definitive conclusions. These patients showed a markedly prolonged PT and APTT as well as an increased INR. In the placebo group, the decreased maximum value reflected the dose reduction in two patients. No attenuation of warfarin doses was required in EGb 761warfarin . The inf®, i.e., the Ginkgo extract did not increase the platelet-inhibiting effect of ASA  daily. The platelet function analysis in 26 participants who took placebo and in 29 participants who took EGb 761® showed no detectable effect on time for clotting and platelet aggregation. A further study with 100 male patients taking 500 mg ASA daily for 7 days also found no effect of EGb 761® on coagulation and bleeding time [® affects platelet aggregation inhibitors binding to the P2Y12 receptor, i.e., clopidogrel.It is noteworthy that the bleeding time seen in the 168 patients taking ASA was not affected by concomitant treatment with EGb 761 ASA see . This ising time . So far,p < 0.001). In individuals with diabetes, aspirin increased the risk of bleeding by 49% (p = 0.13) [ASA as well as warfarin are associated with the risk of additional major or minor bleeding events. The vitamin K inhibitor warfarin effectively prevents stroke, but it has a narrow therapeutic range, is subject to drug and food interactions and requires regular monitoring of the INR, resulting in the frequent need of dose adjustment. Annual rates for any bleeding in patients taking warfarin is about 5%. In inpatient and outpatient settings, the unadjusted incidence rate (per 100 person years) for major critical site bleeding was 13.01 and 4.66 for major bleeding requiring hospitalization, respectively . In heal = 0.13) .The prevalence of bleeding increases with age, since capillaries become frailer. Ecchymoses in the elderly (senile purpura) is estimated to be present in about 12% of individuals after the age of 50 years and up to 30% after age 75, especially those with fair skin . In conc® cannot be discriminated from the background incidence and from adverse effects of the concomitant medication. Overall, the level of evidence is low, unless confirmed by controlled clinical pharmacodynamic or pharmacokinetic studies. Clinical trials are conducted in highly standardized, controlled situations, and deliver results with high-level internal validity. The laboratory values from the randomized and placebo-controlled study presented here are therefore more appropriate than case reports and database analyses of spontaneous reports when it comes to determining the risk of haemorrhage associated with EGb 761®. Postmarketing data generated from high numbers of heterogenous groups may detect rare adverse effects. However, it is especially challenging to assess the causality of spontaneous reports in general and on bleeding due to pharmacodynamic interactions in particular. First, such reports may not be fully documented. On the grounds of limited information, it is also difficult to assess the causal relationship since confounders must be considered. Lack of control group and reporting bias limit their external validity. Since there is no control group, the effect of EGb 761® treatment group. We found no indication that EGb 761® enhances the antiplatelet effect of ASA nor the anticoagulant activity of warfarin. However, the latter group was small. Our findings in this large-scale double-blind study are in line with other studies, thus confirming that neither the use of EGb 761® alone nor its use with ASA is associated with an increased risk of bleeding. ASA represents the class of platelet aggregation inhibitors, and warfarin belongs to the group of vitamin K antagonists; both drugs act as indirect anticoagulants. So far, only very few data are available on potential interactions between EGb 761® and the class of direct anticoagulants. Postmarketing data do not reveal evidence for such an interaction; however, further research is needed.We found that over the 6-month duration of the trial, there was neither an increase in haemorrhage in the treatment groups, nor any alteration in coagulation parameters in either the high- or low-dose EGb 761


Background and Objectives: Ankylosing spondylitis (AS) is a condition that affects 0.1% to 0.5% of the adult population. The aim of this case report was to investigate the possible effects of the drugs taken for treatment of AS as well as mRNA vaccination for COVID-19 on semen quality by performing a highly detailed analysis. Materials and Methods: Sperm characteristics were examined by light microscopy, DNA fragmentation (DFI) was analysed by flow cytometry and morphology was evaluated by transmission electron microscopy (TEM). Results: Semen analysis under therapy with (1) celecoxib and sulphasalazine showed: concentration 47 million/mL, 53% progressive motility, 7% normal morphology and 9.6% DFI, (2) Golimumab and before mRNA Vaccination showed: concentration 108 million/mL, 82% progressive motility, 1% normal morphology and 7.6% DFI, and (3) Golimumab and after 3 doses of mRNA Vaccination showed: concentration 142 million/mL, 85% progressive motility, 1% normal morphology and 6.8% DFI. TEM revealed head, neck and tail abnormalities, as well as the presence of cells with incomplete spermiogenesis white cells and phagocytes in the sample under therapy with celecoxib and sulphasalazine. Golimumab treatment lead to an increased incidence of elongated heads but in general reduced inflammation as no white cells were evident in TEM. Conclusion: The anti-inflamatory drugs celecoxib and sulphasalazine had no adverse effect on sperm quality as all parameters were within normal limits and the patient achieved under that treatment 2 pregnancies following natural conception that lead to the birth of a healthy boy and girl respectively. Anti-TNFa treatment with Golimumab exerted a negative effect on morphology but not on concentration, motility and DFI. After 3 doses of mRNA Vaccination, sperm concentration increased while motility, morphology and DFI remained similar to the values before vaccination suggesting no negative effect of the mRNA vaccine for COVID-19 on sperm quality. Ankylosing spondylitis (AS) affects 0.1% to 0.5% of the adult population and although it has a genetic predisposition, the development of the disease and severity of symptoms highly depend on environmental factors . The HLAAS patients often have reduced fertility potential, reduced sperm motility, increased frequency of varicocele, increased incidence of sperm aneuploidies, higher plasma LH and FSH, and lower T levels compared to controls ,5,7,8. NThe effects of the nonsteroidal anti-inflammatory drug celecoxib and the immunosuppressive anti- inflammatory drug sulphasalazine on the patient’s sperm quality has been previously reported in Chatzimeletiou et al. (2018)  by analyA 43-year-old man with AS first came to the Unit for Human Reproduction at Papageorgiou General Hospital in Greece 6 years ago, while under therapy with celecoxib and sulphasalazine, presenting with primary infertility and seeking fertility treatment. The couple had at that time undergone two previous unsuccessful intra-uterine inseminations and one unsuccessful in vitro fertilisation cycle at a different centre. A detailed semen analysis was performed, and sperm cryopreservation followed on two separate occasions as the patient would start in the future a new therapy with Golimumab, a drug that might have had an effect on sperm quality. During detailed investigation, a female