Ardeotis arabs) populations are declining and are already extinct in some regions. The International Fund for Houbara Conservation (IFHC) has started a conservation breeding program for the Arabian bustard in the United Arab Emirates (UAE). Birds were housed by pairs to allow natural reproduction. Out of 1253 eggs laid, 1090 were incubated, of which 379 were fertile . In total, this led to the production of 251 chicks. Due to lower than desired fertility, we introduced assisted reproduction techniques to increase fertility and develop zootechnical knowledge for the species. This paper presents the results of semen collection, artificial insemination, and semen cryobanking in Arabian bustards. Inseminations with both fresh and preserved semen led to a significant increase in fertility to 84.3% of incubated eggs . Furthermore, we confirmed the viability of cryopreserved semen and its fertilizing capacity. Our results demonstrate the usefulness of artificial reproduction techniques for the conservation of Arabian bustards and suggest that these techniques can be applied to closely related critically endangered species with a minimum of adaptation.Artificial reproduction technologies such as artificial insemination and semen cryopreservation are important tools for species conservation and long-term genetic management. As with many bird species, Arabian bustard , leading to the production of 251 chicks. To improve fertility and acquire crucial knowledge for other endangered large birds, artificial reproduction was implemented in 2018 using fresh, refrigerated, and frozen sperm. A total of 720 ejaculates were collected from 12 birds. We analysed these samples for concentration, volume, motility score (0 to 5), viability (eosin/nigrosine), length, and morphology. The first age at collection was 35.7 ± 18.8 months, mean volume was 89.2 ± 65.3 µL, mean concentration was 928 ± 731 sptz/mL and mean motility score was 2.61 ± 0.95. Morphology analyses revealed a bimodal distribution of sperm length. Five hundred and thirty-five ejaculates were cryopreserved and the initial motility score was 3.4 ± 0.7 and 2.0 ± 0.6 after thawing, while the percentage of normal and intact membrane sperm cells decreased from 88.8 ± 7.5% to 52.9 ± 1%. Sixty-five artificial inseminations were performed, leading to a global fertility rate of 84.3%—more precisely, 85.2% and 83.3%, respectively, for fresh and cryopreserved semen. All methods successfully produced fertile eggs, indicating that artificial insemination is an efficient tool for the conservation and genetic management of the species. Artificial reproductive technologies provide important opportunities for assisting conservation efforts to counter the current biodiversity crisis ,2. ArtifArdeotis is composed of four species: the Australian bustard ; the Kori bustard ; the great Indian bustard . This final species is listed as Near Threatened by IUCN with a global trend of decreasing population owing primarily to hunting pressure and habitat degradation [A. a. lynesi , which is probably extinct, A. a. stieberi , A. a. butleri  and A. a. arabs . The latter is considered extinct in Saudi Arabia, where it was included in a high-priority conservation list, ranking ninth among 102 species [The bustard family (Otididae) is composed of 26 species, eight of which are classified as Threatened within the IUNC Red List of Threatened Species . Within dangered ,14), andradation . Four suChlamydotis spp.) conservation breeding programs [The Arabian bustard Conservation Breeding Program implemented by the International Fund for Houbara Conservation (IFHC) at the National Avian Research Center  was initiated in July 2007 with the arrival of three confiscated birds of Yemeni origin. In 2009–2010, egg collections were conducted in the Tihama region of Yemen, resulting in 11 chicks from nine harvested nests. The founding flock was thus composed of nine males and five females that were housed in groups. The first egg was laid in captivity in 2012 and the first chick produced through natural reproduction hatched in 2013 . Betweenprograms ,20,21,22These techniques are efficient for analyzing and sorting many semen samples and adapted to situations where both cost-efficiency and skill transfer and capacity building are a concern. The objectives of the present work on the Arabian bustard were multiple, with the first to increase the proportion of fertile eggs within the conservation breeding program. Indeed, natural reproduction would not be sufficient to ensure both reproduction in captivity and an efficient long-term genetic management of the species in captivity through pair selection. In addition, this non-model species can be a surrogate to develop, test, and provide techniques that can be applied to other large, endangered birds such as the Great Indian bustard, therefore efficiently contributing to their conservation.Ardeotis genus as well as fertility outcomes resulting from the use of assisted reproduction techniques, including a cryobank of cryopreserved semen. More specifically, we provide information on female laying patterns, semen collection, qualitative and quantitative evaluation of semen, sperm morphology, artificial insemination and subsequent fertilization success and hatching rates.Thus, the present paper aims to present the first data on routine analyses of semen characteristics within the Adult housing facilities consisted of elliptical tunnels measuring 9 × 15 m  where foUpon transfer to incubation, all eggs were disinfected and controlled for defects before being placed in incubators. Water loss was calculated by measuring the change in egg weight throughout incubation. Fertility and viability were measured via egg candling on days 3, 9, 12 and 20 of incubation. Systematic necropsy of non-hatched eggs allowed the team to analyze if the eggs were fertilized or not. Only incubated eggs were considered when evaluating egg fertility results. The hatching rate was evaluated as the proportion of viable live chicks hatched over the total number of incubated eggs. The average incubation duration was 21.8 ± 0.7 days. The rearing of future breeders was developed to minimize stress and promote familiarity with humans; this ensures a limited amount of stress during captures such as artificial insemination or vaccinations. Upon hatching, chicks were transferred to rearing facilities where they were imprinted before being transferred to adult enclosures at about one year old. Imprinting consisted of frequent interactions between chicks and keepers through visual or acoustic contact, hand-feeding, or physical interactions, such as being held. Imprinting reinforcement occurs throughout an individual’s life to reduce stress and allow the implementation Of artificial reproduction techniques.The Abu Dhabi authorities approved the breeding program and captivity conditions under reference 116620140. Founders were transferred to the facilities from Yemen under CITES certificate number 10MEW3077. Operations are conducted in a humane manner, and each breeding protocol is developed while ensuring the appropriate and ethical treatment of birds. To minimize stress during capture, birds are fitted with hoods during manipulation. Furthermore, all operations requiring handling of the birds were performed only by the most experienced keepers under the supervision of veterinarians. The facilities are designed to ensure the well-being of the birds and prevent injuries. On-site veterinary facilities provide the best possible care for sick or injured birds by a team of expert veterinarians, and the standards from sanitary authorities are regularly controlled.Semen collections were performed yearly from 2018 to 2021, between January to September . Semen wSemen analyses methods were based on routine procedures developed for houbara bustard conservation programs where the average number of ejaculates analyzed per day reached 593 with a maximum of 1303 samples in 2021. In such cases, simple but reliable and robust methods were implemented to ensure all ejaculates were analyzed but also to permit an easy transfer of skills and allow for capacity-building. Arabian bustard samples were processed in this same laboratory by experienced technicians. Upon arrival, fresh ejaculates were immediately analyzed, all procedures were done at room temperature, and all used materials were kept in the same conditions RT~24 °C). The mass motility index was assessed by placing a 5 µL droplet on a glass slide (10× phase contrast objective—Nikon E200 phase-contrast equipped microscope) and evaluated using a mass motility index  °C. The ,28,29. S6/mL) [The same slide preparation was used to evaluate the occurrence of contamination within ejaculates ; contaminated ejaculates were discarded and were neither analyzed nor considered to be used for artificial inseminations. Among the remaining non-contaminated samples, the volume of undiluted ejaculates was measured (±1 μL) using an electronic pipette. After dilution with Lake 7.1 diluent (dilution 1:1) , the con6/mL) . The numThe proportion of morphologically normal sperm  and intact membranes was simultaneously accessed by the eosin-nigrosin (EN) method where intact cells appear white while membrane-damaged cells will incorporate the eosin and show pink under staining .Sperm length was accessed placing a 5 µL droplet of diluted and fixed semen sample (Hancock solution ) betweenn = 6). After qualitative and quantitative analyses, including dilution with Lake 7.1 diluent (dilution 1:1), these ejaculates were preserved 24 h at 4 °C in 2 mL Eppendorf Safe-Lock Tubes when the preserved volume was below 250 µL, while 6 mL Sterlin scintillation vials were used otherwise. Motility scores were recorded before and after refrigeration and ejaculates were placed on an orbital shaker plate with gentle shaking to allow the ejaculate to reach room temperature prior to analysis or artificial insemination [p ≤ 0.05).While most unused ejaculates were cryopreserved (cf. below), a few ejaculates were refrigerated for 24 h to be used for artificial insemination the next day to follow the female laying pattern closely and ensure optimal fertility potential (mination . MotilitMost of the collected and non-contaminated ejaculates were submitted to the freezing protocol. Ejaculates were frozen based on Tselutin’s procedure . This prt-test (p ≤ 0.05).During the thawing procedure, pellets were quickly placed on a thermo-regulated stainless conical hotplate  set at 6Based on the experience acquired with houbara bustards, female laying history was taken into consideration to decide the most appropriate time to catch and inseminate the females to optimize fertility while minimizing the number of artificial inseminations performed to reduce stress. Laying histories were carefully recorded, so the artificial inseminations were generally performed within 3 to 6 days before the next clutch . SpecifiBetween 2019 and 2021, semen collection was attempted for 34 males, of which 13 were collected for a total of 720 ejaculates. All collected males were captive-bred individuals that were imprinted. The number of ejaculates collected per bird, age in months at first collection, the ejaculate volume, concentration, sperm number and motility score are summarized in The mean of the total sperm length was 59.74 ± 3.02 µm (52.61 to 68.38 µm ). Each sp ≤ 0.05). No morphology records were taken after refrigeration.Six ejaculates were refrigerated for 24 h for further use for artificial insemination. The mean preserved volume was 231.3 µL ± 75.5 and the mean