Shivering is described as an involuntary, repetitive activity of the skeletal muscles that can have deleterious effects on anaesthetized patients. This study aimed to evaluate the effectiveness of phenylephrine infusion in preventing perioperative shivering in patients undergoing lower segment cesarean section under spinal anesthesia and to observe the change in the patient’s core temperature between the study and control groups.A total of 118 patients scheduled for elective lower segment cesarean section under spinal anesthesia were recruited for this prospective, double-blind, randomized controlled study. The patients were randomized into 2 groups with 59 patients per group. The phenylephrine Group received phenylephrine infusion at a rate of 0.5 mcg/kg/minutes, while the Control Group received normal saline at an equivalent rate. Systolic and diastolic blood pressure, heart rate, core temperature, and the presence and intensity of shivering were recorded before induction and every 15 minutes intraoperatively and postoperatively.P = .044). Postoperatively, the Phenylephrine Group also had a lower incidence of shivering (34.5% vs 42.4%), but the difference was not statistically significant . There were no significant differences in the intensity of shivering between the 2 groups perioperatively, as well as in the systolic and diastolic blood pressure and core temperature. The phenylephrine Group showed a significantly lower heart rate at 15, 30, and 45 minutes after spinal block , and at 0 and 30 minutes  in the recovery room. There were no significant differences in perioperative adverse events such as hypotension, hypertension, and bradycardia.The incidence of intraoperative shivering was significantly lower in the Phenylephrine Group compared to control group (29.1% vs 47.5% respectively; Phenylephrine infusion reduces the incidence of perioperative shivering in lower segment cesarean sections under spinal anesthesia. Studies have demonstrated that the incidence of shivering associated with central neuraxial block is at the range 40% to 70%.,3 The incidence of shivering in obstetric patients with neuraxial blockade is between 40% to 60%.,5Shivering is described as an involuntary, repetitive activity of skeletal muscles. It is activated when behavioral compensation and maximal vasoconstriction are insufficient to maintain core temperature.,6,7 These effects can be detrimental, especially in patients with a poor cardiopulmonary reserve. Shivering also causes patient discomfort and increases the likelihood of postoperative complications, such as infection, pain, and bleeding, leading to delayed wound healing and increased length of hospital stay.,9It is important for anesthesiologists to recognize and treat perioperative shivering and to prevent it, if possible, as shivering can have deleterious effects on anesthetized patients. Shivering can increase intraocular and intracranial pressure, oxygen consumption by 300% to 400%, carbon dioxide production, catecholamine release, metabolic heat production by up to 600%, metabolic rate by up to 400%, and lactic acidosis. Shivering during central neuraxial anesthesia is preceded by core hypothermia and vasoconstriction above the level of the block.Perioperative hypothermia is defined as a core temperature of <33 to 35 °C, whereas the shivering threshold in non-anesthetized patients is 35.5 °C. Central neuraxial anesthesia impairs both central and peripheral thermoregulation as it increases the inter-threshold range by increasing the sweating threshold and decreasing the vasoconstriction threshold and shivering threshold (triggering core temperature) by approximately 0.5 to 0.6℃.,12 Phenylephrine may also attenuate core hypothermia by inhibiting core to-peripheral redistribution of body heat during spinal anesthesia. The aim of this study was to evaluate the effectiveness of phenylephrine infusion in preventing perioperative shivering in patients undergoing lower segment cesarean section under spinal anesthesia and to observe the change in the patient’s core temperature between the study and control groups. The secondary objective of this study was to evaluate hemodynamic changes between the 2 groups. We hypothesized that phenylephrine infusion would reduce perioperative shivering in patients under spinal anesthesia.Phenylephrine is an alpha-1 adrenergic receptor agonist with minimal to no beta-adrenergic activity. Therefore, it is mainly used intraoperatively to treat hypotension.th May 2019 study approval number FF-2019-273). This study was registered at ClinicalTrials.gov (ID NCT04133961). This prospective, double-blind, parallel-group, randomized controlled study was conducted in the maternity operating theater of the Universiti Kebangsaan Malaysia Medical Centre from November 2019 to May 2020 in accordance with the Declaration of Helsinki 2013. All the patients who were screened and met the eligibility criteria were invited to participate in the trial, and all the enrolled patients provided written informed consent. Consent was requested from patients upon arrival to the operating suite for surgery or in the ward if they were admitted the night before surgery.Ethical approval for this study was provided by the Medical Research and Ethics Committee, Universiti Kebangsaan Malaysia Medical Centre, on 302, and height < 150 cm and patient’s refusal to participate in the study.In total, 118 patients were recruited for this study. Inclusion criteria included patients aged 18 to 40 years old, American Society of Anesthesiologists physical status score II, and singleton pregnancy who were scheduled to undergo elective lower segment cesarean section under spinal anesthesia. Exclusion criteria included contraindications to phenylephrine, evidence of fever or infection, hypothermia (<35 °C) preoperatively, body mass index > 40 kg/mUsing a computer-generated table, patients were randomly assigned to 1 of 2 groups, Phenylephrine (Group A) or Control (Group B) with a 1:1 allocation. Patient identifiers were attached to the opened envelopes and secured by a dedicated person, independent of the randomization procedure.All patients fasted for at least 6 hours prior to surgery. They were given gastric acid aspiration prophylaxis prior to the surgery, which consisted of 150 mg oral ranitidine on the night prior to surgery and on the morning of surgery, and 30 mL of 0.3M sodium citrate oral solution on the morning of surgery. Patients and the attending anesthesiologist were blinded to the group to which the patients were allocated. Both phenylephrine  and normal saline were prepared in 20 mL syringes by the investigator, and phenylephrine was diluted to a concentration of 100 mcg/mL. Patients in Phenylephrine Group (Group A) received phenylephrine infusion at rate of 0.5 mcg/kg/minute, which was equivalent to 0.3 mL/kg/hour, whereas Control group (Group B) patients received a saline infusion at a same rate of 0.3 mL/kg/hour. The syringes were labeled based on the same computer-generated randomized numbers, so that the attending anesthesiologist and patient did not know the content of the syringes.Before induction of anesthesia, standard monitors, such as noninvasive systolic and diastolic blood pressure monitoring, continuous electrocardiography, and pulse oximetry, were applied to the patient. Baseline blood pressure, heart rate, oxygen saturation, and core body temperature were measured prior to induction. The ambient temperature of the operating theater was recorded, as measured by a standardized wall thermometer. The baseline core temperature of the patient was measured at the tympanic membrane using an infrared thermometer (Novo Temp TH50Z). Core temperature was measured every 15 minutes intraoperatively. All vital signs were recorded by an attending anesthesiologist who was blinded to the study.0.” The patient was then positioned supine with left lateral tilt to prevent supine hypotension syndrome. Infusion of the study drug was initiated immediately after the block. The level of sensory blockade was tested using loss of sensation to cold, and anesthesia was considered adequate with a sensory block ≥ T4 dermatomal level. After the spinal block, the patient was covered with blankets in the upper body and lower limbs. All patients also received active warming via a forced-air warming device (Bair Hugger temperature set at 38 °C) placed in the upper part of the body. All fluids were prewarmed at 40 °C. Systolic and diastolic blood pressure and heart rate were recorded immediately after the spinal block and every 5 minutes for the first 30 minutes, followed by every 15 minutes thereafter. Other recorded intraoperative data included the total volume of fluids administered and estimated blood loss.A separate intravenous cannula was inserted to administer the study drugs. Co-loading with 500 mL of Hartmann’s solution was initiated in all patients prior to the commencement of spinal anesthesia. The patient was placed in a sitting position for spinal anesthesia. Spinal block was performed at the L3/L4 or L4/L5 levels using a 27-gauge spinal needle by the attending anesthesiologist under aseptic technique. Upon free flow of cerebrospinal fluid, 2 mL of 0.5% hyperbaric bupivacaine mixed with 0.1 mg morphine and fentanyl (15 µg) was administered to the spinal block. The time the spinal block was administered was recorded as “tThe primary outcome of the study was the incidence of shivering and changes in the patient’s core temperature, while the secondary outcomes were changes in hemodynamic parameters such as systolic and diastolic blood pressure and heart rate. All outcomes were assessed by the attending anesthesiologist both intraoperatively (from induction until completion of skin closure) and postoperatively in the recovery room. The Bedside Shivering Assessment Scale score ranges from 0 to 3, where 0 indicates no shivering, 1 indicates that shivering is localized to the neck and/or thorax, 2 indicates gross movement of the upper extremities, and 3 indicates gross movements of the trunk and upper and lower extremities. If the score was 2 or higher, intravenous pethidine 25 mg was administered after the baby had been delivered.The intensity of shivering was recorded by the attending anesthesiologist immediately after the spinal block and every 15 minutes during the surgery using the Bedside Shivering Assessment Scale.Adverse events, such as hypotension, hypertension, and bradycardia, were recorded and managed accordingly. Hypotension (defined as systolic blood pressure < 80% of the baseline) was treated with bolus doses of intravenous ephedrine (6 mg titrated to effect). Intravenous ephedrine were the first choice of rescue drugs to avoid bias. If the blood pressure remained low, an intravenous phenylephrine 100 µg bolus was administered, up to a maximum of 300 mcg. If hypertension (defined as systolic blood pressure > 120% of the baseline) occurred, the study drug infusion was terminated. If bradycardia (defined as a heart rate < 40 beats per minute) occurred, the study drug infusion was terminated, and intravenous atropine (0.5 mg) was administered if it was accompanied by hypotension. The dose and time of administration of all the rescue drugs were recorded. Patients were excluded from the study if they received more than 300 µg of phenylephrine bolus, conversion to general anesthesia, primary postpartum