factor of infertility was identified and hysteroscopic removal of an endometrial polyp (which was missed during the previous IUI/IVF cycles at a different centre) enabled the subsequent establishment of a pregnancy, following natural conception and the birth of a healthy baby boy. Two years later and while the man still under treatment with celecoxib and sulphasalazine, a second pregnancy following natural conception was achieved that lead to the birth of a healthy baby girl. Now, having completed his family, the man consented his frozen samples to be donated for research and additionally gave a fresh sample before and after mRNA vaccination for COVID-19 (under treatment with Golimumab) to be analysed for all parameters.A total of 2 cryovials of sperm , which were stored in liquid nitrogen in the Gamete and Embryo Cryopreservation Bank of the Unit for Human Reproduction at Papageorgiou Hospital, Thessaloniki, Greece, were thawed and processed for standard semen analysis. A total of 3.2 mL of fresh semen collected during the patient’s therapy with Golimumab before mRNA vaccination for COVID-19 and 3.5 mL of fresh semen collected during the patient’s current therapy with Golimumab after 3 doses of mRNA vaccination for COVID-19 were processed for standard semen analysis at Fertilia by Genesis, Thessaloniki, Greece. Sperm characteristics were examined by light microscopy, DNA fragmentation was analysed by flow cytometry at the Department of Immunology and Histocompatibility at Papageorgiou Hospital, Thessaloniki, Greece, and morphology was evaluated by light and transmission electron microscopy (TEM) at the Laboratory of Histology and Embryology, Aristotle University Medical School, Thessaloniki Greece. This study was approved by the Bioethics Committee of the Aristotle University Medical School (1.30/21.11.2018) and Genesis (01/7-2/3056). All analysis was performed following the patient’s informed consent.Semen analysis was carried out according to World Health Organization (WHO) criteria: lower reference limits for volume: 1.5 mL; for concentration: 15 millions/mL; for progressive motility A + B: 32%; and for normal morphology: 4% .The fresh and cryopreserved/ thawed sperm samples were resuspended in 3% glutaraldehyde in phosphate buffered saline (PBS)  (pH 7.4) at 4oC and centrifuged at 1200 rpm for 5 min ,27. The A total of 100 μL of the fresh and cryopreserved/ thawed sperm samples were washed in PBS  and centrifuged for 5 min at 300 g. After removal of the supernatant, the precipitant was incubated in TNE buffer (NaCl (0.15Μ), Tris HCL (0.01Μ), EDTA (0.0011Μ) pH 7.4)  and detergent solution  , for 5 min, acridine orange (Bioline Scientific Athens Greece) was added and a further 5 min incubation followed in the dark. The samples were finally analysed by flow cytometry  separating the intact sperm (green) from the fragmented ones (red) based on the change in color due to acridine orange inserted in the fragmented portion of sperm.Standard semen analysis is shown in The fresh semen sample analysed under current therapy with Golimumab and after mRNA vaccination had all parameters within normal limits according to WHO strict criteria, except morphology, which showed an increased incidence of elongated heads. Golimumab, an anti-TNF monoclonal antibody that minimises immunogenicity, was administered subcutaneously to the patient once a month for the past 4 years. It is unclear why anti-TNFa treatment resulted in a higher incidence of morphologically abnormal spermatozoa, but did not affect concentration, motility and DNA fragmentation, which were all within normal limits. Whether the patient’s genotype for TNF-a or its receptors (TNF-R) is responsible for his semen parameters warrants further investigation. It has previously been shown that TNFR1 36A allele is associated with increased sperm concentration and motility, while TNFR1 36G allele is associated with lower sperm concentration and motility ,29.Sperm elongation refers to a sperm head longer than 5 µm and the width <3 µm or a length of <5 µm and a width of <2 µm  and is aSeveral mechanisms that lead to elongated sperm heads have been proposed, although only cases with a varicocele and sperm head elongation have been studied. These include a higher scrotal temperature, injured Sertoli cells, wrongly positioned spermatids to Sertoli cells and structurally abnormal Sertoli–germ cell junction complexes. An association between elongated sperm head and an abnormally narrow-shaped spermatid microtubular manchette has been found ,34. PrevThe cytokine tumor necrosis factor TNFa was initially recognised as one of the products of activated lymphocytes and macrophages secreted in the female pelvic fluids that inhibit and phagocytose spermatozoa . TNFa isVaccination for COVID-19 with 3 doses of mRNA vaccine  also did not exert a negative effect on sperm parameters. In fact, sperm concentration was increased compared to the initial concentration before vaccination and while the man was under treatment with celecoxib and sulphasalazine . Our resThe cryopreserved sample under treatment with the anti-inflammatory drugs celecoxib and sulphasalazine had all parameters within normal limits. However, the presence of several immature sperm as revealed by TEM may be in agreement with previous reports suggesting that sulphasalazine may have deleterious effects on spermiogenesis/spermatogenesis ,23,24,25The study’s main limitation is that the patient came to our unit while already under treatment with celecoxib and sulphasalazine and thus it was not possible to have a control sample (under no drug therapy). In addition, there was no gap between the commencement of Golimumab and the discontinuation of celecoxib and sulphasalazine in order to obtain a control sample at that time. Future studies involving a case series of AS patients with available control sperm samples and samples under therapy with different drugs may contribute to a better evaluation of the effects of AS treatment on male fertility.6 leukocyte /mL, has an incidence of 15% in the general population and although it is especially common in men with infertility, its effects in semen quality is still controversial in the literature [In the cryopreserved sample under therapy with the anti-inflammatory drugs celecoxib and sulphasalazine, a higher incidence of leukocytes was observed compared to the fresh samples under therapy with Golimumab. This may be attributed to the decrease in inflammation obtained after treatment with the anti-TNFa drug (Golimumab). Leukocytospermia, defined by WHO as >1 × 10terature .Finally, there is a general concern that SARS-CoV-2 may disturb male fertility, and thus vaccination for COVID-19 to protect from potential infection becomes more and more a necessity ,45. AlthWe conclude that for this particular patient the anti-TNFa Golimumab, the anti-inflammatory drugs celecoxib and sulphasalazine and the COVID-19 mRNA vaccine did not exert a negative effect on sperm concentration, motility and DNA fragmentation as they were all within normal limits. Nevertheless, more studies are needed to elucidate the mechanisms leading to alterations in sperm head morphology following anti-TNFa therapy and if this may interfere with sperm’s fertilising capacity or chromosomal constitution. More prospective studies are also needed to evaluate the long-term effects of both the mRNA vaccine and the anti-TNFa and anti-inflammatory drugs on men’s general health and fertility.