motility significantly decreased from 4.08 ± 0.49 to 3.33 ± 0.52 after refrigeration (p ≤ 0.001) after cryopreservation, and the proportion of live/normal sperm decreased significantly from 90.3 to 52.9% . Forty-one inseminations were performed with fresh semen, five with semen preserved for 24 h and 19 with frozen/thawed semen. These artificial inseminations produced 33 eggs; six were preceded by artificial insemination with cryopreserved semen and only five were deemed to be fertile . Twenty-seven eggs were preceded by artificial inseminations with fresh semen, only of which 23 were fertile  .The remaining eighteen eggs were preceded by a combination of artificial inseminations with a different type of semen; fifteen of them were fertile . Thus, 51 incubated eggs were preceded with different semen-type artificial inseminations of which 43 were fertile  at the ninth day of incubation.Ardeotis, a genus where each species in the genus has been the subject of a conservation breeding program [This study is the first to present data on the semen characteristics of the Arabian bustard and for the genus  program ,36,37. HOur results demonstrate the potential for artificial reproduction techniques to conserve Arabian bustards and suggest these can be applied, with a minimum of adaptation, for closely related endangered species, such as the Great Indian Bustard. Indeed, the use of artificial insemination led to a significant increase in fertility compared to natural breeding (an increase from 34.8% to 84.3%). Furthermore, implementing an affordable, easy to execute, and robust pellet cryopreservation protocol allowed the development of a cryobank where all donors from the captive population are represented, acting as a substantial tool for the long-term genetic management of the species.C. undulata) and Asian (C. macqueenii) houbara within IFHC conservation breeding programs, we then decided to proceed with semen collection using a dummy female . Still, the extent of the breeding season associated with small variations in semen characteristics suggests that the species is highly opportunistic and able to take advantage of favorable conditions. A similar pattern was reported for the Great Indian bustard, with breeding recorded in all months of the year (highest between March and September), with rains apparently triggering reproduction events [Little is known about the full extent of the breeding season of the species in the wild. Reproduction has been recorded from April to June in the most Western part of the range, extending possibly to October or November in Yemen , and betn events .Chlamydotis macqueenii; mean length = 47.92 ± 4.69 µm, (unpublished data). It has been hypothesized that inter-species differences in avian sperm length are the result of differences in structural variations related to midpiece mitochondrial composition or in nuclear organization and chromosome elongation [Our results indicate that Arabian bustard sperm is longer than Asian houbara  found that longer sperm were slower, therefore not supporting the sperm morphology–swimming speed hypotheses presented by other groups where longer sperm were faster swimmers [In addition, we observed a bimodal distribution of sperm size in Arabian bustards’ ejaculates, with some sperm being twice as long as others; this difference is primarily due to longer sperm heads. Intra-individual variations in sperm size have been presented as a potential consequence of reduced sperm competition, therefore, relaxing selection on optimal sperm length ,50. Althswimmers . Thus, tWhile the presented results are the only semen characteristics data reported for the genus, data are available from two related species, the North African and Asian houbara, both bred as part of conservation breeding programs managed by the IFHC. Mean motility, volume, and proportion of normal sperm were similar, but both concentration and number of sperm are considerably higher in Arabian bustards . In bustA viable and competent sperm population is important for successful species reproduction, especially considering the necessity of physiological polyspermy for successful avian embryo development ,55. HencWhile using accessible sperm analyses methods, our results demonstrate the efficiency of assisted reproduction techniques in improving reproduction in captive Arabian bustards. Indeed, egg fertility results increased from 34.8% of incubated eggs with natural reproduction to 84.3% with artificial inseminations. These results were comparable to those routinely obtained on a larger scale with houbara bustards where fertility reaches 91.7%. Our results also illustrate cryopreservation’s effectiveness in providing ejaculates with sufficient fertilizing power. Tselutin’s pellet method, using DMA as a cryoprotectant, proved to be a robust cryopreservation method. It has the advantage of being relatively easy and economical to implement, both in terms of freezing and thawing protocols and in terms of required equipment. It then facilitates the implementation of a semen cryobank for the species as for other avian species [Conservation breeding programs play a key role in supporting in-situ conservation, and assisted reproduction techniques are a major enhancing factor in ensuring that demographic and genetic goals can be achieved. Here, we demonstrate the potential of these techniques for the conservation of Arabian bustards and closely related and highly endangered species. Artificial insemination induced a significant increase in the percentage of fertile eggs, including with cryopreserved ejaculates. Further studies will be required to investigate and optimize sperm quality evaluation, eggs fertility, and hatching rate results .On a broader scale, our results also indicate that valuable information for artificial reproduction of endangered bird species can be obtained with a limited laboratory setup. The use of the methods presented here could promote the wider use of assisted reproduction technologies in endangered avian species, from artificial insemination to genome resource banking.


Palmar-plantar erythrodysesthesia (PPE), more commonly known as hand-foot syndrome, is a dermatologic complication following chemotherapy with selective agents. In this article, we present the case of a young lady with stage III breast cancer who developed palmar-plantar erythrodysesthesia following treatment with Herceptin (trastuzumab), an unusual complication of this particular drug. From our review of literature, this is the second known occurrence of PPE secondary to trastuzumab monotherapy. Hand-foot syndrome (HFS) is a common dermatologic complication following selective cytotoxic chemotherapeutic agents. It is characterized by erythema, pain, numbness, and often tingling of the cutaneous layer of the palms and soles . The NatAppropriate protocols were followed, and consent was obtained by all participants in this study.A young, 28-year-old Caucasian female was referred to the oncologist after noticing a lump in her right breast. Mammography revealed two lesions in her right breast; a 1.9 × 2.0 × 1.4 cm solid lesion and a cystic lesion measuring 1.8 × 1.5 × 1.5 cm. Subsequent immunohistochemistry (IHC) diagnosed the patient with an estrogen and progesterone (ER/PR) negative, HER2/neu positive invasive ductal carcinoma. Genetic testing was negative for breast cancer susceptibility gene mutations one and two (BRCA) as well as TP53 gene mutation. A composite risk of mutation probability, using the Hughes model, assigned the patient a 7% risk. Magnetic resonance imaging (MRI) of her breasts revealed a 2.7 cm mass in the upper inner quadrant of her right breast. The American College of Radiology (ACR) Breast Imaging Reporting and Data System (BI-RADS) was six and two of her right and left breasts, respectively.The patient proceeded to bilateral mastectomy and axillary node clearance. She had a grade 3 residual invasive ductal carcinoma of her right breast, with one out of 13 lymph nodes found positive for metastasis.She was started on adjuvant chemotherapy and radiotherapy thereafter. A total of 13 cycles (out of 17) were given, with the initial six cycles comprising of docetaxel, carboplatin, pertuzumab, and trastuzumab (first cycle at 2 mg/kg every week and subsequent cycles at 6 mg/kg every third week); each chemotherapy cycle was followed by subcutaneous Neulasta (6 mg). The latter seven cycles of chemotherapy comprised only of trastuzumab (at 6 mg/kg every three weeks). She appeared to be tolerating this regime well up to her 13th cycle.Trastuzumab administration proceeded uneventfully until a few days after her scheduled 14th cycle of chemotherapy, when she presented with superficial, dry, desquamation on the soles of her feet and palms of her hands with no interference of her daily life activities Figures . She hadShe had not taken any new medication in the past few months aside from her routine trastuzumab. The patient was conservatively managed and counseled to the frequent use of emollients and moisturizers, however, a urea exfoliation product was not used. She was also advised to avoid extreme temperatures, friction, and tight-fitting shoes or gloves. Her chemotherapy was put on hold, and radiotherapy continued as scheduled. Her symptoms began to improve despite continued radiation therapy and resolved with a continued delay in trastuzumab therapy. Trastuzumab was then reintroduced after six weeks without any recurrence of erythrodysesthesia.The National Cancer Institute (NCI) describes hand-foot syndrome (HFS), also called palmar-plantar erythrodysesthesia (PPE), as a condition marked by varying degrees of swelling, pain, tingling, numbness, vesiculation, or erythema predominantly of the hands or feet due to the cutaneous toxicity that can occur from the use of selected chemotherapy agents ,4. PPE iThe most notorious chemotherapy drugs known to cause PPE include: capecitabine-up to 74% of patients, 5-fluorouracil-up to 34% with a continuous infusion and 13% receiving bolus injections, liposomal doxorubicin-up to 48% .The exact mechanism of PPE is unknown, but it is an area of active investigation with several hypotheses put forward. One of these hypotheses postulates that drug accumulation in eccrine sweat glands results in increased concentrations of harmful products damaging the surrounding skin. In severe cases, PPE has been reported to involve the inguinal and axillary regions along with the palms and soles, all areas with a greater presence of eccrine sweat glands. This hypothesis is further supported by reports of an increased concentration of chemotherapeutic agents in sweat, along with separate reports of microscopic damage to eccrine sweat glands in patients with PPE ,10. The The most effective management strategy of PPE is the interruption of treatment by dose reduction or lengthening the interval between drug administrations. This usually leads to a decrease and resolve in symptoms within one to two weeks . SeveralPPE is a common cutaneous toxicity in patients receiving treatment with several chemotherapeutic agents as well as targeted therapies like tyrosine kinase inhibitors (TKIs), though is not typically associated with trastuzumab. The incidence of a severe grade PPE is low; however, it can have a significant influence on a patient's quality of life. Prevention and treatment measures, though effective, still need their efficacy assessed through controlled and randomized studies. The authors urge clinicians to expect and watch out for early signs of dermatologic desquamation in lone trastuzumab therapy.