hemorrhage with blood loss more than 700 mL, surgical time exceeded 120 minutes, or if the drug infusion was terminated earlier during the surgery.Drug infusion was terminated at the end of skin closure. Surgical time was defined as the time from the start of the surgical incision to completion of skin closure. The patient was transferred to the recovery room after surgery, and a warming blanket covering the entire body was applied. The ambient temperature of the recovery room was recorded. The patient’s core temperature, blood pressure, and heart rate were monitored every 15 minutes until the patient was discharged from the recovery room., 22.5%). An estimated sample size of 54 patients in each group was obtained, with the power of this study set at 80% and the α value set at 0.05. Considering a 10% dropout rate for this study, the estimated sample size was 59 patients in each group.The sample size was estimated using the PS Power and Sample Size Calculations program by Dupont and Plummer (2009), based on the incidence of shivering among patients who received phenylephrine . Results are presented as mean ± standard deviation, median with interquartile range, or frequency (percentages), where appropriate. An independent A total of 118 patients were recruited for this study, with 59 patients in each group. In total, 114 patients were included in the final analysis, with 55 and 59 patients in the phenylephrine and control groups, respectively. There were 4 drop-outs in the Phenylephrine Group, where the study drug infusion was terminated before the end of surgery due to bradycardia and hypertension. Figure There were no significant differences in the demographic and intraoperative data between the Phenylephrine (Group A) and Control groups (Group B) Table .P = .044). Postoperatively, the incidence of shivering was lower in the Phenylephrine group (34.5%) than in the Control Group (42.4%); however, this difference was not statistically significant (P = .391).The incidence of perioperative shivering is shown in Figure Table Figure Figure P = .005, .000, and .008, respectively), and at 0 and 30 minutes  in the recovery room.Figure P = .145).Five patients in the Control Group (8.5%) required rescue pethidine for shivering compared to 1 patient in the Phenylephrine Group (1.8%). However, this difference was not statistically significant .Regarding adverse events, 24 patients in the Phenylephrine Group (43.6%) and 21 patients in the Control Group (35.6%) experienced intraoperative hypotension, which was treated with ephedrine. One patient in the Phenylephrine Group (1.8%) had intraoperative hypertension. Postoperatively, 1 patient in the Control Group (1.7%) had hypertension. There were no significant differences between the groups for any adverse event  than in the control group (0.57℃), this data was not statistically significant. This finding was contrasted to 3 of other studies. This was in accordance with our findings, in which patients who received phenylephrine demonstrated a higher systolic blood pressure 15 minutes after surgery. This effect of phenylephrine is beneficial for treating hypotension, and in clinical practice, phenylephrine has been widely used to treat hypotension in cesarean section.,19,20 Hypotension during spinal anesthesia could be due to the vasodilatory effects of the spinal block, which reduces systemic vascular resistance. Spinal block also causes a decrease in venous vasomotor tone, which increases venous pooling and consequently reduces venous return, thereby decreasing cardiac output.Phenylephrine increases systemic blood pressure by increasing the systemic vascular resistance. Patients who received phenylephrine demonstrated a lower heart rate trend throughout surgery, and this effect persisted in the recovery room even after stopping the infusion. Although the difference in heart rate between both groups was statistically significant at 15 to 45 minutes postspinal block, and at 0 minutes and 30 minutes in the recovery room, the heart rate trend in the phenylephrine group was above 70 beats per minute, which suggested that it is safe to use phenylephrine infusion in cesarean section. Previous studies have recommended titrating phenylephrine infusion to maintain blood pressure near baseline levels to better tolerate a relatively lower heart rate.,19Phenylephrine causes bradycardia in response to a physiological baroreceptor reflex.This study had a few limitations. First, this study was conducted only among the maternal population who underwent lower segment cesarean section; hence, it is uncertain whether the type of surgery had any influence on the results. The presence of other factors, such as anxiety, could contribute to shivering, which we did not consider in our study. In addition, the primary outcome (shivering) was measured at 15-minute intervals in our study, but changes might have occurred between measurements, which may have influenced the trend of the results. Finally, we did not determine the optimal phenylephrine dose required to effectively reduce perioperative shivering. We recommend that phenylephrine be studied in different population groups with regard to perioperative shivering. Further studies are required to determine the optimal dose of phenylephrine to prevent shivering.We conclude that phenylephrine infusion reduces the incidence of perioperative shivering in lower segment cesarean sections under spinal anesthesia.Conceptualization: Syarifah Noor Nazihah Sayed Masri.Data curation: Jessica Tan Sook Kuan.Formal analysis: Qurratu Aini Musthafa.Funding acquisition: Jessica Tan Sook Kuan, Farah Hanim Abdullah.Investigation: Jessica Tan Sook Kuan.Methodology: Jessica Tan Sook Kuan, Qurratu Aini Musthafa.Project administration: Jessica Tan Sook Kuan.Software: Qurratu Aini Musthafa.Supervision: Syarifah Noor Nazihah Sayed Masri.Validation: Qurratu Aini Musthafa, Syarifah Noor Nazihah Sayed Masri.Visualization: Farah Hanim Abdullah, Syarifah Noor Nazihah Sayed Masri.Writing – original draft: Jessica Tan Sook Kuan.Writing – review & editing: Farah Hanim Abdullah, Syarifah Noor Nazihah Sayed Masri.


The latest advancements in bone scaffold technology have introduced novel biomaterials that have the ability to generate oxygen when implanted, improving cell viability and tissue maturation. In this paper, we present a new oxygen-generating polylactic acid (PLA)/calcium peroxide (CPO) composite filament that can be used in 3D printing scaffolds. The composite material was prepared using a wet solution mixing method, followed by drying and hot melting extrusion. The concentration of calcium peroxide in the composite varied from 0% to 9%. The prepared filaments were characterized in terms of the presence of calcium peroxide, the generated oxygen release, porosity, and antibacterial activities. Data obtained from scanning electron microscopy and X-ray diffraction showed that the calcium peroxide remained stable in the composite. The maximum calcium and oxygen release was observed in filaments with a 6% calcium peroxide content. In addition, bacterial inhibition was achieved in samples with a calcium peroxide content of 6% or higher. These results indicate that an optimized PLA filament with a 6% calcium peroxide content holds great promise for improving bone generation through bone cell oxygenation and resistance to bacterial infections. The shortage of organ donors is a significant challenge for global healthcare systems. The growing need for tissue and organ transplants has led to a significant gap between the availability of donors and the demand for transplants. This issue is particularly pronounced in the United States, where, as of March 2022, 106,097 people were waiting for organ transplants. Unfortunately, the shortage of donors results in a tragic loss of life. Current statistics indicate that, as of March 2022, an average of 17 deaths per day in the United States were attributed to delays in transplant surgery due to a lack of available organs [Bone is an essential component of our skeletal system that provides structural support, protects vital organs, and enables movement. However, the human body’s ability to repair bone injuries is limited, making bone grafting a crucial procedure for bone fracture healing and regeneration. A bone graft is a surgical procedure in which bone tissue is transferred from one location in the body to another location where bone loss has occurred . The useRecently, there has been a growing interest in fabricating bone scaffolds using 3D printing technologies. This approach allows the design of porous scaffolds with a specific exterior design and porous interior structure to achieve scaffolds with tailored functionality. This enables the scaffold to mimic the natural bone structure and support bone growth. Furthermore, 3D printing technology also allows for the large-scale production of scaffolds, making it more cost-effective and efficient . Fused D3H4O2)n), polycaprolactone n), and poly(glycolic acid) n) are commonly used in bone repairs, tendons, and skin, as they possess favorable characteristics [Materials such as polylactic acid  scaffold, made up of 40% beta-tricalcium phosphate and 60% hydroxyapatite, was fabricated using the robocasting technique. Touri et al. fabricated biphasic calcium phosphate (BCP) scaffold composed of 60% hydroxyapatite and 40% beta-tricalcium phosphate using the robocasting technique. Although an alternative composite filament was investigated, the findings mirror that of our study, with the oxygen release behavior dependent on the concentration of CPO encapsulated in the PLA filaments. It was found that a 3% concentration of CPO was sufficient to produce desirable results in terms of promoting bone ingrowth, increasing the survival of osteoblast cells, and stimulating proliferation in low oxygen conditions .Oversaturation remains a challenge in the preparation of composite filaments, with Zhang et al. observing that an environment with significantly oversaturated oxygen tension was created in the first 48 h in single-walled microspheres. In turn, this may result in the creation of a hyperoxia environment in cases where the oxygen demand by cells is not as high. Double-walled microspheres may better support the oxygen tension, but further research is warranted to determine their efficacy .The available evidence suggests that oxygen-releasing materials are limited by their short release periods of 24 to 48 h ,30,31. HP. aeruginosa bacteria and Gram-negative E. coliS. aureus and MRSA, which has a higher resistant [None of the samples displayed any inhibition zone when tested against esistant , some saesistant ,34.S. aureus and MRSA, indicating that these PLA scaffolds with low CPO content had no effect on the bacteria. A significant inhibition zone was seen at 6% CPO and 9% CPO, indicating that increasing the CPO content to 6% or higher resulted in an antibacterial activity against Gram-positive bacteria, which is consistent with previous research [As shown in the graphs in research , indicatresearch .Porosity is among the most critical properties of biomaterials, particularly in tissue engineering applications, as it is correlated to the biomaterials’ swelling capability. In For the study, 1.75 mm-diameter polylactic acid (PLA) filaments, molecular weight Mw 60,000, were obtained from . Calcium peroxide (CPO) with a particle size of −200 mesh and a purity level of 75% was procured from , a leading supplier of research chemicals and laboratory equipment. Additionally, pure natural bulk enzyme catalase powder  was purchased from . Lastly, dichloromethane (DCM) was obtained from .The process of preparing the PLA/CPO composite filament starts by cutting PLA into 20 g pieces and dissolving it in 100 mL of DCM for 30 min at room temperature with a magnetic stirrer at 700 rpm, see To examine the generated filaments, a scaffold was printed using a commercial Fused Deposition Modelling (FDM) 3D printer—the Creality Ender 3 Pro model manufactured by . The printing process was carried out with the highest CPO ratio (9%) at a temperature of 200 °C and a speed of 75 mm/s. The scaffold was printed successfully in a box shape with dimensions of 8 × 8 × 8 mm for length, width, and height. The scaffold had a porosity of 25% with a pore size of 0.60 mm.The composite filament’s surface and cross-sectional micrographs were captured using field emission scanning electron microscopy (FESEM) . The imaging was performed at an acceleration voltage of 5 keV, and gold sputter coated prior to imaging to enhance the conductivity and provide better resolution. The images were used to study the morphology of the filament and to confirm the dispersion of the CPO particles in the PLA matrix. Additionally, optical images of composite filaments surface were captured using a Canon 1000D camera. This provided a visual representation of the filament’s surface and helped in identifying any morphological changes. Additionally, the XRD was carried out using Cu Ka radiations ULTIMA IV XRD systems . The analysis of material elements was conducted using ICDD, DB card No. 01-071-4107. Additionally, Alizarin Red Staining solution was used to detect CPO particles within the fabricated filament.To prepare the solution, 0.40 g of the Alizarin Red Staining powder was dissolved in 20 mL of distilled water with the aid of a stirrer. The samples were first soaked in an alkaline solution for 20 min and then left to soak in the same solution for 20 h in a dark environment.The degradation of the filament was examined by measuring the mass loss over time. A solution of 2 g of sodium hydroxide was made by dissolving it in 50 mL of distilled water to accelerate the degradation process. Each filament weighing 10 g was immersed in an alkaline solution at a room temperature of 18 °C, and the weight loss and calcium ion release were measured every 24 h during the degradation process for three days. The weight loss of the samples was calculated using Equation (3). The calcium ion release was observed using an ICP optical emission spectrometer .To measure the oxygen released by the filament, the samples were placed in a phosphate-buffered saline (PBS) solution with a pH of 7.4, and their oxygen levels were measured using an oxygen sensor. In addition, 10 mg of catalase were added in the solution.The acidity level (pH) of the samples were measured every 24 h using a digital pH meter.The porosity was characterized using field emission scanning electron microscopy (FESEM)  with an accelerating voltage of 5 keV. For each sample, three SEM images were captured from random spots. From the obtained SEM images, the porosity percentage of the total area was calculated using ImageJ (version 1.53f51). The SEM images were converted to 8-bit binary (white and black) images for ImageJ analysis, where the white spots indicate the solid surface and the black spots represent the pores.Escherichia coli ATCC 11,775 (E. coli), Staphylococcus aureus ATCC 12,600 (SA), Pseudomonas aeruginosa ATCC 9027 (PA), and Methicillin-resistant Staphylococcus aureus ATCC 33,591 (MRSA). We cultivated these bacteria strains on blood agar and stored them on Mueller Hinton agar (MHA)  plates in the refrigerator at 4 °C for later use. When the experiments were performed, the bacteria strains were activated in Mueller Hinton broth (MHB) ; after measuring their turbidity (0.1 ± 0.02), they were spread on the MHA plates via swab cotton. The composite filaments were prepared in a disk shape and then fixed on the surface of the MHA plates containing the bacteria strain beside a standard 30 μg cefoxitin antibiotic disc (FOX) from Mast . Then, all MHA plates were incubated at 37 °C for 24 h. Finally, we measured the diameters of the bacteria growth inhibition zone; the antibacterial performances of the prepared composites were compared with the standard antibiotic FOX disks.The antimicrobial performance of the composite filaments was examined using disk diffusion tests. We utilized the following bacteria strains in the study: Each test was conducted in triplicate, and a mean value was calculated. The data are calculated as the mean with a ± standard deviation (SD). OriginPro 8.0 software  was employed to analyze the data.The field of bone tissue engineering is expanding and focuses on developing new techniques and materials to repair or replace damaged or diseased bones. One promising area of research in this field is the use of 3D printing techniques to create composite filaments for use in bone tissue engineering. PLA is a biodegradable and popular polymer for tissue engineering applications due to its preferable biocompatibility and mechanical properties. CPO, on the other hand, is a material that can release oxygen and calcium ions when it comes into contact with water. This property makes it a useful component in bone tissue engineering, as the increased oxygen levels and calcium ions can help to promote bone growth and healing. This study introduces a novel PLA/CPO composite filament created through wet solution mixing and hot melting extrusion, a process that can control the composition and morphology of the filament. The study examined the effect of varying CPO content on filament performance. The results showed that a 6% CPO ratio provides optimal calcium and oxygen release. However, when increasing CPO content, the composite filament displays poor dispersion of large, agglomerated particles, which can affect its mechanical properties. The composite filament with CPO content above 6% showed excellent antibacterial activity, which can be useful in preventing infections in bone tissue engineering applications. Furthermore, an in-depth study about cytocompatibility is currently being performed and will be published in a separate paper.


Bacillus coagulans Unique IS-2 in a viable but non-germinating form, preventing spoilage during storage and imparting protection against the growth of any other contaminating organism (self-preserved formulation). The gelatin–oil–probiotic suppository showed uniformity in weight and probiotic content (23 ± 2.481 × 108 cfu) with favorable swelling (double) followed by erosion and complete dissolution within 6 h of administration, leading to the release of probiotic (within 45 min) from the matrix into simulated vaginal fluid. Microscopic images indicated presence of probiotics and oil globules enmeshed in the gelatin network. High viability (24.3 ± 0.46 × 108), germination upon application and a self-preserving nature were attributed to the optimum water activity (0.593 aw) of the developed composition. The retention of suppositories, germination of probiotics and their in vivo efficacy and safety in vulvovaginal candidiasis murine model are also reported.The current study describes a suppository base composed of aqueous gelatin solution emulsifying oil globules with probiotic cells dispersed within. The favorable mechanical properties of gelatin to provide a solid gelled structure, and the tendency of its proteins to unravel into long strings that interlace when cooled, lead to a three-dimensional structure that can trap a lot of liquid, which was exploited herein to result in a promising suppository form. The latter maintained incorporated probiotic spores of  Candida albicans and C. glabrata [Gelatin is an affordable and widely accessible polymer due to its biodegradability, biocompatibility, and safety in terms of non-toxic byproducts produced upon enzymatic degradation. Gelatin contains motifs such as Arg-Gly-Asp (RGD), which exhibit cell adhesion, ensuring close contact and hence good biological activity as compared to other polymers . This adglabrata . Opportuglabrata . Almost glabrata .About 1.glabrata , while tglabrata  availablglabrata .There isglabrata .Probiotics inhibit pathogenic Candida species and maintain and modulate microbiota profiles in the vagina ,9,10,11.Delivery of the probiotic through gut to alter vaginal health has previously been reported by many research groups ,21. HoweGelatin and glycerol–gelatin vaginal suppositories are extremely popular, attributed to their ease of use and soothing effect, and provide prolonged and complete release of the active load owing to their slow dispersion in mucosal secretions ,26. FurtThe preservation of the developed system against contaminating organism is another concern with gelatin-based products, as the latter can support the growth of some organisms. Low water activity does not support the growth of such organisms, and thus there is no need to add a preservative into the suppositories. This is of significance for a probiotic product, because the incorporated preservative can affect probiotic viability ,29.Bacillus coagulans Unique IS-2, a nonpathogenic, Gram-positive, spore-forming bacteria, was loaded into the above-described oil–gel suppository base. The selected strain of bacteria has reported clinical efficacy against bacterial vaginitis [® gel) in VVC rat model. The developed probiotic–oil–gelatin suppositories were evaluated for stability (2–8 °C for 3 month) and in vivo 15-day safety studies.In the present study, we thus endeavored to develop a suppository system with an intelligent choice of ingredients containing a self-emulsifying gel base, oil, surfactant, and a soothing agent, which keeps the incorporated probiotic viable, prevents any contamination of the product during storage, aids in the germination of the incorporated probiotic upon application, is acceptable for use and helps maintain/transplant healthy flora in the vaginal cavity for vaginal health. aginitis . The dev8 cfu per suppository. The content uniformity evaluation of the developed gelatin–oil suppositories indicated that the distribution of colony forming units of Bacillus coagulans was uniform in the apex and bottom part of the suppository; however, it was considerably low in the middle part (Bacillus coagulans powder was confirmed by the pour plate method (n = 8) as per the procedure given in the 6 cfu/mg. The cell surface hydrophobicity of Bacillus coagulans was 30.4 ± 5.34%  .The size and size distribution of both the blank and gelatin–oil–probiotic suppositories were determined after dispersion in water. At ambient temperature, the average particle size of the blank suppository was found to be 493.1 nm. The distribution curve for the same a showed The gelatin–oil suppositories prepared here are expected to release probiotic both by melting and dissolution. Small probiotic-loaded oil particles obtained upon the dissolution of the prepared suppositories will not only cover a larger area but will also achieve an intimate contact with the vaginal mucosa . This wiw) is defined as a measure of the energy status of the water present in a sample referred to as free, unbound, or active water. It is different from total water content, as a portion of the total water contained in a sample is at times strongly bound to specific sites of various components in a sample, and hence may not affect the preparation. Active water, on the other hand, influences the shelf life of products as it makes the preparation liable to microbial growth [w at which most bacteria will grow in a product is about 0.90. The aw for mold and yeast growth is approximately 0.61 aw with the lower limit for the growth of mycotoxigenic mold being 0.78 aw [w, whereas during storage for two months (2–8 °C) it increased to 0.593 aw. Although a marginal increase in water activity was recorded for the developed suppositories, the value was still lower than the reported range (0.61–0.90), favoring microbial  growth both of the probiotic, or the contaminating organism. Thus, it may be said that the developed gelatin–oil suppository will be non-reactive, or in other words, will keep the probiotic dormant, i.e., without germination (as confirmed in stability studies). Furthermore, suppositories per se will be endowed with a self-preservation against any harmful bacterial growth during storage and use. It may be noted that the suppository base used here was not a simple gelatin or a glycerol–gelatin base; instead, it was a gelatin base incorporating probiotic-laden small oil globules.Water activity  after storage under refrigerated conditions for three months, as shown in The probiotic spores incorporated into the developed formulation were found to maintain their viability Swelling of gelatin suppositories helps them to snugly fit in the reproductive tract.