Whole-slide imaging and virtual microscopy are useful tools implemented in the routine pathology workflow in the last 10 years, allowing primary diagnosis or second-opinions (telepathology) and demonstrating a substantial role in multidisciplinary meetings and education. The regulatory approval of this technology led to the progressive digitalization of routine pathological practice. Previous experiences on renal biopsies stressed the need to create integrate networks to share cases for diagnostic and research purposes. In the current paper, we described a virtual lab studying the routine renal biopsies that have been collected from 14 different Italian Nephrology centers between January 2014 and December 2019. For each case, light microscopy (LM) and immunofluorescence (IF) have been processed, analysed and scanned. Additional pictures (eg. electron micrographs) along with the final encrypted report were uploaded on the web-based platform. The number and type of specimens processed for every technique, the provisional and final diagnosis, and the turnaround-time (TAT) have been recorded. Among 826 cases, 4.5% were second opinion biopsies and only 4% were suboptimal/inadequate for the diagnosis. Transmission electron microscopy (TEM) has been performed on 41% of cases, in 22% changing the final diagnosis, in the remaining 78% contributed to the better definition of the disease. For light microscopy and IF the median TAT was of 2 working days, with only 8.6% with a TAT longer than 5 days. For TEM, the average TAT was 26 days (IQR 6–64). In summary, we systematically reviewed the 6-years long nephropathological experience of an Italian renal pathology service, where digital pathology is a definitive standard of care for the routine diagnosis of glomerulonephritides. Digital pathology consists of a complex group of technological sources  that are revolutionizing the routine workflow . In the Essential “Electron microscopy was needed to make the primary final diagnosis either changing the preliminary diagnosis or resolving a differential diagnosis in cases where a firm preliminary diagnosis could not be made.”Informative “The ultrastructural findings did not alter the preliminary diagnosis and were not essential to making the primary final diagnosis. However, the ultrastructural findings did provide important information confirming/strengthening this primary diagnosis and/or provided clinically relevant insight into the patient’s historical data, light microscopic findings, and/or immunofluorescence findings related to the primary diagnosis.”Not relevant “Electron microscopy resulted in no change in the preliminary diagnosis, was not needed to confirm this diagnosis, and did not supply other clinically pertinent information related to the primary final diagnosis.”Routine renal biopsies were physically sent for diagnosis or second opinion consultation at the Pathology Unit of ASST Monza, Italy from 14 different Italian Nephrology centers . Upon arrival at the pathology department, the specimens have been processed following a standardized protocol , 7. DepeFor the purpose of the study a final list from January 2014 to December 2019 of 826 complete cases was included. The retrospective analysis of this series allowed the collection of the number and type of specimens processed for every technique , the provisional diagnosis after LM and IF, the definitive diagnosis after TEM, and the turnaround-time (TAT) after LM and IF and after TEM. Numerical continuous variables are reported as median and interquartile range (IQR).Routinely, biopsies were scanned using Aperio CS2 device for LM and Aperio ScanScope FL for IF , in the cases with focal segmental glomerulosclerosis  in which has been performed, as well as in Fabry nephropathy (n = 6), Alport disease/thin glomerular basement membrane (n = 7), fibrillary (n = 3) and immunotactoid glomerulonephritis (n = 1). The technique demonstrated to be informative in many cases  with membranous nephropathy (MN) as well as with lupus nephritis (LN) in which has been performed . In the setting of a preliminary diagnosis of C3 glomerulopathy, TEM allowed to sub-classify the cases as C3 glomerulonephritis (n = 9) or as dense deposits disease (n = 2). Its role has been much more limited or not relevant with entities well definable through LM and IF, such as in the biopsies with IgA nephropathy in which has been performed (n = 29), adding significant informations only in a minority of these cases . Finally, in diabetic nephropathy (DN) and arterionephrosclerosis (ANS) ultrastructural analysis only rarely added useful informations, leading to an unmutated final diagnosis in 80% (n = 8) and 83% (n = 19) of the cases in which has been performed, respectively.The study included 826 cases, 4.5% (37) of which were second opinion biopsies sent as FFPE blocks. IF has been performed on fresh frozen material in 70% (580) of cases, whereas paraffin material has been used for the other 30% (246). Electron microscopy has been performed on 41% (340) of cases, 84% (284) of which with dedicated glutaraldehyde fixed specimen and 16% (56) of which after retrieval from FFPE blocks. In 5% (18) of cases TEM was requested for inconsistency with clinical data and LM/IF findings, in 21% (72) for the detection/exclusion of immune complex deposits, in 38% (130) for the characterization of structure and distribution of the deposits, in 25% (86) for the quantification of the podocyte damage (foot process effacement), in 5% (17) for the measurement of the GBM thickness and in 5% (17) because of LM/IF inadequate samples. The execution of ultrastructural analysis has been essential in 22% (75) of cases, informative in the remaining 48% (163) and not relevant in a further 30% (102). The role of electron microscopy has been considered essential in all cases with minimal change disease  were suboptimal/inadequate for the diagnosis . The remaining 18% (n = 153) were characterized by rarer forms of renal diseases. The incidence of each disease for the whole 6-years period is quite unmutated if the single year frequency is considered, as depicted in Fig. Overall, the most frequent final diagnosis of the series was represented by MN (16%) followed by IgA nephropathy 12%), FSGS , MCD (7%), pauci immune crescentic glomerulonephritis (7%), DN (7%), LN (6%), ANS (5%), amyloidosis (5%) and tubulointerstitial nephritis (4%). Only 4% of cases  to bigger hospitals (hub) . After tn = 34). Paraffin IF has been performed in a minority of biopsies , requiring additional ultrastructural analysis for the final diagnosis only in few cases . Although affected by lower sensitivity for the detection of anti-GBM disease and C3 deposits [Renal biopsies shipping times from distant centers, requiring up to 24 h in some cases, could represent an issue for the central hub for the risk of inappropriate preservation of the samples, especially for the IF fresh specimens. In the absence of a dedicated and properly preserved specimen, that is still considered the gold standard, the previously proposed FFPE retrieval for either IF  and TEM deposits , its empdeposits , 14. Thedeposits . Moreovedeposits . The ultdeposits  as well deposits . ElectroAlthough the initial difficulties in setting up the complex infrastructural system, the digital pathology facility can contribute to the creation of kidney biopsy registries with epidemiologic purposes. In the present study, the most frequent glomerulonephritides were represented, in order, by MN, IgA nephropathy, FSGS and MCD, both in the whole 6-years period and in the single year analysis. These data are substantially concordant with a recent study analysing the incidence of renal diseases in China  as compaFinally, the creation of an integrated network among spokes and hubs, facilitated by the implementation of digital pathology Fig. , allows In the present experience we demonstrated the feasibility and sustainability of the digital switch in renal pathology routine as a possible alternative standard of care in renal pathology. The integrated workflow and the optimized employment of different pathology techniques significantly improved the diagnostic performance and reduced the turnaround-time.