P = 0.02), a smaller baseline DWI lesion volume , an intermediate collateral grade , a good collateral grade , and successful reperfusion  were independently associated with a favorable functional outcome. There was a linear association between the SWI collateral grades and functional outcome (P = 0.008). Collateral estimation using the prominent vessel sign on SWI is clinically reliable, as it has prognostic value.To determine the value of susceptibility-weighted imaging (SWI) for collateral estimation and for predicting functional outcomes after acute ischemic stroke. To identify independent predictors of favorable functional outcomes, age, sex, risk factors, baseline National Institutes of Health Stroke Scale (NIHSS) score, baseline diffusion-weighted imaging (DWI) lesion volume, site of steno-occlusion, SWI collateral grade, mode of treatment, and successful reperfusion were evaluated by multiple logistic regression analyses. A total of 152 participants were evaluated. A younger age (adjusted odds ratio (aOR), 0.42; 95% confidence interval (CI) 0.34 to 0.77; P < 0.001), a lower baseline NIHSS score (aOR 0.90; 95% CI 0.82 to 0.98;  Intravenous thrombolysis (IVT) with tissue plasminogen activator and intraarterial thrombectomy (IAT) has revolutionized the treatment of acute ischemic stroke and been shown to be effective in improving the functional outcome of patients with acute ischemic stroke within 4.5 h and 6 h after symptom onset, respectively8. Time is an important factor in the effectiveness of IVT and IAT since ischemia evolves over time after acute stroke10. However, the treatment effect was not constant even in the optimal time window. Futile endovascular treatment ranged from 29 to 67% in the recent randomized clinical trials8 and hemorrhagic transformation worsen the functional outcome ranged from 2.4 to 8.8%14. Recent studies have shown that endovascular treatment could be effective in select patients up to 24 h after symptom onset16. These differences in the treatment effect beyond the time window are due to individual differences in the pace of infarct progression by collateral circulation. Collaterals are alternative vascular channels for maintaining blood perfusion to the ischemic brain distal to an arterial occlusion; therefore, infarct progression depends on the collateral status, which varies among patients17. Infarction may occur within less than 1 h or may last for hours or days19. Recanalization and reperfusion treatment can be futile or even dangerous when performed in the optimal time window but could be useful if performed at a later time. Meta-analyses showed that better collateral circulation may lead to favorable functional outcome, better recanalization or reperfusion rates, a smaller infarct core, a lower hemorrhagic transformation rate and a lower risk of mortality after IVT and IAT for various durations in patients with acute ischemic stroke21. Collateral estimation is being considered increasingly important to precisely select eligible patients for IVT and IAT. Recently, useful collateral imaging methods using contrast material have been introduced, and they have shown prognostic value in acute cerebral ischemia due to large-vessel occlusion25. However, a noncontrast imaging method for collateral estimation is necessary with consideration of contrast-induced complications which can lead to irreversible kidney damage and an increased risk of morbidity and mortality, especially for patients with chronic kidney disease, which is a major risk factor for postcontrast acute kidney injury28.Acute ischemic stroke is a leading cause of death and major disability worldwide29, studies have shown that the prominent vessel sign  of ischemic brain on SWI can be considered an imaging biomarker representing collateral status and a predictor of functional outcomes35. The prominent vessel sign has been positively correlated with the amount of deoxyhemoglobin in cerebral veins secondary to an increased oxygen extraction fraction of the ischemic brain, which shortens the T2* relaxation and decreases the signal intensity of the veins in the ischemic region with respect to that in a normal brain36. Therefore, it can be hypothesized that ischemic tissue with poor collaterals requires more oxygen and contains more deoxyhemoglobins in tissue vessels, demonstrating a more prominent vessel sign on SWI than that of tissue with good collaterals. However, the results regarding the correlations between the prominent vessel sign and collateral status and the prognosis of ischemic stroke are inconsistent. Verma et al. showed that extensive prominent cortical veins correlated with poor leptomeningeal collateralization, while less pronounced prominent cortical veins correlated with good leptomeningeal collateralization30; however, Park et al. showed that more extensive prominent vessels were associated with better collateral flow37.Susceptibility-weighted magnetic resonance imaging (SWI) is gradient recalled echo T2*-weighted imaging. SWI is an essential sequence in the baseline magnetic resonance imaging protocol used for evaluating patients with acute cerebral ischemia due to its high sensitivity to deoxygenated hemoglobin in the brain and cerebral vessels. In addition to the clinical significance of hemorrhage detectionValidation of the role of prominent vessel signs on SWI as a collateral marker can enable collateral estimation without the use of contrast material or additional imaging. This method is particularly useful in patients who are unable to undergo contrast-based radiological studies. This study was conducted to verify the value of prominent vessel signs for estimating collateral status and for predicting functional outcomes in patients with acute ischemic stroke due to large-vessel occlusion in the anterior circulation.The local institutional review boards of Konkuk University Medical Center (KUMC0114-01-100-007) and Daejeon St. Mary’s Hospital (DC16OIMI0043) approved this prospective observational study, and written informed consent was obtained from all participants. All methods were carried out in accordance with relevant guidelines and regulations of the Declaration of Helsinki and the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for reporting observational study.25. Participants were evaluated for demographic data, medical history, vascular risk factors, routine blood tests, brain imaging, and cardiological tests. The stroke severity was assessed with the National Institutes of Health Stroke Scale (NIHSS). Participants underwent one of the following treatments at the discretion of the attending physician: intravenous thrombolysis, intraarterial thrombectomy, intravenous thrombolysis followed by intraarterial thrombectomy, or conservative treatment. Recanalization and reperfusion were assessed on the basis of conventional cerebral angiograms obtained at the end of the intra-arterial procedure and were classified by using the modified thrombolysis in cerebral infarction, or mTICI, system as follows: 0, no reperfusion; 1, penetration with minimal reperfusion; 2a, < 50% reperfusion; 2b, ≥ 50% reperfusion; 2c, near-complete reperfusion; and 3, complete reperfusion of the affected vascular territory. Reperfusion status was classified as either successful reperfusion (mTICI score of 2b to 3) or failed reperfusion (mTICI score of 0 to 2a). The participants who did not undergo endovascular treatment were also classified as having failed reperfusion. The functional outcomes were assessed on day 90 with the modified Rankin scale; a favorable functional outcome was defined as a modified Rankin scale score of 2 or less on day 90.This secondary analysis used published data obtained from a prospective observational study in patients with acute ischemic stroke due to occlusion or stenosis of the unilateral internal carotid artery (ICA) and/or M1 segment of the middle cerebral artery (MCA) who were evaluated within 8 h of symptom onset. The details of the study population and imaging methods have been described previously24. An experienced neuroradiologist (J.W.C with 14 years of experience), who was blinded to all clinical and other imaging data, measured the infarct volume (in milliliters) by using Medical Image Processing, Analysis, and Visualization 25. By using source data from dynamic contrast-enhanced MR angiography, 3-dimensional rotational arteriography was performed to determine the arterial status, and a multiphase MR angiography collateral map was reconstructed to evaluate the collateral-perfusion status. We generated the multiphase MR angiography collateral map using a MATLAB-based in-house program 24.All participants underwent MRI, including diffusion-weighted imaging (DWI), SWI, arterial spin labeling, dynamic contrast-enhanced MR angiography, dynamic susceptibility contrast-enhanced MR perfusion and fluid attenuation inversion recovery (FLAIR). MRI was performed with 3-Tesla MRI scanners . The acquisition parameters were the same as those used in a previous study35, a collateral grading system was defined according to the scores of the prominent cortical and/or medullary veins as follows: good, intermediate, poor, and very poor . A neuroradiologist  and a neurologist  who were blinded to all clinical data independently compared the prominent cortical and medullary veins between the affected hemisphere and the nonaffected hemisphere in terms of the degree of prominence using the following scoring system: 0, no prominent cortical or medullary vein in the affected hemisphere Fig. ; and 3, P value (< 0.05) in the univariable model and significance in previous studies in multivariable models. To examine the linearity of the relationship between the collateral grades and functional outcomes, the likelihood ratio test for trends was used. Statistical analyses were performed using SAS, version 9.4 . Statistical significance was indicated by P < 0.05.The patient characteristics are expressed as the mean (SD), median (IQR) or number of participants (%). Differences in the distribution of patient characteristics among collateral grades were assessed using the chi-square test, Fisher's exact test, ANOVA and the Kruskal–Wallis test, as appropriate. The interrater reliability for collateral grading was measured by the Cohen weighted κ. Correlation between the collateral-perfusion grade of the multiphase MRA collateral map and SWI collateral grade was assessed using Spearman’s correlation. Multiple logistic regression analyses were conducted to identify independent predictors of favorable functional outcomes. The variables selected for the multivariable model were age, baseline NIHSS score, baseline DWI lesion volume, SWI collateral grade and reperfusion status. We included baseline predictors into multivariable models according to their Of the 154 participants, 2 participants without SWI on baseline MRI were excluded. Hence, 152 participants were included in the current analysis. The mean age was 71 ± 13 years old (range = 34–92 years old), 64% of participants were men (98 men and 54 women), the median baseline NIHSS was 11 , the median onset to door time was 58 min , and the median onset to MRI time was 156 min . Twenty-six participants (17.1%) presented with stenosis (> 50%) of the ICA or M1 segment