(2)Small amount of vaginal fluid is insufficient to initiate the dissolution process, such that the base melts at body temperature to a viscous gel-like consistency, as also observed in the in vitro study. Since the consistency of the melted suppository is high, it stays within the tract and does not leak out.(3)Furthermore, the release of probiotic-loaded oil particles coated with viscous gel cover a larger vaginal area, with vaginal contractions further helping in a widespread distribution.In vivo retention studies indicated that the suppository was intact and maintained its shape even at 10 min after its insertion into the vaginal cavity of the rats . CompletThe vaginal mucosa is highly sensitive to the exposed chemicals and therapeutic agents that may result in irritation and/or inflammation and can make women more susceptible to various infections. Hence, the vaginal irritation potential of feminine care formulations and vaginally administered therapeutic agents is a significant public health concern. The pH of the developed suppository was found to be 4.3 ± 1.0. The pH is very near the normal vaginal pH, thus the suppositories are expected to be non-irritating to the vaginal mucosa. An in vivo vaginal irritation study was performed in rats to evaluate the safety of the vaginal formulations on their multilayered vaginal epithelium. The latter is a characteristic feature of mammalian vagina, too.The results from the histopathological examination  showed tBacillus coagulans spores are reported to form co-aggregates with Candida albicans, and the same was also observed in the present study , free probiotic suspension, probiotic-loaded suppositories, and blank suppositories) were initiated in the infected rats and the fungal burden was monitored every day for seven days. Macroscopic observation  the inhibition follows mechanisms other than the release of bacteriocins/inhibitory substances in the surrounding media; (b) factors such as the immune responses of the host, changes in the immune system induced by C. albicans enzymes or metabolites present in the vaginal lumen cannot be mimicked in the in vitro tests, although they do exert a great influence on the effects demonstrated by probiotics in vivo.The probiotic-loaded suppositories did not show any inhibition by either well diffusion or the streak method. It may be concluded that the vegetative probiotic cells do not inhibit p < 0.001) was observed between the marketed formulation and the free-probiotic-treated group, whereas the probiotic-loaded suppository group showed significantly (p < 0.001) better results. However, on the 7th day of treatment, both the free probiotic group and probiotic-loaded suppository group showed significantly better effects than the marketed formulation. The free probiotic dispersion showed significant effects; however, it tended to leak out following application, clearly concluding that its incorporation into a suppository base, designed herein, is a successful dosage form.Fungal burden studies  indicateB. coagulans spores and vegetative cells did not show any activity against Candida in vitro, the effects obtained in vivo establish the usefulness of both the probiotic per se and the developed gelatin–oil suppository formulation. Thus, it may be said that the observed in vivo effects are mediated by mechanisms such as the competitive exclusion of adherence to epithelial cells by Candida, co-aggregation and auto-aggregation leading to the inhibition of Candida, and the modulation of both the immunological reactions initiated by Candida and the local immunological host response.Furthermore, as is expected of azole antifungals, which are fungistatic in nature, the marketed formulation containing clotrimazole did not show any significant decrease in fungal loads from day four onwards, while the decrease in fungal loads continued with both the probiotic-treated groups. The cfu/animal for the positive control group and blank suppository group did not show any significant inter-day difference, while for the free probiotic group and probiotic suppository group, a significant difference was observed. In the marketed formulation, no significant difference was observed between the 1st and 2nd day and 4th, 5th, and 6th days . What is6 spores, administered per day for seven days, translocated to reach systemic circulation when inserted into the infected animals. This value is clearly insignificant, and it was confirmed that the presently used strain was safe for vaginal administration in infected animals. It confirms that the action of the developed probiotic-loaded vaginal suppositories remains localized.The in vivo translocation study indicated that only 0.007 ± 0.001% of 122 × 10® gel). Unlike the available vaginal probiotic products in the market that contain either extracellular substances or bioactives obtained from probiotics, the currently presented formulation consists of whole-cell probiotics.In the present study, we developed probiotic-loaded gelatin–oil biphasic suppositories for combating vaginal fungal infections. The formulation was tested in an animal model of vulvovaginal candidiasis where it showed effects comparable to a marketed product  are demonstrated by these strains in their active state.The developed gelatin–oil suppositories were safe in terms of pH, vaginal irritation, and translocation/permeation. High patient acceptability is expected, as indicated by in the vivo retention study suggesting no leakiness.The in vivo therapeutic effects of the probiotic-loaded suppositories were confirmed in terms of the decrease in the cfu of Candida in infected rats. The developed probiotic-loaded suppositories can also incorporate other probiotic cells, including their vegetative forms, with equal success, as they have the capability of keeping the loaded probiotic in its dormant state during storage. Furthermore, the components and equipment used in this study are cheap and widely available, which makes it an industrially viable option.Bacillus coagulans  (100 × 106 cfu/mg) was provided by Unique Biotech Ltd., Hyderabad, India. Gelatin  was light yellow to beige in color having pH of 5–6 (1% solution), and gel strength of 156 G bloom. Glycerin , Tween 80® (CDH (P) Ltd., New Delhi, India) and sunflower oil  (linoleic acid 40–80% of unsaturated fatty acids) were used for preparation of probiotic formulations in the present study.All the microorganism cultures employed in this study were procured from Institute of Microbial Technology (IMTECH), Chandigarh, India. Culture medium including PNY, agar, Sabouraud and soyabean casein digest were purchased from Hi Media Laboratories Pvt Ltd., Mumbai, India.Gelatin was dissolved using a magnetic stirrer in the aqueous phase comprising a suitable surfactant and humectant at 60 °C to result in a gel-like matrix. Weighed quantity of probiotic spores was dispersed in sunflower oil, maintained at 60 °C, which was then emulsified slowly with the aqueous phase. The mixture was cooled to 45 °C and poured into previously cooled suppository molds. Suppository-loaded molds were refrigerated overnight, and the formed suppositories were removed, packed in butter paper, and stored in tightly closed containers until further use.Variation in weight of suppositories was recorded by weighing 20 suppositories and calculating average weight.n = 6) can bear without breaking. It also determines the tensile strength of the suppositories to access whether they will be able to withstand the wear and tear during transportation and storage. Hardness of the suppositories was evaluated using Monsanto hardness tester.The test is used to measure the mass (in kilograms) that a suppository (n = 6) were kept in closed containers over a water bath at 37 °C and observed for the lag time before the melting started and the time taken to melt completely.The suppositories (n = 6) was determined by placing a pre-weighed suppository in a 50 mL beaker at room temperature. Fixed quantity (0.75 mL) of simulated vaginal fluid .Suppositories were placed in disintegration apparatus containing 1000 mL of SVF and maintained at 37 ± 0.5 °C. The time taken by the suppository to either melt or dissolve completely in the medium was taken as the disintegration time .n = 6) were dissolved in 10 mL of sterile saline solution, one at a time, and diluted appropriately (105–106). The dilutions (1 mL) were subjected to heat shock for 30 min (to kill any vegetative cells) at 75 °C and plated on sterilized PNY agar medium [Suppositories (r medium . The plan = 3).The test was performed in triplicate (w/v). Samples were diluted appropriately (106) and underwent heat shock at 75 °C. After cooling to about 45 °C, 1 mL aliquots were plated on previously prepared PNY agar medium. The colonies were counted, and results expressed as cfu/g for each part and compared with one another to confirm uniformity of distribution. The test was performed on 6 suppositories.The suppository was cut into three parts: apex, middle, and bottom portion. Each part was weighed and dissolved completely in 10 mL of normal saline solution  were determined by dispersing each suppository in water. Dynamic light-scattering method (DLS), using a computerized inspection system  was used to determine size and size distribution at 25 °C and 37 °C. The light was emitted from a laser diode  at an angle of 160°.The measurements for both probiotic-loaded and blank probiotic dispersions were performed at 25 °C using Delsa Nano C, Beckman Coulter at the electric field strength of 23.2 V/cm. The zetasizer measures the zeta potential based on the Smoluchowski equation:Water activity of freshly prepared suppositories and those stored for 2 months at 2–8 °C for the months May and June 2016  was determined with a computer-controlled Rotronic Hygro Lab , operating between 0.5 and 50 °C. The sample was placed in a sealed container that slowly exchanged moisture with the air inside the container until equilibrium was reached. The equilibration process was monitored by measuring the humidity of the air above the sample with a relative humidity sensor.A cross-section of the developed vaginal suppositories was examined using a scanning electron microscope (Model JSM6100 (Jeol) with Image Analyser). Prior to observation, samples were dried under a vacuum drier and were mounted on metal grids using double-sided adhesive tape.The suppositories were evaluated in terms of change in viability of the loaded probiotic formulation and physical appearance upon storage under refrigerated conditions, i.e., 2–8 °C for 3 months.Sprague Dawley female rats (150–200 g) obtained from central animal house, Panjab University, Chandigarh, India were used for the study. The animals had free access to standard rodent food pellets  and water. They were acclimatized to the laboratory conditions before the experiment. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC)  of Panjab University, Chandigarh, India.The previously described suppository base was poured into small molds prepared using aluminum foil and kept in the refrigerator overnight for solidification. The size of molds was such that it would form suppositories of a size suitable for insertion into the rat vagina.The vaginas of rats were sterilized by washing with sterile PBS solution followed by gavage with 200 µL of 70% alcohol. The opening of the vagina was also washed with 70% alcohol to kill any adhering bacteria. After 1 h, the rat’s vagina was again washed with sterile PBS solutions. The suppositories were inserted into the rat’s vagina followed by swabbing at 4 h, 6 h and 24 h post-insertion. The swabs were rolled onto the glass slides followed by their microscopy. The heat-fixed slides were treated with malachite green (5%) for 5 min in a hot boiling water bath. After 5 min of heating, excess stain was removed by washing under running water. This was followed by the addition of safranin stain to the slides for 30 s and washing off excess stain under running water and blot drying. Stained slides were examined at 100× in oil-immersion lens by light microscopy . Germination of the spores was determined by distinguishing the pink color of vegetative rods from green spores.n = 3) were sacrificed by cervical dislocation at different time points , followed by dissection to observe vaginal cavity and its scoring in terms of intactness of suppository and intensity of blue color at the site, and leakiness, if any.The trypan-blue (0.4% mixed in the aqueous phase)-loaded dark-blue-colored suppositories were prepared and inserted into the rat vagina with the help of forceps. The animals (n = 3) for 14 consecutive days while the control group (n = 3) was administered with only SVF (20 µL) during these times. The rats were observed for any apparent signs of redness, swelling, vaginal discharge or bleeding every day for 14 days. On the 15th day, animals were euthanized; vaginal tissues were excised and fixed in 10% formalin. Tissues were embedded in paraffin and sections were cut using microtome. The sections were stained with hematoxylin and eosin. Each of the three regions, upper (cervico-vagina), middle, and lower (uro-vagina) vagina, was examined for: (i) epithelial exfoliation, (ii) vascular congestion, (iii) leukocyte infiltration, and (iv) lamina propria thickness/edema. Sections were scored  solution. Next, 1 mL of free probiotic suspension, or that obtained from suppository, (108 cfu mL−1) was mixed with 1 mL of Candida albicans suspension (107 cfu mL−1) on a vortex mixer for at least 10 s and then co-incubated for 24 h at 37 °C and kept under agitation using an incubator shaker. A drop of the suspension was then placed on a glass slide and Gram-stained for visual observation of aggregates. The detailed method is described in the Free Candida albicans were performed by two methods:In vitro inhibition studies of probiotic-loaded vaginal suppositories against Candida albicans from cell suspension was spread on already prepared PNY plates to achieve 105 cfu/mL. Wells of 4 mm were formed, a dispersion of probiotic formulation (200 µL) containing 108 cfu/mL was added into the prepared wells, and plates were incubated at 37 °C for 48 h.The appropriate cell density of 5 cfu/mL. Probiotic dispersion was streaked on both sides around Candida albicans, and plates were incubated at 37 °C for 48 h.The appropriate cell density of Candida albicans cells from cell suspension was streaked at the center of the already prepared PNY plates at a cell density of 10Sprague Dawley female rats (150–200 g) obtained from central animal house, Panjab University, Chandigarh, India were used for the study. The animals had free access to standard rodent food pellets  and water. They were acclimatized to the laboratory conditions before the experiment. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC)  of Panjab University, Chandigarh, India.−1 every day for 6 days. On the sixth day lavage was performed using a plastic pipette using 20 µL of sterile saline. The vaginal lavage was smeared on glass slides and observed using optical microscope for the presence of round nucleated epithelial cells, cornified irregular enucleated cells, and small round leukocytes. The proportions of these cells were used to determine the phase of the estrous cycle, and to establish the onset of the pseudo-estrous phase of the estrous cycle of the rats just prior to the first hormonal administration was also determined, through the same procedure.Animals were subcutaneously injected with hormonal injections (β-Estradiol) at a dose of 25 mg kgC. albicans (ATCC 90028) was procured from the Institute of Microbial Technology (IMTECH), Chandigarh, India, and maintained on yeast extract–peptone–dextrose (YPD) agar slants at 4 °C. A single colony from YPD agar plate was inoculated in 50 mL of YPD broth and incubated at 37 °C for 24 h. Cells from the activated culture were aseptically collected by centrifugation at 2000× g for 5 min, washed thrice and resuspended in PBS (pH 7.4). The stock cell suspension was serially diluted and cell density from a suitable dilution was measured using a hemocytometer. Cell suspension with a suitable cell density (105 cfu/mL) was used for further experimentation.Pure culture of 5 cfu/mL) of the previously standardized yeast suspension was introduced into the vagina 1 week after hormonal induction. The hormone injections were continued throughout the study period, but on alternate days. The same volume of sterile saline was used for the control rats. A vaginal lavage was taken two days after the challenge and inoculated onto YPD plates supplemented with chloramphenicol (50 mg mL−1) to confirm the establishment of infection. The infection was considered positive if at least one yeast colony grew for an individual counting, and a mean count for all the cultures from each rat was ≥10 cfu/mL. The same procedure was followed for the rats from the control group. Thereafter, one suppository was administered intravaginally every day to the treatment groups (® gel) for a period of 7 days and the decline in fungal count was compared using the t-test.First, 20 µL  were plated to confirm (cfu/plate) the translocation of any probiotic to the spleen and liver of the rats. The whole procedure was performed aseptically.At the end of 7-day treatment, blood samples were withdrawn from retro-orbital plexus of these animals and collected into sterile microcentrifuge tubes. The collected blood was spread upon the previously prepared PNY agar medium plates with the help of a spreader. Plates were incubated at 37 °C for 48 h and the colonies thus formed were counted. Similarly, the spleen and liver of these animals were isolated aseptically after sacrifice and the tissue homogenates . The


To assess prenatal diagnosis and pregnancy outcomes in twin pregnancies where one fetus has nuchal translucency (NT) above the 95th percentile.In this retrospective analysis, 130 twin pregnancies (260 fetuses) in which one twin had an NT measurement above the 95th percentile while that of the other twin was normal were analyzed. Prenatal diagnostic results such as G bands, chromosomal microarray analysis, ultrasound findings, and pregnancy outcomes were reviewed.p = 0.033, X2 = 4.571). 2 fetuses in DCT  and 4 fetuses in MCT  (p = 0.187) revealed structural abnormalities among the cases with normal prenatal diagnosis. Fetuses in the DCT group had an overall survival rate of 75.4 percent (95/126), whereas those in the MCT group had a survival rate of 60.4 percent (81/134) . According to the findings of Logistics regression analysis, NT thickening, maternal age and method of conception were all significant risk factors for chromosome abnormalities.Karyotype analysis and CMA results revealed that 15  fetuses exhibited chromosomal abnormalities and that 13 fetuses were Variant of Uncertain Significance. Chromosome abnormalities were detected at a rate of 8.9% (5/56) in the DCT group and 25.0% (10/40) in the MCT group (In twin pregnancies with one fetus with NT above the 95th percentile, the prevalence of fetal structural abnormalities of the MCT group and the DCT group were comparable. Pregnant women’s age and mode of pregnancy are risk factors for chromosomal abnormalities. Pregnant women’s age and mode of pregnancy are risk factors for chromosomal abnormalities. The nuchal translucency (NT), maternal age, mode of pregnancy, and chorionicity are the risk factors for fetal survival –3. NotabThe majority of monocytic studies have concluded that a variety of genetic variables, ranging from mutations in a single nucleotide to genetic issues affecting millions of base pairs and structural defects, are responsible for NT thickness , 7, 8. HOur study aimed to investigate the prenatal diagnosis and pregnancy outcomes of twin pregnancies with a fetal NT measurement value > 95th percentile in dichorionic twins (DCT) and monochorionic twins (MCT).The study was conducted in accordance with the ethical guidelines of the Ethics Committee of the Guangzhou Women and Children's Medical Center in Guangdong province. From September 2017 to March 2022, a total of 996 twin pregnancies  came to our prenatal diagnosis center for prenatal ultrasound scans. NT thickening was identified by prenatal ultrasound in 175 cases . The inclusion criteria for this study were: (I) twin live pregnancies; (II) One fetus with an NT above the 95th percentile (NT range: 3.0–6.0 mm); (III) Able to successfully follow up. A total of 45 cases were excluded from this study as they did not meet the inclusion criteria. The specific reasons for exclusion were as follows: 13 cases of one of the twins arrested developing, 8 cases of fetal cystic hygroma, 5 cases of NT thickening in both fetuses, and 19 cases of lost follow-up.A total of 130 cases (260 fetuses) met the inclusion criteria for this study, including 67 MCTs (134 fetuses) and 63 DCTs (126 fetuses), each of which had one fetus with an NT above the 95th percentile. Among the 67 MCT, 28 cases (56 fetuses) successfully underwent invasive prenatal diagnosis, 37 cases of parents refused prenatal diagnosis, and 2 cases failed the test due to poor DNA