When examining infectious samples, rapid identification of the pathogenic agent is required for diagnosis and treatment or for investigating the cause of death. In our previous study, we applied exhaustive amplification using non-specific primers  to identify the causative virus via swab samples from a cat with a suspected viral infection. The purpose of the current study is to investigate suitable methods for the rapid identification of causative pathogens from infected tissue samples. First, the influenza virus was inoculated into mice to prepare infected tissue samples. RNA extracted from the mouse lung homogenates was transcribed into cDNA and then analyzed using the RDV method and next-generation sequencing, using MiSeq and MinION sequencers. The RDV method was unable to detect the influenza virus in the infected tissue samples. However, influenza virus reads were detected using next-generation sequencing. Comparing MiSeq and MinION, the time required for library and sequence preparation was shorter for MinION sequencing than for MiSeq sequencing. We conclude that when a causative virus needs to be rapidly identified from an infectious sample, MinION sequencing is currently the method of choice. When identifying a pathogenic virus from an infectious disease sample, the virus species is first estimated from the clinical course and the presenting symptoms of the patient, various tests and analyses of the pathological findings from the sample, and finally, the application of methods such as immunochromatography, ELISA using a species-specific antibody, and PCR using species-specific primers . In the In the forensic field, various materials, including body fluids, swabs, blood, and tissue, are handled. The methods for the collection and preservation of microbial forensic samples have been reviewed , and man2 in Eagle’s minimum essential medium  supplemented with 10% fetal bovine serum  and 0.1 mM nonessential amino acids (Thermo Fisher Scientific) as instructed.Influenza A virus (H1N1) strain A/PR/8/34 was obtained from the American Type Culture Collection . Madin–Darby canine kidney (MDCK) cell line (RCB0995) was provided by the Riken BRC  through the National BioResource Project of the MEXT/AMED and maintained at 37 °C under 5% CO50 value was calculated using the Behrens–Kärber method [Virus preparation and titration were performed as described previously . Brieflyr method . Viral s5 TCID50) of the stock virus. After inoculation, mice were observed daily. Two mice died seven and nine days after inoculation, and each was dissected on that day (n = 2).Five-week-old BALB/c female mice, which were used for virus inoculation, were obtained from Nippon Bio-Supp. Center  and were housed at room temperature (maintained at 23 ± 1 °C) with a relative humidity range of 50–64%. The mice were fed an MF rodent diet from Oriental Yeast Co., Ltd.  and allowed free access to water. Six mice were used for inoculation, and each mouse was intranasally inoculated with a 20-μL diluent  from 50 μL of supernatant from the lung homogenates. cDNA was synthesized from 2 μL of the RNA extract using a random hexamer as the reverse transcription primer and the PrimeScript RT Reagent Kit . For NGS, double-stranded cDNA was synthesized from 7 μL of the RNA extract using a random nonamer as the reverse transcription primer and a PrimeScript Double Strand cDNA Synthesis Kit (TaKaRa Bio). These procedures were performed in accordance with the manufacturer’s protocols.PB1) gene was developed. The primer sequences and amplicon lengths are listed in PB1 gene were designed using Primer-BLAST . Previously reported primers [PB1 gene: an initial denaturation step of 30 s at 95 °C, followed by 40 cycles of denaturation at 95 °C for 10 s, and annealing and extension at 60 °C for 30 s. Next, a melting curve analysis was performed from 60 °C to 95 °C. A no-template control was used in each batch of PCR mixture as a negative control.To examine whether detection using the RDV method was possible, the viral genes in the samples were quantified. Therefore, a qPCR procedure targeting the influenza A virus polymerase basic protein 1 . For NGS, synthesized double-stranded cDNA was measured using the QuantiFluor ONE dsDNA System .The RDV method was performed as described previously , with a DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit and the Nextera XT Index Kit  according to the manufacturer’s protocols. The libraries were normalized, pooled, and diluted for sequencing. Then, 24 μL of pooled library solution and 576 μL of hybridization buffer were mixed and sequenced using the MiSeq Reagent Kit v2 300 cycles (Illumina). For sequencing using MinION , DNA libraries were prepared using a Rapid Sequencing Kit (Oxford Nanopore Technologies) and sequenced with Flow Cell FLO-MIN106 R9 Version (Oxford Nanopore Technologies) in accordance with the manufacturer’s protocols. For taxonomic classification of reads generated by MinION sequencing, What’s in my Pot?  in the EPI2ME workflow (Oxford Nanopore Technologies) was used. CLC Genomics Workbench 11 (Qiagen) was used to analyze all the obtained reads.t-tests. A p-value < 0.05 was considered significant. Statistical analyses were performed using Microsoft Excel 2016 MSO (version 2111).Copy numbers were analyzed using paired two-tailed Student’s PB1 gene. For each qPCR run, 2 × 103 to 2 × 107 copies of the 742-bp amplicon were included as standards. Fluorescence was monitored throughout the reaction, and the cycle quantification (Cq) value was determined. A standard curve was generated from the Cq value and the copy number. Quantitation was linear over the range of copy numbers examined (data not shown). The copy number of each sample was determined using the standard curve.The positions of nine fragments amplified by the primer pairs presented in PB1 gene in the lung homogenates was quantified by amplicon length. The copy number decreased as the amplicon length increased (p < 0.05) and 402 bp (p < 0.001) but was relatively high at 559 bp.The copy number of the ncreased . The cophttps://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 11 August 2021). The majority of reads (16 reads) were homologous to the 18S, 28S, and 45S ribosomal RNA genes of mammals, including mice . Of the 19 contigs, the minimum, maximum and average lengths were 202, 2191, and 768 bases, respectively. Of the 31 contigs, the minimum, maximum and average lengths were 225, 2535, and 673 bases, respectively. The 50 contigs were searched using the BLAST program. The majority of contigs  were homologous to influenza A virus sequences, although most others were homologous to mouse and bacterial ribosomal RNA genes (data not shown). In total, 45,997 and 142,475 reads were generated by MinION sequencing of the two samples, and these reads were analyzed with WIMP of the EPI2ME workflow. Of the 142,475 reads, 116,371 were host reads. Among the remaining reads, 155 matched influenza A virus sequences and these were detected most frequently. Of the 45,997 reads, 14,783 were host reads. Among the remaining reads, 32 influenza A virus reads were detected, following reads for To confirm their identities, the obtained reads were mapped to the influenza A virus and mouse genomes . The datFinally, the MiSeq and MinION sequencing data were compared . Althoug6 copies of viral cDNA using the RDV method [6, the target influenza virus genome could not be detected from the infected tissue samples. This copy number appeared to have been low to detect using the RDV method without the virus propagation step. Therefore, when the RDV method is used without the virus propagation step, it may require a larger number of samples; however, the number of samples that can be processed is limited. Although omitting the virus propagation step in the RDV method saves time and effort, this step may be necessary to reliably detect the target genome. As the host genome is often detected using the RDV method, the removal of the host genome is important. Although it is difficult to remove the host genome from samples in the RDV method, NGS allows the host genome to be removed during analysis.When a pathogenic virus needs to be rapidly identified from an infectious forensic sample, the RDV method allows for detection of the virus sequence with equipment widely available in most laboratories . In a prV method . AlthougRead analyses of MiSeq and MinION sequencing data revealed that the target virus species could be detected from the infected tissue samples. Although a huge number of reads can be obtained from a small mass of DNA using MiSeq, library and sequence preparation are time-consuming and labor-intensive. Although MinION requires a large mass of DNA, the library can be prepared in approximately 30 min per sample, and reads can be obtained in a shorter time. Furthermore, multiplex samples can be prepared together using the barcoding kit, and the analysis of the obtained reads can be started during sequencing. Although not performed in this study, these adaptations to the method can accelerate the process of pathogen detection. If the sample is a human organ, then a larger mass of DNA can be obtained, which may make MinION the appropriate detection method. Owing to its speed and convenience, MinION is also useful for human identification from skin microbiota and on-site sequencing . In addiHere, the number of reads produced by MinION sequencing was small and varied because the specified DNA mass was not available for library preparation. This may have had the effect of shortening the reads. The proportions of influenza A virus and mouse reads were lower with MinION than with MiSeq , but thiThis study confirmed that target virus species could be rapidly detected from infected tissue samples using WIMP. When a causative virus needs to be rapidly identified from an infectious sample, MinION may be an appropriate method. MinION can also be used to identify pathogens from tissue samples of infectious diseases of unknown cause. If the virus species is ascertained quickly, appropriate treatment can be initiated, or the cause of death can be resolved rapidly. Although PCR confirmation after species detection was not performed in this study, official identification requires PCR confirmation with species-specific primers.We are currently investigating tissue samples harvested from influenza virus-infected mice a number of days after death. We will use MinION to determine whether the target virus can be detected from such samples.