of the MCA, 65 participants (42.8%) with occlusion of the M1 segment of the MCA, 17 participants (11.2%) with occlusion of the ICA, 38 participants (25%) with occlusion of the ICA and M1 segment of the MCA, and 6 participants (3.9%) with occlusion of the anterior cerebral artery accompanied by occlusion of the ICA and/or M1 segment of the MCA. Eleven participants (7.2%) underwent intravenous thrombolysis, 40 (26.3%) underwent intraarterial thrombectomy, 48 (31.6%) underwent intravenous thrombolysis and intraarterial thrombectomy, and 53 (34.9%) underwent conservative treatment. Sixty-eight participants (44.7%) achieved successful reperfusion.P < 0.001). The site of steno-occlusion and mode of treatment varied by the collateral grade (both P < 0.001). Occlusion rather than stenosis and a higher number of occluded arteries were related to a lower collateral grade and more aggressive treatment. The SWI collateral grade was positively correlated with the collateral-perfusion grade determined by the multiphase MR angiography collateral map  , 0.89 to 0.98) was almost perfect. The prominent vessel sign was demonstrated in 134 patients 88.2%). A good collateral grade was obtained in 18 participants (11.8%), an intermediate grade was obtained in 23 (15.1%), a poor grade was obtained in 33 (21.7%), and a very poor grade was obtained in 78 (51.3%). The demographic findings associated with each collateral grade are presented in Table %. A goodP = 0.02), a smaller baseline DWI lesion volume , an intermediate collateral grade , a good collateral grade , and successful reperfusion  were independently associated with a favorable functional outcomes. There was a linear negative association between the collateral grade and functional outcome (P = 0.008) after adjusting for covariates.The results of the multiple logistic regression analyses performed to identify independent predictors of favorable functional outcomes are presented in Table P < 0.001) and a linear negative association between the SWI collateral grades and functional outcomes (P = 0.008). We demonstrated that the significant predictors of a favorable functional outcome in acute ischemic stroke due to occlusion or stenosis of the unilateral ICA and/or M1 segment of the MCA within 8 h of symptom onset were a younger age, lower baseline NIHSS scores, and smaller baseline DWI lesion volume, an intermediate and good SWI collateral grade and reperfusion.Our study showed a positive correlation between the SWI collateral grade and collateral-perfusion grade determined by the multiphase MR angiography collateral map  system, but Verma et al. assessed the degree of prominence. Without considering the degree of prominence, a mildly prominent cortical vein can be associated with good collateral status, although its extensiveness is similar to that found in Park’s study which included subacute-stage infarction. Additionally, as shown in Fig. 44. The use of imaging methods without time resolution leads to the over- or under-estimation of collateral status, especially in patients with late time windows over a day. The multiphase MR angiography collateral map derived from dynamic contrast-enhanced MR angiography is a precise method used for collateral estimation that can provide detailed information about tissue-level, brain parenchymal collateral perfusion and cortical artery information according to an individual’s hemodynamics24. Therefore, it is an optimal method for verifying the value of prominent vessel signs on SWI for collateral estimation. Our study directly showed the association between the degree of prominence of the prominent vessel sign and collateral status estimated with a multiphase MR angiography collateral map , and lack of a collateral grading method44. SWI is an essential sequence with high resolution in MR protocols for hemorrhage detection in patients with acute ischemic stroke and is easily acquired with any MR scanner within 100 s. Our study suggested a practical collateral grading system for SWI and showed the possibility of performing collateral imaging without contrast material or additional acquisition time. Additionally, the susceptibility vessel sign defined as a hypointense signal that exceeds the diameter of the contralateral artery is highly specific for the location of the occluded artery and represents a recanalization prediction after IVT54. The susceptibility vessel sign is also helpful for aiding neurointerventionists in planning the IAT treatment.Recently, developed collateral imaging methods, such as multiphase CT angiographyOne limitation of our study is that the prominent cortical and medullary veins were assessed visually. However, the interrater reliability was almost perfect, and the collateral grading system can be applied without further postprocessing. We expect that the development of an automatic calculation method using a susceptibility threshold to indicate salvageable brains in patients with acute ischemic stroke can provide more precise information on collaterals and the penumbra.In conclusion, collateral estimation using the prominent vessel sign on SWI is clinically reliable, as it has prognostic value. It is particularly useful in patients for whom contrast material is not recommended.


Owing to considerable differences observed in anatomy and physiology between paediatric subsets, it has been well established that children respond to drugs differently compared to adults. Furthermore, from a formulation perspective, there is a distinct challenge to develop a dosage form that is capable of safely, accurately, and reliably delivering the dose across the whole paediatric population. Orally disintegrating mini-tablets (ODMT) have widely been considered as an age-appropriate formulation option that possess the ability for adequate dose flexibility, avoids swallowing difficulties, and exhibits superior stability due to its solid state. Within this study, two strengths (0.5 mg and 2 mg) of carvedilol ODMT formulations were developed using an excipient composition and load that is appropriate for paediatric use. The formulations demonstrated adequate mechanical strength (>20 N) and fast disintegration times (<30 s). Dissolution profiles observed were robust and comparable to the marketed conventional tablet formulation across various parts of the gastrointestinal (GI) tract in both the fed and fasted state, signifying appropriate efficacy, quality, and performance. As such, the formulations developed in this study show potential to address the need of an ‘age-appropriate’ formulation of carvedilol, as highlighted by the European Medicines Agency (EMA) Inventory of the Needs for Paediatric Medicine. In Europe, a product licence (‘marketing authorisation’) is required to market a medicinal product that specifies the agreed terms of use, including medical indication, target age, dose, and route of administration . HoweverThe benefits and limitations of current paediatric formulation platforms have been evaluated, where innovative and flexible solid oral dosage forms have shown superior acceptability among paediatric subsets . Other tIn 2007, the paediatric regulation came into force and aimed to improve the health of children by facilitating the development and availability of medicines for children . The paeCarvedilol is a weakly basic biopharmaceutical class II model drug with a pKa value of 7.8 and aqueous solubility of 4.44 mg/L . It is aThe aim of the present work was to develop an age-appropriate formulation of carvedilol, with paediatric biopharmaceutical considerations for evaluation. Formulation selection was based on API properties, target dose banding, and the suitability of a delivery system for paediatric population needs. A systematic approach including excipient screening, formulation development, characterisation, dissolution studies, and stability evaluation were conducted to generate a holistic preclinical data set. The application of the ODMT technology with paediatric biopharmaceutical consideration for evaluation aims to safely and accurately facilitate the development of more age-appropriate formulations for this vulnerable patient group and serves as a potential candidate for PUMA application.Carvedilol was obtained by Acros Organics . Pharmaceutical grade D-Mannitol (≥98% purity) and magnesium stearate were obtained from Sigma-Aldrich , while MCC as Pharmacel 102 was obtained from DFE Pharma (Germany). Lastly, colloidal silicon dioxide (Aerosil 200) was obtained from Evonik Industries . Pepsin from porcine gastric mucosa (≥400 units/mg protein), sodium hydroxide pellets, sodium chloride, sodium acetate, and sodium monooleate were purchased from Sigma-Aldrich . Sodium taurocholate hydrate, 96%, acetic acid, maleic acid and Lecithin, 60%, eggs were obtained from Alfa Aesar . Glyceryl monooleate as Monomuls 90-O 18 PH was kindly gifted by BASF . Carvedilol tablets, 3.125 mg, were obtained from Bristol laboratories .For sample analysis, Acetonitrile and Methanol (HPLC-grade) were obtained from Fisher Scientific , whereas Trifluoroacetic acid (TFA), for HPLC (≥99.0%) was purchased from Sigma-Aldrich .v/v). TFA was used at a concentration of 0.1% (v/v). Flow rate was set at 1 mL/min and a wavelength of 240 nm was used for detection. A 7-point calibration curve in the range 0.19 to 12.5 mg/mL was prepared via serial dilution in methanol (1 in 2 dilution). All samples prepared for analysis were diluted so as to fall within the calibration range. Method validation was carried out following The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guidelines (Q2(R1)) [All samples were analysed on an Agilent 1260 series , equipped with a reverse-phased Eclipse plus C18, 4.6 × 150 mm, 3.5 μm column  was used. Separation of carvedilol was achieved using an isocratic mobile phase compromising TFA: ACN (55:45 (Q2(R1)) .