quality. Among the 63 DCTs, 20 cases (40 fetuses) successfully underwent invasive prenatal diagnosis, 40 cases of parents refused prenatal diagnosis, and 3 cases failed the test due to insufficient/poor DNA quantity. Overall, invasive prenatal diagnosis was performed in 96 fetuses in 48 cases.The two-dimensional probe frequency ranges from 2.5 to 5.0 MHz and the three-dimensional volume probe frequency ranges from 4.0 to 8.0 MHz on the GE Volusion E10 color Doppler ultrasound diagnostic device. Our fetal anatomy evaluation scan program complies with the 2013 early pregnancy scan recommendations of the International Society of Obstetrics and Gynecology Ultrasound (ISUOG). When ultrasound was used to detect fetal structural abnormalities, we excluded parabiotic twins and one of the twins without a heart. As it is difficult to accurately measure NT and crown-rump length (CRL) when these two complications are combined, they were excluded . We combCMA was performed using the CytoScan 750 K Array , which contains 750,436 25-85mer oligonucleotide probes, including 200,436 single-nucleotide polymorphic (SNP) probes and 550,000 non-polymorphic (NP) probes (0.1 Mb resolution). The copy number variation (CNV) threshold was set to 100 kb, and the number of markers was ≥ 50 kb. According to the ACMG CNV guidelines (specific guidelines), CNVs identified by CMA were divided into pathogenic CNVs, variants with uncertain significance (VOUS), and benign CNVs. CMA was used to evaluate the DNA of both parents to confirm whether the pathogenic CNVs and VOUS were new or inherited. Chromosomal karyotype analysis experiment was performed to select the cells at the vigorous proliferation stage for colchicine treatment so that the cell division was stopped at the middle stage, to obtain enough mitotic cells. After low permeability, fixation, preparation, and staining, the cells were observed under a microscope. Chromosomal banding karyotype analysis is performed on the chromosome specimen obtained by banding technology so that the chromosomes along the longitudinal axis show a certain number of different degrees of coloring, and various widths of light and dark bands. Each chromosome has a unique and constant stripe, depending mainly on the interaction between DNA, proteins, and dyes. Different banding techniques produced different bands on the chromosomes. The G-banding technique involves digestion of chromosome specimens with trypsin and staining with Giemsa.Details concerning the maternal demographics, follow-up prenatal process, and pregnancy outcomes were obtained from the existing medical record system of the medical center. Alternatively, the outcome data of all patients could be routinely obtained from the delivery unit, the patient, or the referral doctor.P < 0.05 (two-tailed test) was considered statistically significant.Statistical software packages SPSS24.0 (IBM) and Minitab19.0 were used for data analysis. All qualitative data were expressed as n (%). All continuous data were retested for normality, and non-normally distributed data were tested using a non-parametric test. The Chi-square test was used to evaluate the qualitative data. We used logistic regression analysis to evaluate the potential independent variables for detecting fetal survival rate and chromosome abnormality rate. This study covered 130 cases of NT thickening (NT range: 3.0–6.0 mm) twin pregnancies (260 fetuses), including 63 cases of DCTs (126 fetuses) and 67 cases of MCTs (134 fetuses) from 1 January 2015 to 1 January 2022 in our center. The demographic characteristics and results of the twin pregnancies are shown in Table 2/46, XY [98] level III, and 1 case mos 46, X, + mar [74]/45, X [26] level III) (see Table P = 0.033). This difference was statistically significant.Table p = 0.698 , with indicated significant differences between the two groups. Detailed information on prenatal outcomes and follow-up is documented in the flowchart in Fig. In this study, there were 31 cases of structural abnormalities, including cardiac malformations, cleft lip, and palate et al. Structural abnormalities were found in 22 of all DCT pregnancies  and 21 of all MCT pregnancies , P < 0.05) (Table Table 5) Table . AdditioP = 0.01, X2 = 6.636). According to the findings of the logistics regression analysis, NT thickness, maternal age, and pregnancy mode were all significant risk factors for chromosomal abnormalities. The NT value, age of the pregnant women, zygosity, and mode of pregnancy were risk factors for unfavorable pregnancy outcomes. NT is a risk factor for fetal structural abnormalities. Overall, these results could provide support and insight to aid in genetic counseling and prognosis in prenatal twins where one fetus has thickened NT.Unlike most previous studies that focused on the association between discordance of increased NT and TTTs in MCT, this study focused on prenatal diagnosis and perinatal outcomes in twin pregnancies with one twin at the NT > 95th percentile in both MCT and DCT. In this study, twin pregnancies where one fetal NT thickening was observed were included. The results of chromosome karyotype analysis and CMA examination showed that the risk of chromosomal abnormalities in the twins with one fetal NT thickening was as high as 15.63% (15/96). Fetuses in the DCT group had an overall survival rate of 75.4% (95/126), whereas those in the MCT group had a survival rate of 60.4% (81/134) . Discordance in NT thickness is commonly related to twin pregnancies. This would be different with increased NT indication in singleton. The level of NT thickness would be related to a different risk of aneuploidy. Many studies have confirmed that NT thickening is associated with fetal aneuploidy chromosome karyotype abnormalities in singleton, with trisomy 21 being the most common. Trisomy 21 was found in both the DCT and MCT groups in our study, which is inconsistent with the results of previous studies [NT thickening is associated with an increased risk of chromosomal abnormalities. Our data showed a higher incidence of chromosomal abnormalities in pregnancies with NT thickening in one of the twins , particularly in the MCT group . With an increase in the NT value, the incidence of fetal chromosomal abnormalities also increased. Logistic regression analysis showed that the risk of chromosomal abnormalities increased 1.891 times for each 1 mm increase in NT value in the twin NT thickening, consistent with the reports in the literature at home and abroad. It is worth noting that because Karyotype analysis and CMA results were not available in every case, the incidence of abnormal karyotype analysis and CMA results could be severely influenced by selection bias, we cannot suppose those cases without karyotype analysis and CMA results were normal.In our study, early pregnancy NT is one of the most important predictors of adverse fetal outcomes, and NT values correlate with maternal age at term and increase with increasing fetal CRL. It has been suggested that pregnancies in which one of the twins has NT thickening are closely associated with fetal chromosomal abnormalities. The results of this study are consistent with those of previous studies , 8, 9. TAdditionally, logistic regression analysis revealed that the risk of fetal structural abnormalities increased with fetal NT thickening in twin pregnancies with NT thickening in one fetus. Ultrasound screening in early pregnancy has a positive effect on reducing birth defects as it can detect certain serious structural abnormalities of the fetus, such as total forebrain, umbilical bulge, abdominal cleft, single ventricle, and severe left heart developmental abnormalities. Special attention should be given to the fact that cardiac anomalies are the most common structural anomalies in twins with one fetal NT thickening. Some studies have shown that the incidence of cardiac structural anomalies is positively correlated with NT thickness. Heart f. This differs from previous studies [CNVs of unclear significance are challenging for clinicians to manage, so physicians must properly consult with the patient before and after prenatal diagnosis. Compared with karyotype analysis, CMA can detect additional microdeletions or microduplications of chromosome structures and genes involved in the mutation section, location, and fragment size. Our results showed that the overall survival rate of pregnant women with DCT was higher than that of pregnant women with MCT. This differs from the results of previous studies . This is2, 13. ThIn addition, due to the hemodynamic imbalance caused by the vascular anastomoses between the two sides of the placenta, MCTs are more likely to experience perinatal death and morbidity than DCTs . MoreoveP = 0.000). Based on these results, the awareness of the increased obstetric risks of IVF may mean that these pregnancies should be managed as "high risk" [Logistic regression analysis yielded a lower survival rate in the in vitro fertilized (IVF) group than in the naturally conceived group (gh risk" , 9. Datagh risk" .In the DCT group, NT thickening may be due to a potential mechanism such as monocytosis. Indeed, around 10 to 13 + 6 weeks into a typical embryo's development, the cervical lymphatic veins and jugular sinus were joined. A small amount of lymphoid tissue was collected from the neck before the cervical lymphatic and jugular sinuses were joined. A transient reflux condition was observed, resulting in brief thickening of the transparent layer of the neck. After 14 weeks, a typical fetus is no longer viable , 13 If tUnlike most previous studies , 7, 15 tSome limitations of this study must be examined. First, this was a retrospective single-center cohort, with some inherent risk of bias and a small sample size. Second, in the case of early fetal loss, no pathological examination of the fetus was performed, so the cause of fetal death or whether the fetus was abnormal was unknown. Furthermore, monogenic genetic disorders including Noonan syndrome have been previously reported to be associated with fetal NT thickening in early pregnancies, but this study did not use a WES line to further validate fetuses with normal karyotype and CMA results. Therefore, data related to monogenic NT thickening disorders in one of the associated twin fetuses are lacking, and some monogenic genetic disorders may have been missed. Finally, it is a retrospective study- that may have recall bias, and it is difficult to confirm IVF contributed to nearly 50% of DCDA twins or PGT-A contributed to less commonly aneuploidy or genetic defects. Additional multicenter prospective studies are necessary to assess the prenatal and perinatal outcomes of twin pregnancies with abnormal NT.In summary, we have demonstrated in twin pregnancies that when one twin's NT is above the 95th percentile, the incidence of chromosomal abnormalities is lower in the DCT group than in the MCT group. Furthermore, the overall survival rate of the DCT group was higher than that of the MCT group, while the risks of fetal structural malformations were comparable. NT thickening and zygosity are high-risk factors for chromosomal abnormalities and adverse pregnancy outcomes. The low survival rate of fetuses with NT thickening in the MCT group was mainly due to the high incidence of chromosomal abnormalities, fetal structural abnormalities, and unique complications such as sIUGR.