The effects of reinforcement content , chemical treatments  and treatment cycles (1 and 3 cycles) on the mechanical and thermal properties of the biocomposites were investigated. The biocomposites of treated hemp hurd powder exhibited enhanced thermal stability in the temperature range commonly used to process PLA (130–180 °C). The biocomposites containing 15 wt. % hemp hurd powder prepared using a single-cycle alkaline/peroxide treatment (PLA/15APHH1) exhibited a Young’s modulus of 2674 MPa, which is 70% higher than that of neat PLA and 9.3% higher than that of biocomposites comprised of PLA containing the same wt. % of untreated hemp hurd powder (PLA/15UHH). Furthermore, the tensile strength of the PLA/15APHH1 biocomposite was found to be 62.6 MPa, which was 6.5% lower than that of neat PLA and 23% higher than that of the PLA/15UHH sample. The results suggest that the fabricated PLA/hemp hurd powder biocomposites have great potential to be utilized in green and sustainable packaging applications.Sourced from agricultural waste, hemp hurds are a low-cost renewable material with high stiffness; however, despite their potential to be used as low-cost filler in natural fiber reinforced polymer biocomposites, they are often discarded. In this study, the potential to add value to hemp hurds by incorporating them into poly(lactic acid) (PLA) biopolymer to form bio-based materials for packaging applications is investigated. However, as with many plant fibers, the inherent hydrophilicity of hemp hurds leads to inferior filler-matrix interfacial interactions, compromising the mechanical properties of the resulting biocomposites. In this study, two chemical treatments, alkaline (NaOH) and alkaline/peroxide (NaOH/H Environmental issues associated with both plastic waste and depletion of petroleum resources have been a major driving force for the production and utilization of biodegradable biopolymers. Among biodegradable biopolymers, poly(lactic acid) (PLA) is attractive for a variety of applications owing to its biocompatibility , renewabIn recent years, natural fiber-reinforced biopolymers have received extensive interest due to the “green” nature of these biocomposites. Moreover, natural fibers have positive characteristics including desirable mechanical properties, low density, and low cost, which make them a promising candidate as reinforcing fillers ,11. AmonAmong surface modification techniques, chemical treatments of natural fibers have been studied extensively in the past few years. Alkaline treatment is a common chemical treatment to remove non-cellulosic constituents from natural fibers due to its simplicity and low cost ,24. Nume2O2) is an alternative chemical treatment that is extensively used in the textile industry [v/v) on the mechanical properties of PLA/kenaf biocomposites. They reported that the biocomposites formed with the bleached fibers showed higher tensile strength, Young’s modulus, and elongation at break compared to biocomposites containing unbleached fibers [Bleaching with hydrogen peroxide . If the cost of this polymer can be reduced through the incorporation of an inexpensive biodegradable filler without compromising the properties of the material overall, PLA may find more use in applications such as packaging. Most of the studies in the literature used bast fibers  as reinforcing agents for PLA-based biocomposites. While the use of these reinforcing agents can often result in improvements in the thermal and/or mechanical properties of PLA-based composites, due to the high cost of the fibers themselves , the resulting composites may have limited use. Very few studies have reported the use of hemp hurds—an underutilized part of the hemp plant that is often considered a by-product —as reinfIn this work, we prepared PLA/hemp hurd powder biocomposites, and characterized their thermal and mechanical properties as a function of hemp hurd powder treatment and concentration to: (1) add value to hemp hurds by using them as low-cost filler, 2) find suitable end-of-life application for by-products of the hemp industry, (3) develop bio-based and potentially biodegradable material that can be used in commercial applications. Hemp hurd powder was prepared by grinding hemp hurds to obtain a powder with a size of less than 500 microns. The hemp hurd powder was subjected to two different types of chemical treatments , which were selected to achieve good interactions between the inherently hydrophilic hemp hurds  and the  find sui3 at 20 °C, purity: ≥98%) and hydrogen peroxide (H2O2) solution  were purchased from Sigma Aldrich®, Oakville, ON, Canada, and used as received without any processing.Poly(lactic acid)  was purchased from NatureWorks LLC, Minnetonka, MN, USA. Hemp hurds were provided by the Vegreville Decortication Facility, Vegreville, AB, Canada. Required chemicals such as sodium hydroxide (NaOH) pellets  for 3 min; the resulting powder was sieved using a 500 µm size screen . This material was denoted as untreated hemp hurd powder (UHH). Once prepared, the ground hurds were stored in sealed plastic bags at room temperature until their use.The visible dirt of the received hemp hurds was removed by vigorous washing with 60 °C water for 2 h. After drying at 90 °C for 24 h, washed hemp hurds were ground using a blade coffee grinder  to dissolve water-soluble hemicellulose. Afterward, the mixture was vacuum filtered using a 20 µm size screen. The filtered wet powder was then put in 100 mL of 8 wt. % NaOH solution and sonicated at room temperature for 1 h. The solution was then kept at room temperature (without sonication) for 47 h. Then, the sample was vacuum filtered using a 20 µm size screen and washed with distilled water containing 1% acetic acid to neutralize the remaining caustic soda, and then thoroughly washed with distilled water until a pH of ~7 was reached. Finally, the hurd powder was dried at 90 °C for 24 h. The resulting powder was denoted as single-cycle alkaline-treated hemp hurd powder (AHH1). The same treatment procedure was repeated twice more to obtain three-cycle alkaline-treated hemp hurd powder (AHH3).v/v hydrogen peroxide solution was added in the mixture, which was vigorously stirred for 90 min at room temperature. This treatment process was inspired by Zhang et al., who observed that performing alkaline hydrogen peroxide treatment improved the properties of citrus dietary fiber [After washing and vacuum-filtering of 5 g of hemp hurd powder using the procedure explained in the alkaline treatment ry fiber . The samBefore biocomposite fabrication, hemp hurd powder and PLA were dried at 70 °C in an oven overnight to minimize the moisture content. The compounding was carried out in a co-rotating twin-screw extruder  at 160 °C with a rotor speed of 75 rpm. All batches were cycled through the extruder channel for 3 min (the residence time) to ensure proper melt-mixing and then extruded through a rectangular nozzle. The extruded filaments were cooled to room temperature and then pelletized manually using scissors. The resulting pellets were rectangular in shape, typically 2–3 mm in length, 2 mm in width, and 0.5–1 mm in thickness. As per the ASTM D638-14 test , standarThroughout this work, each biocomposite is referred to in the format of “PLA/filler type”, where the number before the filler type refers to hemp hurd powder content; for instance, PLA/15APHH3 denotes PLA/hemp hurd powder biocomposites containing 15 wt. % of three-cycle alkaline/peroxide-treated hemp hurd powder. The designated names for hemp hurd powders and prepared biocomposites are summarized in −1 with a scanning resolution of 4 cm−1. A total number of 120 scans were taken for each sample and averaged. The chemical analysis of untreated and treated hemp hurd powders was performed using an FTIR spectrometer  equipped with ATR  to determine the interfacial bonds. Each spectrum was obtained in the spectral range of 4000 to 400 cmThe morphological analysis of untreated and treated hemp hurd powders was carried out using SEM analysis . Samples were placed on aluminum holders with double-sided conductive tape. Prior to carrying out