−1) using sealed flat-bottomed TZero aluminium pans and pierced lids (sample mass 1–2 mg) at a heating rate of 10 °C min−1 in the range of 50 to 400 °C. For samples containing both the drug and excipient, 1 mg of each was measured in a weighing boat and gently mixed with a spatula for one minute before being transferred into the sample pan. All studies were conducted in triplicate and data are presented as mean ± standard deviation.Excipient and drug compatibility were studied using a TA DSCQ200 apparatus  with TA Instruments Universal Analysis 2000 software. Data was collected under nitrogen atmosphere . A total of 1 g of sample was placed on the VIBRI/I feeder and fed through the RODOS disperser. Pressure was set at 2 bars and measuring range was set between 0–175 μm. Measurements were taken in triplicates and presented as ±SD. Volume mean diameter (VMD), X10, X50, and X90 were the values obtained. Both starting materials and final blends were analysed to give an indication of flow based on particle size and to ensure acceptable flow properties are prevalent for successful direct compression.Angle of repose was determined using the method outline in the U.S. Pharmacopeia monograph, <1174> . A knownMeasurements were carried out in triplicates and presented as mean ± standard deviation.Bulk (ρbulk) and tapped densities (ρtapped) were also used to assess powder flow. A Sotax tap density tester USP II  was used as outlined in the USP monograph, <616> . A 25-mLCarrs’s index and hausner ratios were calculated using the following equations:An Erweka AR403s Multi-Lab , equipped with a cube mixer attachment was used to mix a total of 50 g of powder that included mannitol, MCC, magnesium stearate, colloidal silicon dioxide, and carvedilol. Different speed rates, mixing times, and blending order were evaluated to analyse any consequent effects on content uniformity. In each case, magnesium stearate was added at the end and allowed to mix for a total of 1 min, whilst colloidal silicon dioxide (CSD) was added second to last and allowed to mix for a total of two minutes. A Sartorius LA220S balance  was used to accurately weigh out samples.Content uniformity was assessed using a random sampling technique, where 10 samples of 50 ± 1 mg were taken out from different parts of each powder blend. Each sample was dissolved with 50 mL of methanol in a volumetric flask and sonicated for 30 min. Post sonication, the samples were filtered through a 0.45-μm syringe filter and analysed via High Performance Liquid Chromatography (HPLC).w/w and a tablet mass of 50 mg were produced using a Specac semi-automatic hydraulic press  equipped with a 4-mm multi-tip (three) with concave faced punches at a compression force of 10 KN with quick release. After step wise fine-tuning of the formulation composition and tabletting parameters, two formulations for ODMT production were finalised  was used to measure the force required to break the tablets produced. Hardness values were measured in Newton’s. Measurements and carried out in triplicates and presented as mean ± standard deviation.Friability testing was carried out using a Sotax F2 Friabilitor  that measures the capacity of tablets to resist mechanical stress. Six tablets were carefully de-dusted using a soft brush and an initial weight was taken. Tablets were then placed in the rotating drum and rotated for 4 min at a speed of 25 rpm . The tablets were removed, dusted, and a final weight was taken. The percentage friability was calculated using the following equation:Disintegration testing was carried out as stated in the official USP disintegration monograph <701>. An Erweka ZT3  disintegration tester was used. A tablet was placed in one of the six vessels and allowed to oscillate at a rate of 30 cycles per minute. In this instance, a disk was not used in order to allow for a more direct comparison of the oral cavity environment. A total of 800 mL of distilled water was used as the disintegration medium that had a constant temperature of 37 °C. Measurements were achieved for a single tablet at a time to enhance accuracy. The disintegration time was noted until there was no more residue of tablet aggregates left on the upper side of the vessel mesh. Measurements were carried out in triplicates and presented as mean ± standard deviation.Adult simulated gastric and intestinal media , were prepared as presented by Jantratid et al.  and emplThe saturation solubility of carvedilol in simulated dissolution media was determined according to the reported method . BrieflySink value (S/C) was calculated as a ratio of solubility (S) to drug concentration (C) to determine whether sink conditions were achieved. A sink value greater than 3 indicates sink conditions . PercentMinimum dissolution volume was calculated using sink condition (Sc), determined by the ratio of solubility (S) to drug concentration (C), denoted by S/C. A value of greater than 3 is considered as sink condition . All disGraphPad Prism version 8  was used to carry out one-way analysis of variance (ANOVA) with a posthoc Tukey’s test to identify statistically significant differences in solubility between adult and paediatric biorelevant media. An alpha value of 0.05 was used.Although carvedilol has been approved since 1995 and its clinical need has been established, no attempt has been made to reformulate carvedilol into an age-appropriate formulation; the only licensed medicinal form that is available is a conventional tablet, while forms available from special-order manufacturers include oral suspension. In addition, the EMA Inventory of the Needs for Paediatric Medicine has also listed carvedilol in need of an ‘age-appropriate’ formulation. To address this formulation gap, the objective of the current work was to develop two strengths (0.5 mg and 2 mg) of an age-appropriate carvedilol formulation that possess increased dose flexibility, palatability, safety, quality, and efficacy. Carvedilol is a low dose potent compound with low aqueous solubility. It is gastric stable and does not exhibit any taste concerns. For such reasons and with an aim to minimise the total number of excipients within the formulation, the selection of an ODMT was most suitable. The dose of carvedilol is based on child weight and is usually titrated when initiated. The availability of multiple strengths ensures to cover a wide range of therapeutic doses and therefore, the novel ODMT formulation is aimed at birth up to the age of 12 years.In order to limit the total number of excipients within the formulation, one of the objectives of this study was to exploit excipient characteristics that provided multiple functions including palatability, compressibility, and disintegrating properties.Drug and excipient compatibility is an important part of pre-formulation study, where concomitant use of excipient and active pharmaceutical ingredients (API) are assessed. Commonly used excipients in ODT formulation were scanned alongside carvedilol to determine compatibility. However, for the purpose of this study, carvedilol:excipeint compatibility is displayed for excipients that were included in the final formulation only.Carvedilol displayed a sharp endothermic peak at 117.33 °C, indicating its melting point and crystalline structure . The phaThe sharp melting peak of carvedilol remained consistent when combined with magnesium stearate, suggesting compatibility between the two components . Howeverw/w), the success of direct compression is dictated by the excipients to ensure content uniformity, flow, and compressibility [For low concentration drugs, as in the case of carvedilol  indicated carvedilol to possess poor flow (46.47°) , which iAccording to Carr’s index and Hausner ratio, mannitol exhibited very poor flow (33.33% and 1.5) whereas MCC shows passable flow (23.08% and 1.3) . These rMixing of particulate solids is an important process step in achieving homogenous blends and key to assuring that each resulting unit dose possesses the specified amount of drug to achieve effective therapeutic levels. For low concentration drugs, as in the case of carvedilol, the success of direct compression is dictated by the excipients which are incorporated into the powder blend, with an aim to optimise flow and compressibility. It is essential for the powder to flow evenly, ensuring that the right amount of homogeneous powder enters the tableting die, ensuring consistent tablet weight and content uniformity .It was expected that longer mixing time would provide better content uniformity, since the API particles would have more contact time with other particles, increasing the chances of collision and resulting in improved homogeneity ,40. HoweBoth formulations produced a balance between mechanical strength and fast disintegration times . FriabilCarvedilol is a weakly basic compound with a pKa value of 7.8, exhibiting the distinctive pH-dependent solubility profile of a weak base, with low solubility in dissolution media of high pH values and high solubility in media of low pH values due to changes in ionisation  and as sAll adult and paediatric biorelevant media prepared for dissolution testing are categorised as level II media (biorelevant) that mimic various parts of the GI tract in regards to adjusted osmolality, pH, buffer capacity, bile components, and phospholipid concentration, and therefore, are superior to conventional USP/BP dissolution methods as they better reflect the solubilisation capacity of adult and paediatric GI fluids both during the fed and fasted state .p value > 0.05). It was expected that the dissolution rate to decrease in adult and paediatric FeSSGF due to the higher pH values of 5 and 5.7. This was true as the percentage release of carvedilol was only about 30–40% after the first 10 min. Nonetheless, no significant difference between adult and paediatric biorelevant media was observed.The initial rate of release of carvedilol from the novel ODMTs in a pre-prandial state was immediate, with the carvedilol percentage release exceeding 80% after the first 10 min . The absAlthough the fasted state of the gastric media is ideal for rapid and complete dissolution, it has been advised that heart failure patients take their medication with food to avoid a sharp increase in plasma concentration and limit the risk of orthostatic hypotension . Due to On the other hand, a clinically relevant dissolution profile between FeSSIF-V2 and paediatric FeSSIF was observed, where both ODMT formulations displayed a faster dissolution rate and an overall greater extent of percentage carvedilol release in FeSSIF-V2. This may be owing to the larger concentration of bile salt (sodium taurocholate) and lipid (lecithin) in FeSSIF-V2. Lecithin enhances the solubility of drugs through complexation, whilst sodium taurocholate holds the ability to produce micelles. Other than micellar solubilisation, bile salts may alter drug polarity and therefore solubility through ion pairing, when interacting with drug molecules . Based up > 0.05) differences observed. The choice of excipients and concentrations utilised, alongside fine-tuned production parameters for the manufactured carvedilol ODMT, ensured a robust dissolution profile, comparable to the marketed formulation without including dedicated functional disintegrants. Tablet disintegration is a prerequisite for dissolution, where orodispersible formulations must disintegrate rapidly however, the use of superdisintegrants in and as a model formulation constituent is not preferred, since many are incompatible with various drugs compounds, moisture sensitive (leading to instability), and lack toxicity data in both adults and paediatrics [diatrics . Upon coTwo strengths of carvedilol ODMT formulations were developed using an excipient composition and load that is appropriate for paediatric use. The novel ODMTs displayed all relevant features of an age-appropriate formulation, including enhanced dose flexibility, palatability, excipient safety, and concordance to development and ability for safe administration. Dissolution profiles observed were robust and comparable to the marketed formulation across various part of the GI tract in both the fed and fasted state, signifying appropriate efficacy, quality, and performance. No significant difference in dissolution profiles between adult and paediatric biorelevant media were established. The systematic selection of drug candidates listed within the EMA inventory of paediatric therapeutic needs, alongside paediatric specific formulation approach, provides an encouraging starting point that ultimately aims to increase the availability of more age-appropriate formulations for children through PUMA applications.