Secretion analysis is a useful tool in forensic genetics, since it establishes the (cellular) origin of the DNA prior in addition to the identification of the DNA donor. This information can be crucial for the construction of the crime sequence or verification of statements of people involved in the crime. For some secretions, rapid/pretests already exist  or can be determined via published methylation analyses or expression analyses . To discriminate nasal secretion/blood from other secretions , assays based on specific methylation patterns at several CpGs were set up in this study. Out of an initial 54 different CpG markers tested, two markers showed a specific methylation value for nasal samples: N21 and N27 with a methylation mean value of 64.4% ± 17.6% and 33.2% ± 8.7%, respectively. Although identification or discrimination was not possible for all nasal samples , 63% and 26% of the nasal samples could be unambiguously identified and distinguished from the other secretions using the CpG marker N21 and N27, respectively. In combination with a blood pretest/rapid test, a third marker (N10) was able to detect nasal cells in 53% of samples. Moreover, the employment of this pretest increases the proportion of identifiable or discriminable nasal secretion samples using marker N27 to 68%. In summary, our CpG assays proved to be promising tools in forensic analysis for the detection of nasal cells in samples from a crime scene. Short tandem repeats (STRs) are commonly used in forensic casework for the identification of victims or perpetrators as well as the analysis of family relationships . In the So far, enzymatic or immunological methods, which are based on the presence of proteins, as well as microscopic detection methods have been routinely used for trace characterization, especially regarding blood and semen samples . HoweverIn addition to proteins, RNA can be used to identify body fluids , 6. HoweSpecific CpGs in the context of body fluid analysis have already been described in the literature for the body secretions saliva, blood, semen, menstrual blood, and vaginal secretion –19. The But to the best of our knowledge, nasal discharge has never been investigated in this context. The proof of presence or absence of nose secretion or nose blood in a forensic trace can serve to confirm or refute a described crime scene scenario thus being of great help in reconstructing a crime scene scenario.This study aims to set up methylation assays for the identification of nasal secretions based on specific CpGs to distinguish not only nasal secretion but also blood derived from nose bleeding from other fluids including venous, arterial, and menstrual blood.The study included 182 samples of 67 adult individuals (age range 18–94 years) comprising 35 nasal mucus samples, 39 oral mucosa/saliva samples, 35 blood samples, 29 vaginal fluid samples, 21 menstruation blood samples, and 23 semen samples. No information was available about diseases or operations like vasectomy or hysterectomy. Samples were collected between 2021 and 2022 in the Institute of Legal Medicine, University Hospital Essen, Germany.All samples were obtained after informed consent and with approval of the Medical Ethics Committee at the University of Duisburg-Essen in accordance with the Declaration of Helsinki and national laws (ethic vote number: 21–9843-BO).For a discrimination of nasal mucus, 27 CpGs associated to 19 different genes were chosen , resulting in an extraction volume of 50 μl. DNA concentration of samples was established by real-time PCR using the PowerQuant™ System (Promega) according to the manufacturer’s instructions providing a reproducible and reliable detection threshold at least down to 25 pg DNA . Using 2Sequence analysis was established in a PyroMark® Q48 Autoprep instrument using the PyroMark® Q48 Advanced CpG Reagent Kit according to the manufacturer’s instructions (Qiagen) . In addix and air pollution exposure. In all industrialized countries all over the world, people’s nasal mucosa is more or less constantly exposed to exhaust gases [In order to find nasal mucus markers, specifically regulated CpGs had to be found. Since no CpGs were mentioned in the literature in the context of body fluid identification and nasal mucus, it was decided to investigate CpGs in which methylation pattern changes have been described after NOst gases . Therefost gases  so that Due to the demand of downstream methods, especially bisulfite conversion, all samples included in this study had a DNA concentration between 2.5 ng/μl and 50 ng/μl. For all markers, identically prepared samples with regard to extraction method or bisulfite treatment were used so that an impact of incomplete bisulfite conversion problems can be excluded.Amplification and pyrosequencing could be successfully demonstrated for every locus included in this study. Duplicate analysis of samples showed a maximum deviation in methylation rate of 5%.A DNA methylation marker that allows traces to be assigned to specific cell or tissue types should ideally show hypermethylation (> 90%) in the target and hypomethylation (< 10%) in the nontarget or vice versa . To deteIn the four remaining markers N2 (cg23602092), N10 (cg09080874), N21 (cg16518142), and N27 (cg20864568), DNA methylation percentage in nasal secretion varied between 11 and 26% (N2), 61% and 88% (N10), 38% and 99% (N21), and 18% and 52% (N27), respectively  . Overlaps to saliva, blood, vaginal secretion, and menstrual blood methylation values could be seen  which is highly specific and sensitive  is very A negative result of the blood test excludes the presence of nasal blood, blood, and menstrual blood. Then, our CpG assays N10, N21, and N27 could be able to identify nasal secretion if present. Here, in N21, the amount of identifiable samples does not change regardless of the pretest result. Regarding CpG marker N10, all blood negative samples with < 75% methylation include nasal cells. In this study, this allowed identification of 53% of all analyzed nasal samples (Fig. Moreover, the presence of seminal fluid could be determined directly, because its methylation values did not overlap with any other fluid in CpG marker N27. If nasal secretion and seminal fluid were excluded, a saliva pretest as well as further methylation analysis to identify vaginal secretion should be done.In summary, our workflow allows several outcomes in that an unknown sample may be identified directly Fig. , e.g., aAn even greater problem is the identification of mixture samples of several body fluids , 34. SinIn this study, it was possible to identify nasal mucosa-specific CpG markers and to set up methylation assays for the identification or discrimination of nasal samples. Even if an unambiguous determination of nasal secretion is not possible in 100% of samples, the results obtained so far are applicable to legally relevant questions in many cases. By optimizing or extending our workflow with additional CpG markers specific for other secretions, the unambiguously determinable proportion of unknown secretion samples can be increased.


Weaning is a stressful period for pigs that causes gastrointestinal disruption and low growth rates. For a long time, zinc oxide at pharmacological doses, along with different antibiotics, has been used prophylactically during this phase to reduce the incidence of these gastrointestinal problems. Nowadays, the increasing concern about the environment, along with the global and constant growth of bacterial resistance to antibiotics, has led to the prohibition of the prophylactic use of zinc oxide in diets for piglets in the EU, along with the implementation of new regulations on the use of antibiotics. Consequently, the pig sector faces the important challenge that supposes developing alternatives to the classical system based on the use of these antimicrobial compounds. This study is the first step to achieving this goal by minimizing the use of various antibiotics and zinc oxide in weanling pigs by supplementing citrus flavonoids and only one antibiotic (amoxicillin). Accordingly, the influence of zinc oxide plus antibiotics and citrus flavonoids plus amoxicillin in weaned pigs has been investigated and its impact on growth performance, gut microbiology profile, gut signaling, intestinal architecture, and serum biomarkers indicative of stress and inflammatory responses have been evaluated. Citrus flavonoids plus amoxicillin improved growth performance and gut health, evidencing a positive microbial modulation, stress status reduction, and a positive effect on the gastrointestinal barrier, and other digestive functions. Additionally, the expression of some bitter taste receptors in the intestine has been increased when supplementing both dietary strategies, the one based on zinc oxide or the one based on citrus flavonoids supplementation. Consequently, the present study shows that in weanling piglets, the supplement of citrus flavonoids with amoxicillin might be a promising alternative to the dietary use of pharmacological doses of zinc oxide with more than two antibiotics, therefore minimizing the use of antimicrobial compounds without detrimental effects on performance.p < 0.05), whereas ZnO pigs evidenced intermediate results. Jejunum tissue analysis showed that pigs fed the FLAV diet overexpressed genes related to barrier function, digestive enzymes, and nutrient transport compared to those pigs fed the CON diet (p < 0.05). An increase in the abundance of bacterial genera such as Succinivibrio, Turicibacter, and Mitsuokella (p < 0.05) was observed in the FLAV compared with the CON and ZnO piglets. ZnO and FLAV increased the expression of TAS2R39, while ZnO pigs also expressed greater TAS2R16 than CON (p < 0.05) in the intestine. FLAV treatment improved the gut function, possibly explaining a higher performance at the end of the nursery period. Consequently, citrus flavonoids supplementation, together with amoxicillin, is a promising alternative to the use of zinc oxide plus amoxicillin and apramycin in weanling pigs, minimizing the use of antibiotics.Since citrus flavonoids have antioxidant and anti-inflammatory properties, it was hypothesized that these compounds would become a suitable alternative to the use of therapeutic doses of zinc oxide at weaning. A total of 252 weaned pigs ([LargeWhite × Landrace] × Pietrain) were distributed according to BW (5.7 kg ± 0.76) into 18 pens . Three experimental diets for the prestarter (0–14 d postweaning) and starter (15–35 d postweaning) period were prepared: (i) a nonmedicated (CON) diet, (ii) a CON diet supplemented with zinc oxide at 2500 mg/kg, amoxicillin at 0.3 mg/kg and apramycin at 0.1 mg/kg (ZnO), and (iii) CON diet with the addition of a commercial citrus flavonoid extract at 0.3 mg/kg and amoxicillin at 0.3 mg/kg (FLAV). Pig BW, ADG, ADFI, and FCR were assessed on d7, d14, and d35. Samples of intestinal tissue, cecal content, and serum were collected on day seven (18 piglets). FLAV treatment achieved greater BW and ADG during the starter and for the entire experimental period compared with the CON diet ( Among livestock production, the swine industry provides one of the most important protein sources for human nutrition . Even thDifferent feed additives have been studied and proposed as an alternative to zinc oxide to improve postweaning pigs’ health and reduce the incidence of diarrhea . Citrus The experiment was planned, designed, and conducted at the end of the year 2018, aiming to deal with the reduction of antimicrobials in swine production (antibiotics and zinc oxide) and facing a new scenario and anticipating the ban on zinc oxide in 2022.Experimental procedures were approved by the Ethics Committee of the Universitat Autònoma de Barcelona  based on the European Union guidelines for the use of animals in research .2) allotted entire males and females. The commercial weaned facility owned by the Universitat Autònoma de Barcelona had a fully slatted floor, unlidded hoppers , and nipple drinkers. Temperature and ventilation rates were monitored using thermostatic heaters and exhaust fans adjusted depending on the age of the pigs (28 to 22 °C).At weaning (21 ± 1.6 d), 252 pigs ((Large White × Landrace) × Pietrain) with an initial average BW of 5.7 ± 0.76 kg were used in a 35-d study. Animals were identified through an