SEM characterization, a Denton sputter unit was used to coat the samples with a thin layer of gold.cI) was calculated using the Segal method [I002) and the intensity observed at the local minimum at ~18° (amI) using Equation (1):The X-ray powder diffraction analysis of untreated and treated hemp hurd powders was performed using an X-ray diffractometer . The diffracted intensity was recorded at a scanning rate of 0.5°/min between 5 and 40° diffraction angle at 40 kV and 30 mA using locked couple mode. The crystallinity index  equipped with an ultra-micro balance cell and differential thermal analysis (DTA) sensors. An approximately 10 mg sample was placed in an alumina crucible and heated from 25 to 799 °C at a heating rate of 10 °C/min under a nitrogen atmosphere with a flow rate of 20 mL/min.cX) of PLA and its biocomposites were calculated using Equation (2):w is the weight fraction of PLA in the biocomposites, ΔmH corresponds to the melting enthalpy of biocomposite, and Thermal analysis and crystallization behavior of PLA and the biocomposites were investigated by use of a differential scanning calorimeter . Samples with a mass of approximately 5 mg were placed in sealed aluminum crucibles and then heated from −20 to 200 °C at a heating rate of 5 °C/min. Following the first heating scan, samples were held at 200 °C for 5 min and then cooled down to −20 °C at a cooling rate of 5 °C/min. Finally, samples were heated again to 200 °C at a heating rate of 5 °C/min to obtain the second heating cycle behavior. The degree of crystallinity  equipped with a 1 kN load cell at a crosshead speed of 5 mm/min. At least 5 samples were measured for each composite type and the average value is reported. As shown in −1  [−1 (CH stretching in cellulose) [−1 (C=O stretching of acetyl groups in hemicellulose) [−1 (C-O stretching of acetyl groups in lignin) [−1  [−1 (C-O and C-O-C stretching vibrations of cellulose) [−1  [−1 can be clearly observed in FTIR spectra of untreated, alkaline, and alkaline/peroxide treated hemp hurd powders are shown in llulose) , 2913 cmllulose) , 1734 cmllulose) ,40, 1239 lignin) ,42, 1158llulose) , 1100–10llulose) ,45, 896 llulose) . For all−1 corresponding to hemicellulose and lignin, respectively, suggesting the removal of these groups. The vanishing of these peaks has been noted previously in other studies: Kathirselvam et al. [−1 vanished after NaOH treatment of cellulose fibers from Thespesia populnea bark, and attributed this change to the removal of hemicellulose. Viscusi et al. [−1 after ball mill + NaOH treatment of hemp fibers, and attributed the change to the removal of both hemicellulose and lignin. Oza et al. [−1, attributed to the removal of wax and pectin. In studies [−1 were observed after NaOH treatment, corresponding to the removal of lignin. Overall, the changes in the spectra of the treated hemp hurd powder show that lignin and hemicellulose were at least partially removed during the chemical treatment. The main changes in the spectra observed after alkaline and alkaline/peroxide treatment were the notable decrease in the intensity of the peaks at 1734 and 1239 cmm et al.  noted thi et al.  observeda et al.  studied  studies ,42, simiThe effect of alkaline and alkaline/peroxide treatments on the morphology of the hemp hurd powder can be seen by comparing the SEM images of untreated and treated hemp hurd powders shown in The results of the alkaline treatment (which used an 8 wt. % NaOH solution) are in agreement with the literature. Kathirselvam et al. treated raw cellulose fibers with 2, 5, and 8 wt. % of NaOH solution for 60 min at room temperature . They obThe XRD patterns of untreated, alkaline, and alkaline/peroxide treated hemp hurd powders are presented in m was selected at the local minimum near to 18°. For untreated hemp hurd powder, this minimum was observed at 2θ = 18.7° and this feature was slightly shifted to higher or lower diffraction angles for treated hemp hurd powders. The crystallinity index of each type of hemp hurd powder was calculated using the Segal method, and the results are summarized in cI = 53.28%), corresponding to an approximately twofold increase in crystallinity with respect to untreated hemp hurd powder (UHH). The increased crystallinity index of treated hemp hurd powders can be attributed to the removal of amorphous content of hemp hurd powder including hemicellulose, lignin, and pectin during treatments, which facilitated the rearrangement of cellulose chains [I002) is increased in the XRD pattern of treated fibers, resulting in a higher crystallinity index as compared to untreated fibers. They attributed this observation to the removal of non-crystalline components such as hemicellulose, pectin, and wax.In terms of the type of treatment, hemp hurd powder treated with alkaline/peroxide solution exhibited peaks with larger magnitude at 2θ = 22.5° compared to the alkaline-treated hemp hurd powder, indicating that the combined treatment is more effective at removing non-cellulosic components. For all samples, other noticeable peaks are observed at 2θ angles around 14.8, 16.2, and 34.5° corresponding to the crystallographic planes (1 1 0), (1 1 0), and (0 0 4), respectively ,47. The e chains . Similare chains ,49. For e chains . A.N. BaT10, corresponding to the temperature at which 10% weight loss has occurred, and T50, the temperature at which 50% weight loss has occurred, were used as the reference parameters to evaluate the thermal stability of the hemp hurd powders. Extracted T10 and T50 values of untreated and treated hemp hurd powders are summarized in T10 and T50 values increased for treated hemp hurd powders, indicating that the thermal stability of the treated hemp hurd powders increased in comparison to the untreated hemp hurd powder. This might be due to the removal of thermally unstable contents of hemp hurd powder such as hemicellulose, pectin, and wax during the treatments [T10 values of the hemp hurd powders, indicating continued removal of hemicellulose and pectin during the successive treatments. TGA results of alkaline and alkaline/peroxide treated hemp hurd powders are shown in eatments ,41,50. MThe derivative of thermogravimetric (DTG) thermograms of alkaline and alkaline/peroxide treated hemp hurd powders are presented in iT, corresponding to the temperature at which the sample mass has decreased by 5%), final decomposition temperature , the temperature of maximum mass loss rate , and the percentage of char residue for each sample were determined from TGA and DTG thermograms and are represented in iT of 322 and 320.4 °C, respectively; indicating better thermal stability in terms of initial decomposition temperature compared to PLA/15APHH1 and PLA/15APHH3 samples, with iT values of 317.3 and 303.5 °C, respectively. However, in terms of the final decomposition temperature, PLA/15APHH1 exhibited the highest fT value of all biocomposites (373.6 °C). The PLA/15APHH3 biocomposite was found to be the least thermally stable sample, as it exhibited the lowest initial and final decomposition temperatures. This observation could result from excessive delignification during the three cycles of successive alkaline/peroxide treatment process, which may have degraded the structure of the hemp hurds [TGA and DTG thermograms of neat PLA and PLA/hemp hurd powder biocomposites containing 15 wt. % of hemp hurd powder are presented in mp hurds .Char residue  at 799 °C for all of the biocomposites was found to be higher than that of PLA, as presented in iT values of 327 and 322 °C, respectively. PLA/5APHH3 and PLA/15APHH1 yielded higher final degradation temperatures (fT) of 376.1 and 373.6 °C, respectively. For all biocomposites, increasing the filler content resulted in a higher char residue at 799 °C. This char corresponds to the constituents of the hemp hurd powder (cellulose and lignin), which typically leave residue even at these high temperatures [TGA and DTG curves corresponding to the composites containing 5 and 15 wt. % of treated hemp hurd powders are shown in the eratures . These oTo ensure the processibility of PLA/hemp hurd powder biocomposites