SPINT2 was also measured in the plasma and placentas of women with established placental disease at preterm (<34 weeks) delivery. Using first-trimester human trophoblast stem cells, SPINT2 expression was assessed in hypoxia/normoxia (1% vs. 8% O2), and following inflammatory cytokine treatment . Placental SPINT2 mRNA was measured in a rat model of late-gestational foetal growth restriction. At 36 weeks, circulating SPINT2 was elevated in patients who later developed preeclampsia , or delivered a small-for-gestational-age infant . SPINT2 was elevated in the placentas of patients who required delivery for preterm preeclampsia (p = 0.025). Though inflammatory cytokines had no effect, hypoxia increased SPINT2 in cytotrophoblast stem cells, and its expression was elevated in the placental labyrinth of growth-restricted rats. These findings suggest elevated SPINT2 is associated with placental insufficiency.Biomarkers for placental dysfunction are currently lacking. We recently identified SPINT1 as a novel biomarker; SPINT2 is a functionally related placental protease inhibitor. This study aimed to characterise SPINT2 expression in placental insufficiency. Circulating SPINT2 was assessed in three prospective cohorts, collected at the following: (1) term delivery ( Aberrations in placentation, particularly those amounting to restricted vascular remodelling, are associated with a constellation of obstetric complications, with significant implications for mothers and babies. This includes foetal growth restriction (FGR), in which affected foetuses fail to achieve their unique growth potential in utero, owing to inadequate uteroplacental perfusion. This confers an increased risk of perinatal morbidity and mortality upon the foetus ; in factCurrently, there is an absence of effective and targeted treatments for both preeclampsia and FGR, and the latter, in particular, eludes precise diagnosis. Consequently, there are no interventions to rescue a poorly functioning placenta; except for iatrogenic preterm delivery, which has its own associated risks. In the search for circulating biomarkers of placental insufficiency, SPINT1, a serine protease inhibitor, also called HGF activator inhibitor 1 (HAI-1), has been identified as a promising candidate . SPINT1 Spint1 knockout mouse models, Spint2-deficient mice suffer from placental defects. However, the effects of the loss of Spint2 extend beyond the placenta, causing embryonic lethality, unless matriptase is simultaneously ablated, in which case there are impairments to the neural tube closure [The validation of this serine peptidase inhibitor as an indicator of placental dysfunction justifies the investigation of its analogue SPINT2/HAI-2. To date, SPINT2 has not been thoroughly assessed in the human placenta; thus, its application in FGR diagnosis is yet to be explored. SPINT2 has a comparable tissue distribution to SPINT1 , and con closure .Given the previous findings of dysregulated SPINT1 in FGR, it was hypothesised that the expression of SPINT2 would be similarly deranged in the placenta and maternal circulation of pregnancies that are complicated by FGR, even prior to diagnosis. Further, the expression of SPINT2 in placental trophoblasts was expected to be regulated by hypoxia and inflammation, which are signature contributors underlying placental insufficiency. Therefore, the aim of this study was to characterise the expression of SPINT2 at the mRNA and protein level in placental and plasma samples from pregnancies that have been affected by preeclampsia and/or FGR, as well as in a rodent model of placental insufficiency, and to observe any hypoxic- or inflammation-mediated changes in expression in vitro.Given that SPINT2 has not previously been analysed in the human placenta, this study first characterised its expression in pregnancies that were known to be compromised by FGR and/or preeclampsia.n = 14), SPINT2 mRNA expression (n = 19). In contrast, the placentas from pregnancies that were complicated by both preeclampsia and FGR (n = 20) had significantly decreased SPINT2 mRNA expression , whereas those affected by preeclampsia only (n = 60) had significantly increased SPINT2 mRNA levels .In preterm FGR placentas (pression a was higp = 0.0171) compared to the controls , with a similar result observed in the placentas from pregnancies that were affected by preeclampsia alone .The level of SPINT2 protein expression was also measured in these placentas b, which We next sought to measure SPINT2 within the maternal circulation in prospective cohorts, prior to any diagnoses.p = 0.0507) in those women whose infant was born small for the gestational age , compared to appropriate for the gestational age  controls . Circulating SPINT2 was significantly elevated (p = 0.002) in the SGA  samples that were collected at 36 weeks’ gestation . This association was lost, however, earlier in gestation, where 24- to 34-week plasma from women with underlying vascular disease  and SGA (n = 58) levels of SPINT2. In this cohort, SPINT2 did not vary across gestation (p = 0.054) in SPINT2 across gestation in those women who were destined to birth an SGA infant (R2 = 0.0645).In maternal plasma  in women who were destined to develop term preeclampsia , relative to the controls . In the 24- to 34-week plasma from women attending the MAViS clinic , while secreted SPINT2 was unchanged (p = 0.008). Given SPINT2 is also likely expressed in syncytiotrophoblast, we measured expression and secretion in syncytialised first-trimester human trophoblast stem cells  in the syncytiotrophoblasts that were exposed to hypoxia.In primary trophoblasts that were isolated from term placentas, SPINT2 mRNA transcripts were significantly increased in response to hypoxia , compared to that of dams who underwent sham surgery. Interestingly, the labyrinth layer—akin to the chorionic villi  of the human placenta—of restricted placentas had modestly upregulated SPINT2 mRNA expression .The derangement of SPINT2 expression in response to hypoxia was also demonstrated in placentas from a rat model of uteroplacental insufficiency, induced by ligating the uterine vessels, thereby impeding placental perfusion. There are distinct morphological differences  betweenp < 0.01) in syncytiotrophoblasts , it was apparent that SPINT2 expression is not dysregulated in FGR-like functionally related homologue SPINT1 . NeverthThe expression of SPINT2 mRNA in the preterm placentas was decreased in concurrent FGR and preeclampsia, but elevated where only preeclampsia is present ; whereas, SPINT2 protein expression was increased in all cases of preeclampsia (with and without FGR). It is interesting that SPINT2 mRNA and protein are both significantly elevated in preeclamptic (without FGR) placentas, whereas the placentas plagued by concurrent preeclampsia and FGR had decreased SPINT2 mRNA, but elevated protein. This is an unusual phenomenon, and the reason for this disparity has not been elucidated by the present study. It may suggest that there are post-transcriptional modifications in the preeclamptic/FGR placentas, to reduce SPINT2 protein turnover, as a compensatory mechanism for the low transcript levels; however, further study is needed to confirm this hypothesis.n = 6), and the high variability in expression for the samples that were available. A larger sample size would aid in verifying whether there are bona fide alterations in mRNA, and/or protein in the placentas and plasma of FGR-affected pregnancies. In the cases of FGR, the circulating levels of SPINT2 were modestly elevated at term and at 36 weeks’ gestation; however, at earlier gestations, there were no observed changes in expression. Similarly, there was elevated circulating SPINT2 at 36 weeks in those women who were destined to develop preeclampsia, while earlier gestation levels were unchanged, relative to the controls. Interestingly, SPINT2 was not altered in preterm disease, raising the possibility that derangements in circulating levels only arise in later-onset disease. Alternatively, the lack of statistical significance in the established disease plasma cohort may be attributable, in part, to having relatively few FGR samples  . Having Mouse models have previously established the importance of SPINT2 in placental development , and, coTo characterise the expression of SPINT2 in the maternal circulation and placenta of FGR- and/or preeclampsia-complicated pregnancies, human specimens were obtained. All studies were approved by the Mercy Health Human Research ethics committee (R11/34).n = 14), PE (n = 60) or both (n = 20), according to preeclampsia guidelines from ACOG 2020 [n = 19) were gestation-matched and obtained from women who were delivered preterm due to other complications not associated with placental insufficiency or hypertensive disorders of pregnancy, such as placenta praevia or spontaneous preterm rupture of membranes. Although the control sample comprises pregnancies with complications, gestation-matching is important when investigating proteins highly expressed in the placenta, as the pattern of expression commonly varies with advancing gestation. Samples in both the case and control group were excluded if there were congenital anomalies and/or histopathological evidence of congenital infection. Patient characteristics are detailed in Placental tissue samples were collected from consenting women, delivering by caesarean section at less than 34 weeks’ gestation, with decision for delivery made independently by the treating obstetric team. The samples were classified as FGR  for 48 h, then snap frozen and stored at −80 °C. Subsequently, RNA or protein was extracted from tissue lysates.n = 6), PE (n = 40) or both (n = 11). These were compared to gestation-matched blood specimens collected from control pregnancies delivered at term (n = 26). These samples were aliquoted and stored at −80 °C until future analysis. Patient characteristics of this cohort are detailed in Plasma samples were also collected on the day of delivery at less than 34 weeks’ gestation from women delivering prematurely with FGR . The FLAG2 study was approved by the Mercy Health Research ethics committee  and written informed consent was obtained from all participants. The total number of remaining samples used for SPINT2 analysis was 227, comprising 152 controls  and 75 cases (SGA). Patient characteristics are shown in The Fetal Longitudinal Assessment of Growth 2 (FLAG2) study recruited 562 unselected women on the day of elective caesarean section at the Mercy Hospital for Women . Women who were aged over 18 years with a well-dated singleton pregnancy, at 36n = 198) according to the GROW Bulk centile calculator , all cases delivering with preeclampsia  and a cohort of controls (n = 182). Patient characteristics detailed in The Biomarker and Ultrasound Measures for Preventable Stillbirth (BUMPS) study is a large prospective cohort collection at MHW, with samples collected from an unselected population at 28 and 36 weeks’ gestation. Women were screened for eligibility and invited to participate at their oral glucose test, universally offered to non-diabetic pregnant women around 28 weeks’ gestation to test for gestational diabetes mellitus. Following written informed consent, women aged over 18 years, with a singleton pregnancy and normal mid-trimester foetal morphology examination were eligible to participate. The BUMPS study was approved by the Mercy Health Research ethics committee . For this study, a case-cohort of 364 samples was selected from the first 1000 BUMPS participants, including all cases delivering an infant <10th centile . Women are referred to