ear tag, individually weighted, blocked according to the initial BW, and distributed into three experimental diets in 18 pens  within the same weanling room. Each pen  at 0.3 mg/kg and amoxicillin at 0.3 mg/kg (FLAV). During the ST phase, diets remained the same except for the ZnO diet. It consisted of the addition of zinc oxide at 1500 mg/kg plus the addition of amoxicillin at 0.3 mg/kg, neomycin at 0.19 mg/kg, and tiamulin at 0.10 mg/kg. The antimicrobial plan used in the positive control was applied based on the in-farm therapeutic strategy already conducted in the commercial conditions according to the health status and inherent challenges . In the FLAV treatment, amoxicillin was used throughout the study due to the broad spectrum of this antibiotic and the high prevalence of Streptococcal meningitis on the farm. Two-phase diets  were forFor each dietary treatment, the feed was offered in mash and pellet form during the PS and ST phases, respectively. Both feed and water were provided ad libitum.In order to calculate the average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR), the individual BW of the pigs, as well as feed disappearance from each feeder, was recorded on days 7, 14, and 35. Pigs were daily monitored for mortality and illness incidents.Proximate analytical determination of diets was performed following the Association of Official Agricultural Chemists International (2005) methods: dry matter (AOAC 934.01), crude protein (AOAC 968.06), ether extract (AOAC 920.39), and ash (AOAC 942.05). Neutral detergent fiber content was determined using the Ankom nylon bag technique .g for 10 min to obtain serum. Aliquots of serum were stored at −80 °C until chemical analyses. A PigMap analysis was performed on the Olympus AU 400 analyzer [From each pen, three pigs (average BW) were selected to take blood samples on days 0, 7, and 14 of the trial. The same animal was sampled during the experimental period. Samples were taken by jugular puncture and collected in an individual vacutainer tube without anticoagulant (10 mL). Tubes were subsequently centrifuged at 1500× analyzer . Two comn = 6). Selected animals exemplified the average pig BW within the pen. Pigs were sedated using zolazepam and xylazine and subsequently euthanized by an overdose of pentobarbital. Samples were fixed in 4% paraformaldehyde and then embedded in paraffin. Then, sections were stained with a hematoxylin-eosin solution. Villus height, crypt depth, villus height to relative crypt depth ratio (V:C), and the number of goblet cells and lymphocytes were measured by using a light microscope. Only full and vertical-oriented villi were considered within the analysis.On day 7, samples of the jejunum and ileum tissue (about 5 cm) were collected from one pig per pen . Samples were stored at −80 °C until analysis. An Open Array Real-Time PRC Platform  was self-designed as described by González-Solé . BrieflyIn addition, four genes related to bitter taste receptors were individually analyzed  from jejunum and ileum samples. RNA was extracted by homogenizing tissues in TRIzol  using a Polytron Instrument . PrimeScript RT Reagent Kit  was used to transcribe RNA to cDNA, following the manufacturer’s instructions. The quality of RNA was assessed by a NanoDrop instrument  at 260, 280, and 230 nm. Quantification of gene expression was performed as described in Paniagua et al. [® Reagent Kit v2  for the V3–V4 regions of the bacteria. Sequence reads were processed on the QIIME v.1.9.1 pipeline [On day 7, from the same selected animals, cecal content (500 mg) was collected for 16-S microbial sequencing analysis. Samples were immediately frozen and stored at −80 °C until analysis. Bacterial DNA was obtained by following the manufacturer’s instructions for the commercial QIAamp DNA Stool Min Kit . After the proper verification of DNA concentration and purity  it was eluted in 200 μL of Qiagen buffer AE ad stored at −80 °C until analysis. Samples were amplified (500 cycles) using the MiSeqpipeline  and assipipeline  at a 90%pipeline .Prior to ANOVA analysis, the normality and homogeneity of the data were assessed using the Shapiro–Wilk test. Data were analyzed through the MIXED procedure of SAS  considering a randomized complete block design. The effect of the experimental diet was considered a fixed effect, whereas the BW block counted as a random effect. The pen was the experimental unit. In the case of acute phase proteins, histomorphological measurements, gene expression, and microbiota community data corresponded to the individual pig selected that represented the average BW in each pen, therefore the animal represented the pen . For acute phase proteins and stress markers, the initial measurements (d 0) were used as covariables. Digesta bacteria diversity was analyzed at the OTU level using a vegan package . Shannonp-values to those obtained by an ANOVA of transformed data. They were analyzed using a mixed-effects model , where the model included treatment, tissue, and the interaction between treatment and tissue as fixed effects. For both, gene expression and microbiota analysis, the Benjamini and Hochberg false discovery rate (FDR) multiple testing correction was performed [p ≤ 0.05 and tendencies were considered when the p-value was between >0.05 and <0.10.Biostatistical analysis of gene expression and microbiota community data was performed in R Studio v.3.5.1 software. The means and SEM values for bitter taste gene expression data presented in the figures correspond to nontransformed data and erformed . Signifip < 0.10). Interestingly, pigs from the FLAV treatment achieved greater BW and ADG during the ST and for the entire experimental period compared to the CON diet (p < 0.05). However, BW and ADG from the ZnO animals showed no differences when compared to CON and FLAV treatments at the end of the study. Although both treatments, FLAV and ZnO, improved ADFI during the ST and for the entire experimental period, no influence of experimental diets was observed on FCR (p > 0.10).Pig performance response for the experimental diets is shown in p = 0.752).Mortality was 1.59% and was not related to any dietary treatment . Ileum analysis showed that pigs fed the CON diet had an upregulation of genes involved in barrier function (MUC2 and TFF3), inflammatory response (IFNGR1 and IL8), and antioxidant enzymes (GPX2 and SOD2m) but a downregulation of the PPARGC1α gene (immune response) compared to the FLAV diet  and villus height to crypt depth V:C ratio (p = 0.059). Pigs fed the ZnO diet tended to have longer ileum villi and a higher V:C ratio than those fed the CON diet.Ileum histomorphometry parameters are shown in p = 0.007). No influence of experimental diets was observed on serum TNF-alpha and PigMap levels (p > 0.10).As shown in The main objective of this study was to explore a commercial approach to minimize the use of multiple antimicrobial substances in weanling diets, such as zinc oxide and antibiotics, through supplementation with citrus flavonoids. As the trial was performed under commercial farm conditions, the FLAV treatment also included amoxicillin in addition to citrus flavonoids due to the historical sanitary status of the farm and ZnO treatment included more antibiotic molecules following their conventional strategy to reduce postweaning health disorders. Although the major function of antibiotics is to act as antimicrobial substances against bacteria modifying the normal microbial community of the intestine, their anti-inflammatory effects have been also described . ConsequWeaning, in addition to activating stress signaling pathways, can also alter the normal expression of intestinal genes in pigs ,24. AfteOne of the hypotheses of this work was that citrus flavonoids may be a suitable alternative to pharmacological doses of antibiotics and zinc oxide probably led by the effect of bitter compounds on bitter taste receptors (TAS2R). These taste receptors have been described along the gastrointestinal tract constituting the chemosensory system, modulating the proper digestive response , food intake, and behavior ,31,32,33No statistical interaction was found for treatment and tissue (jejunum and ileum) when TAS2Rs’ gene expression was analyzed, however, the gene expressions of TAS2R16 and TAS2R39 also showed different expressions depending on the tissue, jejunum, or ileum, whereas TAS2R7 and TAS2R38 were not affected. Colombo et al. ,34,36. ILactobacillus and Lachnospira compared to the CON diet. Interestingly, FLAV diets increased the abundance of these same bacteria genera together with Roseburia compared to the control diet. The importance of the intestinal microbiota for gastrointestinal function and their association with animal health and growth has been previously shown [Lactobacillus, Bifidobacterium, Lachnospira, or Roseburia genera are known as beneficial functional microbes which have great potential to contribute to the reduction of the growth of pathogenic bacteria, stimulation of the immune system, feed intake, and feed efficiency [Among all parameters that can influence animal growth performance, intestinal integrity, along with a stable microbiota, is particularly important to enhance feed efficiency and maintain overall health. Curiously, when intestinal morphology was evaluated, no differences were observed between experimental diets. However, taking a deep look into gut health parameters, diets offered to pigs modulate the microbiome community composition. Although alpha diversity indices showed a similar richness within experimental diets, the beta diversity analysis showed dissimilarities among those microbial communities. This finding suggests that the difference is not due to the presence of specific bacteria but to the larger abundance of certain bacteria . Indeed,ly shown ,40,41,42ficiency . Furtherficiency . It is wficiency , as wellficiency . TherefoMoreover, it is worth noting the lower plasma concentration of cortisol found at day seven postweaning in pigs fed the FLAV diet. Cortisol, in addition to being one of the widest biomarkers of stress in pigs, is considered a powerful suppressor of the immune response (production of cytokines and immunoglobulins) and whose secretion predominates under the prolonged exposition to a stressful stimulus . As mentLactobacillus and Lachnospira, whilst only a few nutrient transporter genes were better expressed in the ilium of these animals compared with the CON piglets. On the contrary, as previously discussed, the FLAV piglets evinced better intestinal maturity together with a greater microbiome gut community, increasing the relative abundance of some genera known as beneficial functional bacteria (Lactobacillus or Roseburia), which might explain the greater performance observed in the FLAV animals at the end of the study. Consequently, although the ZnO group evinced greater ADFI, only numerical improvement was observed for BW and ADG at 35 days postweaning compared with the CON animals, whereas the FLAV piglets achieved greater BW, ADFI, and ADG.In the present study, pigs fed the FLAV diet showed improved performance during the first 35 days postweaning compared to the CON diet, whereas the ZnO pigs were intermediate. Interestingly, ZnO negatively affected the abundance of some beneficial genera, such as Taking these results together, it must be stressed that feed and its associated nutritional components influence directly the major components of gut health such as gene expression, microbiota environment, and intestinal integrity. Consequently, supplementing feed with functional additives such as citrus flavonoids together with antibiotics like amoxicillin can positively impact intestine digestive and immune function and, thereby, on pig growth along with lesser inflammatory and pro-oxidative status, representing thus a first step to minimize the use of antibiotics and avoiding pharmacological doses of zinc oxide. Additionally, TAS2R seems to be involved in the multifactorial mechanism of action of these substances (zinc oxide and citrus flavonoids) supplemented with antibiotics. The next steps should be to evaluate the effect of the supplementation of these flavonoids without any dietary antibiotics added to confirm them as a promising alternative to dietary antibiotic use as well, improving health and performance in weanling pigs.