using melt-processing techniques, the thermal stability of hemp hurd powder within the processing temperature range is required. Considering the initial degradation temperature of biocomposites containing 15 wt. % of treated hurd powder exceeds 250 °C, it can be inferred that melt processing of PLA/hemp hurd powder biocomposites at the melting temperature range of PLA (130–180 °C) [gT) of the biocomposites. This reduction was most prominent for the samples containing hemp hurd powder that underwent a single cycle of treatment (PLA/15APHH1 and PLA/15AHH1).DSC thermograms corresponding to the second heating cycle of PLA and its biocomposites containing 15 wt. % of hemp hurd powder are given in ccT), and a melting temperature (mT). However, a ccT peak was not present in the thermogram of PLA, reflecting the amorphous nature of this material. Similarly, neat PLA exhibited only a small melting peak at around 149.1 °C. For each of the biocomposites, only very small variations in melting temperatures were observed (ranging from 148.8 to 149.7 °C). Biocomposites containing three-cycle treated hemp hurd powder showed lower cold crystallization temperatures compared to the ones comprised of single-cycle treated hemp hurd powder. A reduction in cold crystallization temperature was observed previously for PLA upon the addition of increasing concentrations of pulp fibers, which were reasoned to act as heterogeneous nucleation sites [Each of the thermograms corresponding to the biocomposites exhibited peaks corresponding to a cold crystallization temperature  of the biocomposites, where the lowest gT was observed for the biocomposites with 15 wt. % filler content. It was also observed that increasing filler loading increased the crystallinity of the biocomposites.PLA (without hemp hurd powder) exhibited the lowest crystallinity 1.3%), while the addition of treated hemp hurd powder resulted in a higher crystallinity. Biocomposites containing three-cycle treated hemp hurd powder yielded the highest crystallinity values of 18.7% and 18.5%, corresponding to PLA/15APHH3 and PLA/15AHH3, respectively. The increase in crystallinity of samples that occurred due to the incorporation of hemp hurd powder supports the idea that the treated hemp hurd powder acts as a nucleating agent in the PLA matrix, leading to the alignment of the PLA chains %, while . DSC resTensile properties of PLA and its biocomposites containing various proportions  of untreated and treated hemp hurd powder were evaluated. To better understand the impact of hemp hurd powder on mechanical properties of biocomposites, samples were prepared with 15 wt. % filler loading of untreated and treated hemp hurd powders, and the Young’s modulus, tensile strength, and elongation at break of the samples are presented in The addition of 15 wt. % hemp hurd powder (prepared using the various treatment methods) resulted in a decrease in both elongation at break and tensile strength. The measured value of elongation at break for neat PLA was 5.7%, which was decreased to the range of 3.8~2.8% upon the addition of 15 wt. % of hemp hurd powders. In the case of tensile strength, the value for neat PLA was determined to be 66.9 MPa. For the PLA/15UHH sample, the obtained value of tensile strength was 50.7 MPa, corresponding to a 24% reduction in comparison to neat PLA. The reduction in tensile strength exhibited by biocomposites formed with untreated hemp hurd powder (UHH) with respect to PLA can be ascribed to inferior filler-matrix interaction caused by the high hydrophilicity of UHH. This powder contained hydrophilic components such as hemicellulose and lignin, which are incompatible with the hydrophobic PLA matrix.Performing chemical treatments on the hemp hurd powder improved the filler-matrix compatibility to some extent, although the tensile strength values of the biocomposites were all lower than those of neat PLA. Nonetheless, each of the biocomposites with treated hemp hurd powders showed higher tensile strength values compared to the PLA/15UHH sample. The chemical treatments increased the surface roughness of hemp hurd powder (as shown in SEM results), which is expected to improve the filler-matrix interfacial interaction. This can be achieved by enhancing mechanical interlocking mechanism between filler and polymer matrix , caused In addition to exhibiting the highest tensile strength of the biocomposites, PLA/15APHH1 was determined to have the highest Young’s modulus (2673.6 MPa), indicating an increase of 70% over neat PLA and a 10% increase as compared to PLA/15UHH. The tensile properties of the fabricated PLA/hemp hurd powder biocomposites are in agreement with the literature. B.A. Khan et al. evaluated the mechanical properties of PLA/hemp hurd biocomposites for various proportions of untreated hemp hurds  . They foThe tensile strength values of PLA/15AHH1 and PLA/15APHH1 samples were found to be comparable. However, it was noted that the incorporation of three-cycle treated hemp hurd powder yielded lower tensile strength for the same filler loading in either case of alkaline and alkaline/peroxide-treated hemp hurd powder. This observation can be attributed to the excessive delignification and degradation of cellulose during the third cycle of treatments, resulting in poor mechanical properties as stated in the work of Malenab et al., who observed that increasing the immersion time of abaca fibers in NaOH solution resulted in lower tensile strength of fibers ,62. HereThe effect of filler loading on tensile properties of the biocomposites is shown in The aim of this study was to determine whether hemp hurd powder could be used as a low-cost filler (at 15 wt. %) in PLA without compromising the overall properties of the polymer. It was found that when untreated hemp hurd powder (ground to less than 500 μm) was blended with PLA, the tensile strength of the resulting composite decreased. By utilizing one or three cycles of alkaline and alkaline/peroxide treatments on hemp hurd powder before compounding with PLA, a higher tensile strength was achieved than for composites prepared using untreated hemp hurd powder. Of all of the biocomposites, the one containing 15 wt. % hemp hurd powder treated with a single cycle of alkaline/peroxide treatment (PLA/15APHH1) exhibited the best mechanical results in terms of tensile strength and Young’s modulus, exhibiting a 23% increase in tensile strength and 10% increase in Young’s modulus in comparison to the samples containing 15 wt. % untreated hemp hurd powder, and a 6.5% decrease in tensile strength and 70% increase in Young’s modulus as compared to the neat PLA. One drawback of the biocomposites is that the elongation at break—which is already relatively low for neat PLA—is further reduced through the addition of each type of the filler, particularly the untreated hemp hurd powder. DSC results indicated that treated hemp hurd powder could act as a nucleating agent leading to increased crystallinity of the PLA/hemp hurd powder biocomposites compared to PLA. Thermogravimetric analysis revealed that biocomposites containing treated hemp hurd powder exhibited proper thermal stability in the processing temperature range of PLA (130–180 °C) indicating the fact that this range of temperature does not pose any detrimental risk to the thermal stability of treated hemp hurds, and that these materials could subsequently be processed using thermal techniques such as injection molding, compression molding, and 3D printing without degrading the biocomposites.Overall, these results suggest that treated hemp hurd powder can be used as a low-cost additive to reduce the amount of PLA required without compromising the overall properties of the material. Additional studies on both degradability and total cost of the composites  would be beneficial towards establishing the applicability of these materials in applications such as packaging. Moreover, further studies on the barrier properties, such as water vapor transmission rate and oxygen transmission rate of the prepared biocomposites, are required to evaluate their potential to be used in food packaging applications.