the clinic in early pregnancy for monitoring across gestation based on hypertensive disease, which predisposes to preeclampsia and/or FGR, allowing for longitudinal sampling between 24- and 34-weeks’ gestation. The inclusion criteria for women in the MAViS study were as follows: 1. chronic hypertension ; 4. history of ischaemic heart disease; and 5. previous early onset preeclampsia. A case-cohort of 294 participants was recruited between October 2011 and December 2016, with a plasma sample obtained between 24 and 34 weeks, and complete outcome data were included in the current study. These participants were selected from an overall cohort of 518 participants and included 179 control women and 115 who either delivered with preeclampsia, FGR or both. The study was granted ethics approval by the NRES Committee North West (11/NW/0426). Patient characteristics are listed in In order to assess the in vivo expression of SPINT2, samples were obtained from a previously established rodent model of FGR. The placental deficiency in this model was induced during late gestation (at day 18 of 22), providing an in vivo model of late-onset, placental-derived FGR . UteroplThe placentas were collected, weighed and the labyrinthine layer separated from the basalis layer before being immediately frozen in liquid nitrogen, then stored at −80 °C for later analysis. RNA was extracted from both regions. The development of this model of uteroplacental insufficiency in rats was approved by the La Trobe animal ethics committee (AEC: 12–42), in accordance with the National Health and Medical Research Council’s (NHMRC) Australian code for the care and use of animals for scientific purposes.To examine SPINT2 in response to hypoxia and inflammatory stressors, first-trimester human trophoblast stem cells (hTSCs) were obtained from the RIKEN BRC through the National BioResource Project of the MEXT/AMED (Japan), as previously detailed in the manuscript from Okae et al., 2018. This cell line was isolated from first-trimester placentas under ethical approval from Tohoku University School of Medicine . The celPrimary cytotrophoblast cells were isolated from term placentas according to the protocol optimised by Kaitu’u-Lino et al., 2014 . In summHypoxic conditions were simulated for first-trimester hTSCs and term trophoblasts to assess the effect of inadequate oxygen perfusion during placentation and approaching term, respectively. After a 24-h incubation in 8% oxygen at 37 °C, allowing cells to adhere to the basement membrane , cells were incubated in different oxygen concentrations. Given the physiologically relevant oxygen tension in utero is 8%, those cells designated normoxic were incubated at 37 °C in 8% oxygen for 48 h. Hypoxia involved exposure to 1% oxygen for the same duration. The media was collected for subsequent analysis and cells lysed for RNA extraction.Two inflammatory cytokines, tumour necrosis factor alpha  and interleukin-6 , were added to the media of primary trophoblasts and first-trimester hTSCs  to simulate the inflammation common to the preeclamptic placenta. These cells were incubated at 37 °C for 24 h after plating, followed by treatment with 0 ng/mL, 0.1 ng/mL, 1 ng/mL or 10 ng/mL of the recombinant cytokine, diluted with fresh media. After being cultured for a further 24 h in the treatment media, cells and media were collected.Protein was isolated from placental tissue and syncytiotrophoblast, using RIPA buffer containing protease inhibitor cocktail  and Halt™ phosphatase inhibitor cocktail  to lyse cells and centrifugation to pellet debris. To quantify the protein content of each sample, a Pierce™ BCA assay (Thermo Fisher Scientific) was performed according to the manufacturer’s protocol. Equal protein amounts were loaded for ELISA.The Genelute™ mammalian total RNA miniprep kit  was used to extract RNA from cultured hTSCs, primary term trophoblasts and placental tissue, as per the manufacturer’s protocol.2O). The iCycler iQ™5  protocol was run according to kit specifications, being held at 4 °C after completion until collection and storage at −20 °C for later PCR analysis.RNA extracted from samples was converted to cDNA using the Applied Biosystems™ high-capacity cDNA reverse transcription kit , following the manufacturer’s guidelines. The reaction comprised 150 ng RNA solution  specific to the genes of interest are detailed in t of sample duplicates were normalised to appropriate reference genes before being calibrated to the average Ct of experimental controls, allowing the results to be expressed as percentage relative to controls.Quantitative PCR was carried out to ascertain the mRNA expression of SPINT2 protein levels were measured in maternal plasma samples, hTSC media, and placental lysates via ELISA. The large cohort analyses were analysed using an ELISA kit for SPINT2 , following manufacturer’s specifications. The <34 week plasma was diluted 1:12 for SPINT2. ® ELISA . Then, 5 μg of each sample was loaded, diluted in 1% BSA in PBS according to the concentration determined by the BCA assay. Cultured hTSC media was also analysed using the R&D Systems SPINT2 kit. Media samples were undiluted, with the exception of the hypoxia studies, which were diluted 1:2 with 1% BSA in PBS. Cellular SPINT2 in placental lysates was analysed using a SPINT2 DuoSett-test or Mann–Whitney U test was used according to Gaussian distribution. For more than two groups, a one-way ANOVA or Kruskal–Wallis test was used, according to Gaussian distribution, and post hoc analyses ascertained by Dunn’s multiple comparisons test. Outliers were identified and accounted for using a ROUT test.In vitro experiments were carried out in technical triplicate and repeated 3–5 times. The results of in vitro experiments were normalised to controls so data could be expressed as % control. Using GraphPad Prism 8 , statistical analyses were carried out, with data first assessed for Gaussian distribution, then analysed using appropriate statistical tests. Maternal characteristics and birth outcome data (Unlike SPINT1, circulating SPINT2 is not consistently dysregulated in diseases of placental insufficiency—in preeclampsia or foetal growth restriction. We have shown that SPINT2 is unlikely to be a clinically useful biomarker; however, we did identify changes in placental SPINT2, which suggest it may be functionally involved in human placentation; this is a role yet to be explored.


The microbial population, diversity and interactions along the intestinal tract  were assessed in two producing types of pigs, where castrated Duroc pigs were used as heavy pigs, whereas entire F2 crossbred pigs (Pietrain sires × (F1: Duroc × Landrace) dams) were used as lean ones. Half of the animals belonged to two production phases (growing vs. fattening) and were subjected to a moderate crude protein restriction (2%). The producing type of pig and the production phase were the effects that most affected the parameters studied, where the fattening pigs and the lean ones showed higher alpha diversity indices and microbial network complexity. However, the lower dietary crude protein content had only a slight effect on microbial networks. These findings provide further understanding about how different effects  interact and affect gut microbiome, which could be taken into account for the optimization of pork production efficiency.Lactobacillus was the predominant genus, and along with Streptococcus and Clostridium, their relative abundance decreased throughout the intestine. Although low CP diet did not alter the microbial diversity, it increased interaction network complexity. These results have revealed that the moderate CP restriction had lower impact on intestinal microbiota than PP and PT of pigs.Characterization of intestinal microbiota is of great interest due to its relevant impact on growth, feed efficiency and pig carcass quality. Microbial composition shifts along the gut, but it also depends on the host , diet composition and environmental conditions. To simultaneously study the effects of producing type (PT), production phase (PP) and dietary crude protein (CP) content on microbial populations, 20 Duroc pigs and 16 crossbred pigs (F2), belonging to growing and fattening phases, were used. Half of the pigs of each PT were fed a moderate CP restriction (2%). After sacrifice, contents of ileum, cecum and distal colon were collected for sequencing procedure. Fattening pigs presented higher microbial richness than growing pigs because of higher maturity and stability of the community. The F2 pigs showed higher bacterial alpha diversity and microbial network complexity (cecum and colon), especially in the fattening phase, while Duroc pigs tended to have higher Firmicutes/Bacteroidetes ratio in cecum segment.  Optimizing feed efficiency is one of the current challenges for the swine industry that also promotes the reduction of both feeding costs and environmental impact ,2. WithiTogether with management  and genetic plans, implementation of new production strategies should also consider the animal’s physiology ,6. In thSince there is high potential to modulate gut microbiome in different managing scenarios , it woulProtocols and experimental procedures were approved by the Ethics Committee for Animal Experimentation of the University of Lleida, under Project License CEEA 09-05/16. Care and use of animals were in accordance with the Spanish Policy for Animal Protection RD 53/2013, which meets the European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes.2) for the first 9 days, and then placed individually in metabolic cages during the last 6 days, as was previously described in [A total of 36 male pigs belonging to two production phases , were housed in the same environmentally controlled room at the Swine Research Center located in Torrelameu . Twenty of these 36 pigs were surgically castrated purebred Duroc pigs, of which 12 were in the growing phase (mean ± standard error: 26.4 ± 3.11 kg of body weight (BW) at sacrifice) and 8 in the fattening phase (86.1 ± 2.74 kg BW), while the remaining 16 pigs were entire crossbreds (F2) (Pietrain sires × (F1: Duroc × Landrace) dams), 8 in the growing phase (30.5 ± 1.36 kg BW) and 8 in the fattening phase (91.1 ± 1.23 kg BW). Moreover, two experimental diets were formulated for each PP, which were provided ad libitum for 15 days (4 days of dietary changeover plus 11 days of adaptation) prior to slaughter. Both diets were isoenergetic and identical in covering the nutritional requirements but differing in 2% of CP concentration. Half of the pigs of each PT were allotted to one of the two dietary treatments, standard protein (SP) diet and low protein (LP) diet, formulated with 17% and 15% CP in the growing phase and 15% and 13% CP in the fattening phase, respectively. All diets were supplemented with synthetic amino acids to meet the nutrient requirements recommended by FEDNA . The ingOn the last day of the trial, pigs were sacrificed, and the contents of ileum, cecum and distal colon were collected simultaneously for microbial characterization. Samples were immediately frozen in dry ice and stored at −40 °C until further analysis.Microbial genomic DNA was extracted using DNeasy PowerLyzer PowerSoil Kit  following the manufacturer’s instructions. Mock community DNA was included as positive control for library preparation .Samples were amplified using primers 341F and 805R, which target the V3–V4 region of the bacterial and archaeal 16S rRNA. PCR was performed in 10 μL final volume with 0.2 μM primer concentration. The PCR included: 3 min at 95 °C  followed by 25 cycles of 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C, and a final elongation step of 5 min at 72 °C. PCR products were purified using AMPure XP beads  with a 0.9× ratio according to the manufacturer’s instructions.The paired-end sequencing was conducted following an Illumina Miseq sequencing 300 × 2 approach. Quality control filtering and OTU binning of the resulting sequences were executed using DADA2 software . FinallyAnalysis described below were performed in duplicate aiming to assess: (i) differences in ileal, cecal and colonic microbiota between PTs (Duroc vs. F2) in both PPs; and (ii) differences in ileal, cecal and colonic microbiota between experimental diets (SP vs. LP) in both PPs.p < 0.05 and tendency to difference at p between 0.05 and 0.10.Sequence data were normalized to the same mean and alpha diversity indices were calculated  to measure the variability of species within a sample. Data were then analyzed with a linear model including PT × PP and diet × PP as fixed effects . Data from the three intestinal segments  were analyzed separately to decrease datasets sparsity . Contrasts between either PT or diets were performed by Tukey’s test . Individual samples out of three standard deviations of the mean were discarded and not included to the statistical analysis. Significant effects were declared at To circumvent the compositional bias problem ,19, we aSequencing generated a total of 3,709,916 high-quality sequences obtained from the 108 digestive content samples from the three intestinal regions. The mean number of sequences per sample was 34,251 for the ileum, 31,167 for the cecum and 37,635 for the distal colon. These sequences resulted in a total of 181 OTUs in ileum (33 OTUs per sample), 395 OTUs in cecum (107 OTUs per sample) and 453 OTUs in colon (136 OTUs per sample). The unclassified rate of OTUs at genera level increased along the gut segments, with 8.9%, 11.3% and 14.2% for ileum, cecum and colon samples, respectively.Microbial diversity indices along with the Firmicutes/Bacteroidetes ratio were analyzed in both PTs of pigs in each PP . The Firp = 0.014 and 0.009 for Shannon and Simpson indices, respectively), cecum  and colon . Moreover, F2 pigs showed higher microbial richness in the cecum (p < 0.010), and higher evenness in both ileum and distal colon segments (p = 0.011) compared to Duroc. In all three intestinal segments, fattening pigs presented a significantly higher microbial richness than growing ones (p < 0.05). A significant interaction was also found in ileum between PT and PP effects (p = 0.014), where fattening F2 pigs had significantly higher microbial richness than growing F2 pigs, although no differences were detected between growing and fattening Duroc pigs.The PT had a significant influence on microbial diversity . F2 pigsp = 0.089) with no variation with PP. Although no significant differences between PTs nor PPs were obtained in the distal colon, the Firmicutes/Bacteroidetes ratio in Duroc pigs numerically decreased from growing to fattening phases, whereas in F2 pigs it remained constant.The Firmicutes/Bacteroidetes ratio between both PTs in growing and fattening phases was only calculated in cecum and distal colon. In the cecum, Duroc pigs showed a slight tendency to have higher ratio than F2 pigs (p < 0.001). In addition, no significant differences between the cecum and distal colon were detected in each PP, except for microbial richness in the growing phase that was significantly higher in the distal colon. This is graphically evidenced in p < 0.05), as well as a slight tendency to have higher Simpson index (p = 0.084). However, only Simpson index in ileum and microbial richness in the cecum presented significant differences.Alpha diversity indices were also obtained between intestinal segments in the two PPs , in whicLactobacillus was the predominant genus in the ileum of growing and fattening pigs, and it decreased progressively from ileum to distal colon. The same dynamics were observed in Streptococcus and Bifidobacterium , while no significant differences were found for the dietary CP content (p > 0.433), although it seemed to be more evident in the growing phase than in the fattening phase. The beta diversity of the two PTs belonging to both PPs is represented in p < 0.001). In a similar manner, differences in microbial community composition between the two PTs were found in ileum and cecum segments , although the microbial communities of the ileum appeared to be more distinct than those of the more distal segments.The beta diversity analysis of microbial OTUs in ileum, cecum and distal colon is graphically represented in Microbial networks were built to test interactions among microbial genera in each intestinal segment. Degree of interaction was studied through the number of genera (nodes) that established significant interactions (edges) with other genera, as well as the number of interactions established per node (node degree).The overall microbial network complexity increased across the intestinal segments: the mean number of nodes and edges in each intestinal segment were 12 and 13 in ileum, 50 and 155 in cecum and 68 and 230 in distal colon . In the Betweenness centrality, which measures the possible influence of an individual node on other nodes, was also calculated . This meAuthors are aware of the limiting number of animals per group with the 2 × 2 × 2 factorial design to reach consistency in both results and conclusions. The present study was part of a complex trial in which animals’ protein and lipid metabolisms were also studied . In addiDifferences in intestinal bacterial composition between the two PPs were studied since animal age regulates changes in nutrient digestibility and metabolism, immunity and hormone status, and tissue development with a direct impact on microbiome. Intestinal tract samples from growing and fattening phases were obtained from pigs of two PTs, at about 83 and 154 days of age, respectively. Among all alpha diversity indices analyzed, microbial richness was the unique one that showed significant differences between growing and fattening phases. In all intestinal segments, fattening pigs achieved higher values than growing pigs. This is in agreement with previous reports ,23, whicIn the present study, the ileum microbiota presented a significant interaction (PP × PT) in their richness. While microbial richness increased with age in F2 pigs, it remained constant between growing and fattening phases in Duroc pigs. In a previous report, ileum microbiota showed high variability in diversity indices across ages , suggestGut microbiota of both PPs clustered separately in the three intestinal segments in the PLS–DA graphical representations, evidencing a differential microbial composition throughout the productive period, as was previously described . In addiAt the phylum level, although no significant differences were detected in Firmicutes/Bacteroidetes ratio throughout the intestinal tract, it numerically decreased with age. The lower ratio correlates with the increasing proportion of Bacteroidetes phylum as pigs aged found in previous studies ,30.The study of gut microbiota between the two extreme PTs of pig aimed to elucidate certain microbiota traits responsible for the phenotypic differences. Duroc pig is a preferred breed for producing premium dry-cured products due to its high intramuscular fat content, and excellent fatty acid composition . To promPT also impacted the alpha diversity, with F2 pigs having significantly higher microbial diversity than Duroc pigs across the three intestinal segments, according to Shannon and Simpson indices. Moreover, F2 pigs also presented higher microbial richness (in cecum) and evenness (in ileum and colon). This implies that F2 pigs host a higher number of different taxa with more similar abundances. Considering that the rearing conditions throughout the experiment were controlled, these differences may be correlated with physiological traits. These results are in agreement with those obtained by several studies ,33,34 in2 = 0.63). Although no significant differences were found in their study, Firmicutes phylum was numerically higher in obese pigs [Regarding Firmicutes/Bacteroidetes ratio, the balance between these two dominant phylogenetic types in the gut microbiota has been closely related to adiposity and fat metabolism . Howeverese pigs , suggestese pigs  who founLactobacillus genus in high quality meat breeds [Clostridium is found in higher proportion in fattier pigs than in their lean counterparts [Streptococcus and Bifidobacterium genera, which were also higher in Jinhua pigs [Microbial community in ileum and cecum segments clustered separately between both PTs of pig through beta diversity analyses, although no genera with significantly different abundance could be identified. However, numerical differences in genera abundance between fatty and lean pigs are in accordance with previous studies. Other authors have already found slightly higher levels of t breeds ,44. Moreterparts ,33. Durohua pigs  than in Although breeding selection is generating highly efficient animals, there is still some questions over their gut microbiota adaptation to nitrogen-restricted diets. Two percentage units were reduced in the experimental LP diets with respect to SP. All diets were supplemented with essential amino acids to meet the nutritional requirements for both growing and fattening phases . This apAlthough gut microbiota diversity was not affected during the 15-day experiment, LP-fed pigs presented more complex microbial networks than SP-fed pigs along the three intestinal segments, especially in ileum and colon segments. In the cecum, this effect was registered only during the growing period. These results suggested that, although the limited impact of a 2% CP restriction, microbiota underwent a controlled adaptation process, revealing new relationships between microbes that may result in new metabolic pathways. A similar phenomenon was previously described in ruminants  and malnSeveral studies have described the variation in microbial populations across the intestinal segments, which are anatomically and functionally distinct. In addition, microbial populations are modified by inherent variations in the environmental conditions , transit time, substrate and the presence of gut receptors throughout the intestinal tract . The morIn addition, microbial composition is subjected to other factors such as genetics, age, diet and environmental conditions, therefore, it is difficult to compare the ratio of species abundance between different studies . FirmicuLactobacillus genus. Lactobacillus was also the most predominant genus of the core microbiota of all intestinal segments [Terrisporobacter and Clostridium) was also abundant, especially in the fattening phase, and showed similar pattern of change across intestinal segments [Bifidobacterium was more abundant in the upper intestine [As was previously defined , the mossegments ,51, withsegments ,51. Bifintestine , while Rntestine ,52. The The microbial community was mainly affected by the PP and PT of pig. Fattening pigs showed higher microbial richness than growing pigs, which is attributable to the higher maturity and stability of their microbial community. In addition, F2 pigs presented higher bacterial diversity and microbial network complexity, especially in the fattening phase, while Duroc pigs tended to have higher Firmicutes/Bacteroidetes ratio in cecum. The moderate restriction in dietary CP content increased the complexity of microbial interaction network, whereas it had limited impact on microbial community composition.