However, our understanding of these mechanisms is incomplete. Sahay et al. addressed this gap by examining the effect of PAH exposure on epigenetic and transcriptional regulation of genes in the Erα pathway in a mouse cohort exposed to aerosolized PAH at proportions measured in urban air. In addition to alterations in the Erα signaling pathway in the pregnant mice and in their offspring and grandoffspring, the investigators observed higher body weights in mice exposed to PAH compared to the control. Given that associations between mammary tissue adiposity, systemic adiposity, and breast cancer risk have been observed previously, the finding of higher body weight in the PAH exposure group raises the possibility that body weight might influence the association between PAH exposure and breast cancer risk. Along with new analyses, we discuss the possibility that body weight may modify the association between PAH exposure, mammary cellular proliferation, and mammary gland ductal hyperplasia in offspring and grandoffspring mice and future research that may be needed to delineate these associations.Breast cancer risk remains incompletely explained, and higher incidence rates of breast cancer over recent times and in urban and industrialized areas suggest environmental causes. Polycyclic aromatic hydrocarbons (PAH) are ubiquitous in the environment and epidemiological and rodent studies have shown associations between exposure to PAH and breast cancer incidence as well as mammary tumorigenesis. In addition,  Breast cancer is the most commonly diagnosed cancer in women in the United States . Breast Emerging evidence indicates that environmental exposures contribute to current breast cancer risk. The proportion of breast cancer risk attributable to traditional risk factors, such as weight, height, age at menarche, parity, age at first birth, duration of breastfeeding, menopausal status, menopausal hormone therapy, family history of breast cancer, history of benign breast biopsy, greater breast density (the ratio of dense fibroglandular tissue to nondense adipose tissue), alcohol consumption, and physical activity, account for 53 – 70% of breast cancer incidence ,5. Risk Studies of the importance of ambient air pollution to breast cancer risk have been inconsistent ,10. The Many studies, though not all, have shown epidemiological associations between measures of PAH or surrogates of PAH exposure, and the development of breast cancer. For example, in the Long Island Breast Cancer Study Project (LIBCSP), a population-based case-control study of mostly postmenopausal women, the odds of breast cancer were elevated 44% in association with long-term vehicular traffic benzopyrene (BER) positive breast cancer cell line, that were treated with 5 μM of B[a]P for 24 hours, expression of the tumor suppressor breast cancer gene (Brca) 1 was downregulated and protein expression of the tumor suppressor gene P53 was upregulated [P53 gene and treated with 5 μM of B[a]P for 24 hours, B[a]P administration did not lower Brca1 transcription [Brca1 occurs through P53 [Brca1 mutation repair mechanisms [in vitro study, treatment for 24 hours with 6 nM of a mixture of 36 PAH compounds derived from sediment from an EPA superfund site raised aryl hydrocarbon receptor (Ahr) and Erα protein levels and increased cell proliferation in MCF-7 cell lines [Ahr may cause downstream upregulation of Erα, resulting in cellular proliferation, a phenotype previously associated with breast carcinogenesis [A few mechanistic studies in cell lines have addressed the associations between PAH exposure and breast cancer risk. In one, in MCF-7 cells, an estrogen receptor  who were exposed to ambient PAH, as well as in the offspring (F1) and grandoffspring (F2) mice [Arnt) mRNA expression and lowered Brca1 mRNA expression in the mammary glands of the pregnant mothers at postpartum day (PPD) 28, the offspring and grandoffspring at postnatal day (PND) 60, and the grandoffspring mice at PND28 [Ahrr) mRNA expression and raised Ahrr methylation at CpG sites in the mammary glands of the pregnant mothers at PPD28, the offspring and grandoffspring mice at PND60, and the offspring and grandoffspring mice at PND28 [Erα promoter methylation at CpG sites in the mammary glands of the pregnant mothers at PPD28 and the offspring and grandoffspring mice at PND60 and PND28 [Erα mRNA expression in the mammary glands of the pregnant mothers at PPD28, the offspring and grandoffspring mice at PND60, and the grandoffspring mice at PND28 [Erα and Ahrr mRNA expression and methylation for some CpG sites in the mothers predicted transcriptional and epigenetic regulation of Era and Ahrr in the offspring and grandoffspring mice at PND60 [Erα pathway observed in the mothers may drive the changes observed in offspring and grandoffspring mice [As only a few studies have coupled observations of the association between PAH exposure and mammary tumorigenesis with underlying mechanisms y et al.  stands oogenesis ,23,27 wiogenesis . PAH expF2) mice . The appF2) mice . Sahay eat PND28 . PAH alsat PND28 . In addind PND28 . Concomiat PND28 . PAH-indat PND28 . In addiat PND60 . This fiing mice .Ahr [Arnt [Brca1 and Erα mRNA expression. Consistent with Jeffy et al. [Brca1, presumably through regulation of Ahr. The finding of upregulation of Erα methylation and downregulation of Erα transcription contrasts with the findings of Gearhart-Serna et al. [in vivo in mice and in vitro in a cell line) can explain the opposite findings of raised and lowered Erα expression with PAH exposure. Further, both Khanal et al. [Erα and resulting downregulation in Erα mRNA expression in response to PAH exposure, which suggests a role of epigenetic regulation in mediating PAH-induced mammary tumorigenesis [The findings in Sahay et al. are consistent with PAH ligand binding to Ahr , dimerizhr [Arnt , and dowy et al.  and Romay et al. , exposura et al. . 8 of tha et al.  were alsa et al. . Despitea et al.  used 28 a et al. , and it a et al.  and Sahaa et al. , as welll et al.  and Sahal et al.  reportedigenesis ,33. Sahaigenesis .Sahay et al. also provided evidence that PAH exposure increased the body weight of the offspring and grandoffspring mice at PND60 , an age The relationship between systemic adiposity and breast cancer risk is more complex and varies by menopausal status and disease subtype. Measures of body size have been correlated positively with breast cancer risk in postmenopausal women ,47–49 anER, progesterone receptor (PR), and human epidermal growth factor receptor (HER) [ER positive breast cancer but positively associated with triple negative breast cancer, a subtype that lacks expression of ER, PR, and HER [ER positive breast cancer and a reduced or unchanged risk of triple negative and ER negative breast cancer [ER positive breast cancer among postmenopausal people in larger bodies is thought to be caused by the production of estrogen in adipose tissue, which supports the survival of ER positive cancer [Differences in breast cancer risk by body size are further divided by disease subtype. Breast cancers are classified by the expression of or (HER) . Among p and HER . Converst cancer . After mt cancer . Specifit cancer . Thus, te cancer . The role cancer . Even whe cancer ,54, weige cancer , and race cancer ,61, thatPparγ) promoter and increased mRNA expression of Pparγ, C/EBPα, and Cox-2, which are transcription factors involved in adipocyte differentiation and function [Notably, PAH exposure has been associated with higher body weight in experimental and epidemiological studies. In a mouse cohort in which pregnant mice were exposed to the same PAH mixture and levels used in Sahay et al. , PAH expfunction . In a bifunction  and Yan function , was assfunction . Similarfunction .Studies showing an association between PAH exposure and breast cancer risk and between PAH exposure and systemic adiposity raise the question as to whether the links between air pollution or PAH specifically and breast cancer may be influenced by the presence of systemic adiposity. In new analyses, body weight in offspring and grandoffspring mice at PND60 did not differ by the extent of cellular proliferation  or presence of ductal hyperplasia in mammary tissue  . In anal2 strengthened the association between PAH exposure determined from self-reported grilled / smoked meat consumption and environmental tobacco smoke (ETS) exposure and breast cancer, but did not modify the association between estimated PAH exposure from active smoking or from residential vehicular traffic and breast cancer [2 weakened the association between PAH exposure from self-reported indoor synthetic log burning and breast cancer [Erα pathway signaling [To our knowledge, only a few other studies have examined effect modification by BMI on the association between exposure to PAH and mammary or breast cancer. In the LIBCSP cohort, BMI ≥ 25kg/mt cancer . In contt cancer . Given tt cancer  and has t cancer . Thus, gt cancer . Howevert cancer ,62,63 ant cancer . PAH expignaling , which hignaling .Erα pathway and subsequent alterations in gene expression in this pathway may contribute to mammary carcinogenesis. A unique strength of this study is the focus on physiological delivery of ambient PAH over time in animals to mimic human exposure during pregnancy [ERα signaling.Despite the apparent complicated relationship between PAH, systemic and breast adiposity, and breast cancer risk ,24, littregnancy . In addiregnancy  also wasregnancy  and systregnancy , influen